Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors.Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide  was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh.Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh.Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of somatostatin through SST2 receptors, resulting in enhancement of the acid response mediated by M3 receptors on parietal cells. These rcretions . It is gcretions . Howeverecretion . HoweverSomatostatin, a peptide hormone, regulates many physiological functions in the gastrointestinal tract . This peecretion . It is tIn the present study, we examined the effects of CCh on gastric acid secretion in wild-type (WT) and mAChR KO mice lacking M1–M5 receptors, and investigated the involvement of M4 receptors in the stimulatory action of CCh. We demonstrated the importance of M4 receptors in the cholinergic stimulation of gastric acid secretion and showed how this secretion can be modulated by the activation of M4 receptors, particularly focusing on the relation to D cell/somatostatin.ad libitum. All experimental procedures used were carried out in accordance with the Helsinki Declaration and have been approved by the Committee for Animal Experimentation established by Kyoto Pharmaceutical University.Age-matched male C57BL/6J mice, weighing 25∼30 g of WT and those lacking M1, M2, M3, M4, or M5 receptor, were used. The generation and characterization of each subtype of mAChR KO mouse strain has been previously described by others . AnimalsAcid secretion was measured in acute fistula stomachs of both WT and M1–M5 KO mice according to a previously published method . Under uTable 1. An aliquot of the RT reaction product served as a template in 35 cycles of PCR with 0.5 min of denaturation at 95°C and 1 min of extension at 68°C using the Advantage 2 polymerase mixture  in a thermal cycler . Total RNA was reverse-transcribed with a first strand cDNA synthesis kit . The sections were exposed to 3% bovine serum albumin solution for 1 h to reduce the non-specific binding of anti-sera. The sections were exposed to each primary antibody for 16 h at 4°C, and incubated with the appropriate secondary antibody for 2 h at a room temperature. The sections were mounted with VECTASHIELD mounting medium, including 4,6-diamidino-2- phenylindole . The preparations were observed using a fluorescence microscope  and photographed using an Olympus digital camera. The following primary antibodies were used: rabbit anti-mAChR M4 and goat anti-Somatostatin . Alexa Fluor 488 conjugated donkey anti-rabbit IgG and Alexa Fluor 546 conjugated donkey anti-goat IgG  were used as secondary antibodies.Expressions of mAChR M4 receptor and somatostatin were immunohistochemically examined in the gastric mucosa of WT or M4 KT mice. The stomachs were excised, rinsed with ice-cold PBS, and embedded in O.C.T. compound  iced with liquid COSerum levels of somatostatin were measured in both WT and M4 KO mice before and after the s.c. administration of CCh (30 μg/kg), according to a previously published paper . Thirty Drugs used were urethane , carbamylcholine chloride [carbachol: CCh], octreotide, CYN154806 , and atropine sulfate . CCh and atropine were dissolved in saline, while octreotide and CYN154806 were dissolved in dimethyl sulfoxide  and diluted with distilled water to desired concentrations. Each agent was prepared immediately before use and administered as a single injection s.c. or i.p. in a volume of 1 ml per 100 g body weight.t-test. Differences between multiple groups were evaluated with analysis of variance followed, when necessary, by a Dunnett’s multiple comparison test. Values of P < 0.05 were considered statistically significant.Data are presented as means ± SE. Differences between two groups were evaluated with the Student’s Figure 1A). Pretreatment of the animals with atropine  dose-dependently inhibited the increase of acid secretion in response to CCh, and the net acid output at 0.1 and 0.3 mg/kg was 2.8 ± 0.3 and 0.3 ± 0.1 μmol/2 h, respectively, both of which were significantly lower than that (7.9 ± 1.3 μmol/2 h) of control mice given CCh plus vehicle .Under urethane anesthesia the stomachs of WT mice spontaneously secreted acid secretion in almost negligible amount of less than 0.1–0.2 μmol/20 min. Subcutaneously administered CCh (30 μg/kg) significantly stimulated acid secretion; the acid secretion reached a peak value of 3.8 ± 0.5 μmol/20 min 20 min after the administration, followed by a gradual decrease to near basal levels 100 min later  of mAChRs involved in the regulation of gastric acid secretion, we examined the effects of CCh on acid secretion in the stomachs of mAChR KO mice lacking M1, M2, M3, M4, or M5 receptors.+ as basal secretion, and no significant difference was observed in the basal rates of the stomachs of these animals (data not shown). The stimulatory action of CCh  was similar between mAChR KO mice lacking M1 and M2 receptors and WT mice, with the net acid outputs being 8.0 ± 0.8 μmol/2 h and 6.8 ± 0.9 μmol/2 h, respectively, which were almost equivalent to that (8.1 ± 0.7 μmol/2 h) in WT mice . Although a slight decrease in the acid response to CCh was observed in M5 KO mice, the net acid output (6.4 ± 0.7 μmol/2 h) was not statistically significant from that of WT mice. In contrast, the acid response to CCh was significantly decreased in M3 and M4 KO mice, with the net acid outputs being 2.3 ± 0.9 μmol/2 h and 2.1 ± 0.5 μmol/2 h, respectively. As shown in Figure 3A, CCh-stimulated acid secretion was markedly decreased in M4 KO mice. In WT mice the secretion of acid reached a peak value of 4.7 ± 0.7 μmol/20 min with the net acid output being 8.5 ± 1.2 μmol/2 h, while in M4 KO mice the peak value was 1.0 ± 0.2 μmol/20 min with the net acid output being 1.8 ± 0.3 μmol/2 h, which was significantly lower than that in WT mice .The stomachs of both WT and various mAChR KO mice consistently secreted 0.1∼0.3 μmol/20 min of HSomatostatin has been shown to inhibit secretory and motor functions in the gastrointestinal tract and antagonizes the actions of several hormones . Since tFigures 4A,B). This inhibitory effect of octreotide was completely abrogated when CYN154806 (0.1 mg/kg), a SST2 antagonist, was given 20 min before the administration of octreotide; the net acid output in response to CCh was 9.5 ± 0.8 μmol/2 h, which was almost equivalent to that of WT mice.The basal secretion of acid in WT mice was very scanty with less than 0.1∼2 μmol/20 min and did not show significant changes following the s.c. administration of octreotide (20 μg/kg). However, the gastric acid response induced by CCh (30 μg/kg) was markedly reduced by the prior administration of octreotide; the net acid output was 0.7 ± 0.6 μmol/2 h, which was highly significantly different from that (8.5 ± 1.9 μmol/2 h) of WT mice . By contrast, CCh did not increase acid secretion in the stomachs of M4 KO mice; the peak value of acid secretion was 0.9 ± 0.1 μmol/20 min, while the net acid output was 1.0 ± 0.8 μmol/2 h, which was almost equivalent to that in the stomachs of M4 KO treated with saline. However, when M4 KO mice were pretreated i.p. with CYN154806 (0.1 mg/kg), the administration of CCh potently increased acid secretion; the peak value was 5.1 ± 0.3 μmol/20 min, while the net acid output was 10.1 ± 2.5 μmol/2 h, which was significantly greater than that of M4 KO mice without pretreatment of CYN154806. The decreased acid response to CCh in M3 KO mice was not affected by CYN154806 (data not shown).Subcutaneously administered CCh (30 μg/kg) markedly increased acid secretion in the stomachs of WT mice, the peak value of acid secretion was 4.2 ± 0.2 μmol/20 min and the net acid output was 8.2 ± 1.3 μmol/2 h . On the other hand, the immunostaining of M4 receptors was also observed in the same area of the stomach; the higher magnification showed the co-existence of M4 receptors with somatostatin (a left figure of the lower panel), suggesting the expression of M4 receptors on D cells. No expression of M4 receptors was detected in the stomach of M4 KO mouse (a right figure of the lower panel), although the immunostaining of somatostatin was observed in this animal similar to WT mouse (data not shown).To examine the presence of M4 receptors in D cells, we performed the immunostaining of the gastric mucosa with anti-somatostatin and anti-M4 receptor antibodies in WT mice. As expected, the expression of somatostatin was clearly observed in the stomach of WT mouse; it was stained red . On the other hand, the serum levels in WT mice were slightly but significantly decreased after the s.c. administration of CCh (30 μg/kg), the values being 0.22 ± 0.01 ng/ml. By contrast, the administration of CCh in M4 KO mice markedly increased the levels of somatostatin to 0.57 ± 0.04 ng/ml, the values being significantly higher than those in WT mice or those in M4 KO administered vehicle.Serum levels of somatostatin in WT mice under urethane anesthesia were 0.33 ± 0.04 ng/ml. The levels of somatostatin in M4 KO mice were slightly higher (0.40 ± 0.04 ng/ml) than those in WT mice, although the difference was not statistically significant . The expressions of mAChRs were also observed in the stomachs of KO mice lacking M1–M5 receptors, except lacking a respective mAChR subtype in the corresponding KO mice.Since it was found that both M3 and M4 receptors were involved in the stimulatory action of CCh on gastric acid secretion, we examined the gene expressions of mAChR subtypes, M1–M5, in the mouse stomach. The expressions of M1–M5 mRNAs were all observed in the stomachs of WT mice, though the intensity slightly differed depending on the subtypes , are widely expressed in many peripheral organs to include the gastrointestinal tract . In the We found that CCh, a muscarinic agonist, dose-dependently increased acid secretion in the stomach of WT mice, and this response was almost completely inhibited by the prior administration of atropine, confirming the mediation of this secretion by the activation of mAChR. Cholinergic stimulation of gastric acid secretion is thought to involve M1 receptors in addition to M3 receptors, because acid secretion was inhibited by pirenzepine, a selective M1 receptor antagonist . HoweverIn the present study, we found using KO mice lacking M1–M5 receptors that CCh stimulated acid secretion in M1, M2, and M5 KO animals as effectively as in WT mice, but the stimulatory effect was markedly attenuated in M3, and M4 KO mice. We observed a slight decrease in the acid response to CCh in M5 KO mice, but the net acid output was not significantly different from that of WT mice. The reason for the different results between the study of 2+ that mediates the secretion of acid as well as many hormones . It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells to result in enhancement of the acid response by removing the negative influence of somatostatin via the activation of SST2 receptors.Given the findings of the present study, we conclude that the cholinergic stimulation of gastric acid secretion is mediated by the activation of M3 receptors in parietal cells and modified indirectly by M4 receptors in D cells (KT authored the paper and designed the study; TE, SH, and TA performed the experiments; TE and SH performed data analysis and coauthored the paper; KT contributed to critical revision of the paper. All authors approved the submission of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-methyl-d-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18.The GluN2B subunit of the The effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a–GluN2A or GluN1a–GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50  values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a–GluN2B (84%) than those mediated by the GluN1a–GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a–GluN2B (62%) and the GluN1a–GluN2A (55%) subunits.The newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A. N-methyl-d-aspartate receptors (NMDAr) are inotropic glutamate receptors (iGluRs) that contribute to multiple neuronal functions such as [ such as –3. Howev such as , 5. Func such as , 6–8. Th such as , 10. The such as . An incr such as . Increas such as .NMDAr are similar to other iGluRs in that each subunit has four domains, which include the amino-terminal domain (ATD), an agonist/ligand binding domain (ABD), a trans-membrane domain (TMD) and a carboxyl terminal domain (CTD) –15. In GLQDNQDLIRDKSN) and EAR18 (GEDDYQDAQDLIRDKSN) were designed in the Bioinfo Grip laboratory at the National University of Colombia  to interact with the ligand binding domain (LBD) of the GluN2B subunit. The peptide design and their in silico evaluation was achieved by using point mutations on the conantokin-G sequence and evaluating their binding capacity with AutoDock Vina [http://pepcalc.com) and have similar hydrophobicity levels: 19.36 and 19.65, respectively . Permeability was predicted using CellPPD (http://crdd.osdd.net/raghava/cellppd/index.html) which provided negative CPP values for these peptides , indicating that they are non-cell permeant . The peptide synthesis was made by a commercial supplier . These peptides show no modifications at either terminal, their N terminal is a free amine and the C terminal is a free acid  as previously described [For each peptide a three-dimensional structure model was generated using UCSF  Chimera software , and a escribed .Rattus norvegicus). This amino acid sequence includes the amino terminal (ATD) and the ligand binding (LBD) domains. The GluN2B structural model was predicted using I-TASSER , this sch.edu/, . This tech.edu/, . The C-sch.edu/, . The TM-ch.edu/, , the selhttp://www.ebi.ac.uk/pdbsum/, [The selected GluN2B model (using I-TASSER) was further evaluated using PDBsum , that ppdbsum/, . The selpdbsum/, . The modpdbsum/, , includipdbsum/, .http://vina.scripps.edu/, [Molecular docking was performed with the obtained three-dimensional structures of the peptides and of the GluN2B subunit by using “Autodock Vina” . A Gridhttp://flexpepdock.furmanlab.cs.huji.ac.il/index.php, [http://www.cgl.ucsf.edu/chimera/, [http://accelrys.com/products/discovery-studio/visualization.html).The Docking results were then analyzed with the Rosetta FlexPepDock web server , which dex.php, . The dochimera/, ).Interac2 followed by cervical dislocation. Day 18 embryos (E18) were removed by caesarian section. Dissociated hippocampal cultures were prepared as previously described [4 cells/well, 24 well plates) with neurobasal medium supplemented with B27  or with 2% bovine fetal serum and used between 7 and 37 days in vitro; the data was collected from 57 cells that had on average 14.9 days in vitro (14.9 ± 10.8 mean ± SD). Coverslips were pre-treated by placing 50 µL of poly-d-Lysine  and Laminin  in the middle of the coverslip for 5 min at room temperature (RT).Pregnant rats were maintained and used following the guidelines approved by the New York University Langone Medical Center Institutional Animal Care and Use Committee. Pregnant rats were killed using COescribed , 30. CelThe plasmids pEGFP-NR1a (GluN1a) (Addgene plasmid # 17926), pEGFP-NR2B (GluN2B) (Addgene plasmid # 17925), and pEGFP-NR2A (GluN2A) (Addgene plasmid # 17924) were a gift from Stefano Vicini . HEK-2932, 10 Hepes, 10 d-glucose, pH 7.4. Mg2+ was omitted to prevent the voltage-dependent block of the NMDAr ion currents [+ channels [Electrophysiological recordings were performed under voltage-clamp by using the whole cell conformation , 33. Intcurrents , 36. Thechannels ; 20 μM CCurrents were measured using a 2-electrode voltage clamp amplifier Axopatch 200B . Output from the voltage clamp amplifier was sent to a microcomputer using a data acquisition interface . Currents were filtered at 2.5 or 5 kHz (Axopatch 200B) and sampled at 10–50 kHz. For analysis the current traces were filtered at 1 kHz. Pclamp 10.4 (Axon Instruments) was used for data acquisition and data analyses.2+ currents of similar magnitude over time  followed by a 10 s pulse of bath solution through a puffer (flow rate: 250 μL/min) located directly on top of the cells . Successive stimulations with NMDA were done every 4 min, which under control conditions resulted in NMDA-evoked CaWhen comparing two groups with various stimulations we used Two-way ANOVA  followed by Sidak’s multiple comparison post-test. When comparing more than two groups with various stimulations we used Two-way ANOVA followed by Dunnett’s multiple comparison post-test, as recommended by GraphPadPrism 7.02. Results are expressed as mean and standard error mean (mean ± S.E.M.).2+ currents was similar between consecutive stimulations  is conserved (71.9% identity) and of the LBD is highly conserved (95.5% identity) between GluN2A and GluN2B , 41, 42.Figure As observed in hippocampal neurons, in HEK-293 cells transfected with recombinant NMDAr the simultaneous removal of the NMDA + EAR16 (or NMDA + EAR18) also resulted in a short-lived increase in the NMDA-evoked current, suggesting that the off rate of EAR16 (and EAR18) was fast enough that would allow binding of NMDA early on during washout.Figure In the case of EAR18, the interactions involve 9 hydrogen bridges Fig. , 3 of thIn this study we demonstrated that the peptides EAR16 and EAR18 can inhibit NMDA-evoked currents in hippocampal neurons in a dose-dependent and highly reversible manner. We also demonstrated that these peptides inhibited NMDA-evoked currents evoked by recombinant NMDAr containing the GluN1a–GluN2B subunits. However, only EAR16 showed higher selectivity for GluN1a–GluN2B over GluN1a–GluN2A.Conantokin-G is a 17 amino acid peptide (MW 2264.2 Da) blocks NMDA-evoked currents in hippocampal  and cortThe prediction for the three-dimensional structure for EAR18 and EAR16 was performed using a hydrophilic environment. Figure The Met 739 residue in GluN2B, is not present in the GluN2A subunit and has been reported to be a molecular determinant for the selective antagonist of conantokin-G on GluN2B over the GlunN2A subunit , 51. ThePreviously by testing various conantokin analogs, it was found that the presence and location of Gla appeared to be important to confer to the conantokin analogs selectivity for the GluN2B subunit over the GluN2A subunit . We founThere are only two amino acids difference between EAR18 and EAR16, in EAR16 these are a Leucine (Leu 5) and an asparagine (Asn 8), which in EAR18 correspond to tyrosine (Tyr 5) and alanine (Ala 8). Our results are consistent with previous observation in which it has been reported that replacement of Leu 5 with Tyr in conantokin-G results in a reduction on the conantokin-G selectivity for GluN2B .To determine whether the amino acid substitutions in EAR16 results in selectivity for only the GluN2B, additional studies are required measuring its selectivity for GluN2B over the other two GluN2 subunits, the GluN2C and GLuN2D.With respect to the predicted free energy of binding, one would expect that EAR18  would have a higher affinity and potentially a higher interaction with GluN2B than EAR16 . However, their affinity is comparable, and EAR16 shows an apparent higher selectivity for GluN2B than GluN2A, than EAR18. Hence, neither the lower free energy of binding or the higher number of interactions of EAR18 over EAR 16 predict selectivity for GluN2B with respect to GluN2A.The IC50 value for EAR16 and EAR18 is high as compared to that displayed by conantokin-G. The reported IC50 for conantokin-G is 0.48 µM in hippocampal  and cort2+. Second, we measured the selectivity of these peptides between GluN2B and GluN2A. This was done in part because the amino acid sequence corresponding to that used for the GluN2B model (1-818 amino acids) is conserved (71.9% identity) and of the LBD (44 amino acids) is highly conserved (95.5% identity) between GluN2A and BluN2B [This study has several limitations. First, we did not measure whether the peptide block was voltage-dependent; neither whether the peptide block was affected by the presence of physiological levels of Mgd BluN2B , 41, 42.We found that EAR16 and EAR18 block in a reversible manner NMDA-evoked currents in hippocampal neurons. Moreover, EAR16 showed selectivity for the GluN2B over GluN2A. The high reversibility of both of these peptides and the selectivity of EAR16 for GluN2B, make them attractive as potential pharmacological agents.


The dielectric properties were investigated in the temperature region of 26–650 °C. The dielectric diffuse anomaly in the dielectric relaxation was found in the high temperature region of 600–650 °C with increasing the measuring frequency, which was related to the dynamic thermal process of ionized oxygen vacancies generated in the high temperature. Two phase transition points were detected during heating, which were found to coexist from 150 to 200 °C. Electric field induced ferroelectric to antiferroelectric phase transition behavior of the (Pb0.97La0.02)(Zr0.95Ti0.05)O3 ceramics was investigated in this work with an emphasis on energy storage properties. A recoverable energy-storage density of 0.83 J/cm3 and efficiency of 70% was obtained in (Pb0.97La0.02)(Zr0.95Ti0.05)O3 ceramics at 55 kV/cm. Based on these results, (Pb0.97La0.02)(Zr0.95Ti0.05)O3 ceramics with a large recoverable energy-storage density could be a potential candidate for the applications in high energy-storage density ceramic capacitors.(Pb Microstructure and electrical properties of PLZT2/95/5 ceramics can be improved by powder processing or sintering. Generally, the AFE to FE phase transition in PZ occurs around 225 °C on heating [OA) with eight formula units in the unit cell, whereas the FE phase has a rhombohedral structure (FR) possibly with two formula units [FR phase transforms into a cubic paraelectric (CP) phase (PE) on heating above 231 °C [3+, Ba2+, Sr2+ at Pb2+-site and Ti4+, Sn4+ at Zr4+-site in PZ, such as in (Pbx1-Bax)ZrO3 ceramics, Pb0.95Sr0.05(Zr0.5Ti0.5)O3 ceramics, O3 ceramics [Electrical capacitors display an extremely high powder density but their energy storage density needs further improvement ,2,3,4. Aructures . In the elements ,8,9,10.  heating . The stre 231 °C . This phe 231 °C , electrie 231 °C  or hydroe 231 °C . For exaceramics ,12. 1/3Nb2/3)O3-PbTiO3, (Bi0.5Na0.5)TiO3-Ba0.85Ca0.15Ti0.9Zr0.1O3, Ba0.95Ca0.05Zr0.3Ti0.7O3, etc. [Currently, the energy-storage behaviors of AFEs in bulk ceramics have attracted increasing attention. Compared with FE materials, AFE materials possess more abundant phase transition behavior, which is larger than those of FE and linear dielectrics ,16. BaseO3, etc. ,18,19,203 and efficiency of 70%. These results demonstrated that the (Pb0.97La0.02)(Zr0.95Ti0.05)O3 ceramics may be quite promising candidates for dielectrics in high energy density electrical capacitors due to the reversible electric field-induced FE to AFE phase transition. In this work, high temperature dielectric relaxation behavior was observed in PLZT2/95/5 ceramics, and the relaxation mechanism related oxygen vacancies was discussed. The recoverable energy-storage density calculated from hysteresis loops reached about 0.83 J/cm0.97La0.02)(Zr0.95Ti0.05)O3 (PLZT2/95/5) ceramics were fabricated by a conventional mixed oxide solid-state reaction method. Raw materials of Pb3O4, TiO2, ZrO2 and La2O3 were weighed according to the formula PLZT2/95/5 in the required stoichiometry with 5 wt % excess Pb3O4. Precursor oxides were mixed by ball milling in ethanol for 24 h, then dried and presintered at 850 °C in an alumina crucible for 5 h. After being remilled and dried, the calcined fine powder with 5 wt % PVA as a binder was cold pressed into cylindrical pellets of size 12 mm diameter and 1–2 mm thickness using a hydraulic press. The ceramics were fabricated by sintering at 1250 °C for 5 h. In order to avoid the vaporization, Pb3O4 atmosphere for the sintering was maintained using PZT95/5 as spacer powder.(PbP-E) loops were characterized by Radiant Technologies Precision premier II  over the temperature range of 30–200 °C. Ceramic samples were polished to the thickness of 0.8 mm for the measurement of electrical properties. Both sides of samples were electroded with silver paste and sintered at 650 °C for 2 h. The crystal structure and orientation of the ceramic samples were characterized by using an X-ray diffractometer  with working current and voltage of 20 mA and 36 kV, respectively. The temperature dependence of dielectric and impedance properties were measured by Agilent E4980A in the temperature range of 26–650 °C with the heating rate 3 of °C/min. The polarization-electric field (*), which matched with the pyrochlore phase. These phases arose due to the volatization of PbO and over sintering at high temperature [2+ (0.149 nm) are larger than that of La3+ (0.136 nm), which indicated smaller lattice parameters of PLZT2/95/5 ceramics.perature . The phaε' and imaginary ε'' parts of relative permittivity for PLZT2/95/5 ceramics at various frequencies are plotted in ε' displayed almost the same under different frequencies, as we can clearly see in To = 155 °C), whereas the maximum anomalous peak was linked with the transformation of FE to PE phase (CT = 225 °C) at the higher temperature. The samples exhibited FE and AFE coexistence state when T exceeds oT and PE state when T exceeds CT [CT = 225 °C) obtained in PLZT2/95/5 bulk materials [ε'' parts of relative permittivity were observed with hollow circles, which is consistent with the dielectric measurements. The first anomaly occurred near the AFE to FE transition temperature, whereas the second one took place around the FE to PE transition temperature. The similar phenomenon was reported by some researchers [Temperature dependence of the real ceeds TC . The dieaterials ,25 or abaterials . No dielaterials ,25. In Fase TC = 5 °C at tearchers . Moreoveearchers . The samearchers . The higearchers . P-E loops. The hysteresis loops were observed with an electric field 55 kV/cm and this obviously manifested the FE and AFE coexistence phase. For example, EFE-AFE declined slightly from 55 to 50 kV/cm, which was attributed to the free energy barrier between FE and AFE with the rising temperature [P-E hysteresis loops, which was ascribed to the AFE to FE phase transition.In order to gain a better study of FE and AFE phase transition behaviors, the hysteresis loops of the PLZT2/95/5 ceramics are shown in perature . Under tperature . The samperature ,23, whicJreco (the blue area) of the PLZT2/95/5 ceramics at 200 °C. Generally, the energy storage density could be estimated from the P-E loops, which was calculated with the following equations [E is the applied electric field, and Pr and Pmax represent the remanent polarization and maximum polarization, respectively. From these equations, it was evident that Jreco values of certain materials could be improved by increasing their operating electric-fields and polarization. For the application of dielectric capacitors in practice, a higher energy-storage efficiency η is also always desired. The energy storage efficiency η was calculated as the following formula:Jloss is the energy loss density in quations :(1)Jst=Jreco value for the PLZT2/95/5 ceramics was changed from 0.12 to 0.83 J/cm3, and the corresponding η value was varied from 40% to 70%, showing temperature stability of the energy storage performance. In practical application, people often desire a higher energy-storage efficiency η and lower energy-loss density Jloss. Jreco values and has the lowest Jloss values over the measurement range. For example, the Jreco value for the PLZT2/95/5 ceramics was varied from 0.28 to 0.83 J/cm3, and the corresponding η value was varied from 40% to 70% as the electric field increases from 30 to 55 kV/cm. Under the same conditions of applied electric field, the experimental data showed much higher values of Jreco than those reported in BaTiO3-SrTiO3 composites, BaSrTiO3 ceramics, and BaTiO3 ceramics [P-E data were taken from 30 to 200 °C (which was less than the transition temperature). Hence, energy storage properties over 200 °C have not been obtained.The energy storage density and the energy-storage efficiency measured at 10 Hz are presented in ceramics ,34,35. H3 ceramics [In order to analyze the high temperature dielectric behavior, impedance analysis is conducted as a powerful technique which has been effectively used for probing into electrical properties of the electro-ceramic materials, such as conductivity, dielectric behavior and relaxation characteristic ,37. The ceramics . Z″/Z″max) with frequency at different temperatures is shown in Z″/Z″max value exhibited a peak at each temperature and moved to higher frequency with increasing temperature, which can be as an evidence of hopping mechanism, in which the numbers of polaron will gradually decrease as it hopped with gradual decrease of electron lattice coupling [T,ω0a, E, and βk are the absolute temperature, characteristic frequency, activation energy, and the Boltzmann constant, respectively. The experimental data were fitted with the above equation, as shown in aE was about 1.61 eV. Based on our result of aE value, it can be reasonably concluded that the high-temperature dielectric behavior of PLZT2/95/5 ceramics may be ascribed to the dynamic thermal process of ionized OVs [The variation of normalized imaginary part of impedance (coupling . In perocoupling :(4)ωp=ωized OVs . The resized OVs . The movized OVs ,43. The ized OVs . It is taE value, it can be reasonably concluded that the high-temperature dielectric behavior of PLZT2/95/5 ceramics may be ascribed to the dynamic thermal process of ionized OVs. The energy-storage density and efficiency calculated from hysteresis loops achieved about 0.83 J/cm3 and 70%, respectively. It indicated that these PLZT2/95/5 ceramics may be a promising material for energy storage ceramic capacitors applications. In summary, PLZT2/95/5 ceramics were successfully prepared by a conventional mixed oxide solid-state reaction method. The hysteresis loops were observed in temperature range of 150–200 °C, where FE and AFE were found to coexist. The high temperature dielectric relaxation behaviors related to OVs of PLZT2/95/5 ceramics were investigated, and the relaxation behavior was analyzed with activation energy about 1.61 eV. Based on our result of


Bradyrhizobium elkanii strains BLY3-8 and BLY6-1, which are incompatible with Rj3 genotype soybean cultivars. The genome sequences of these strains will be useful to identify a causal gene for this incompatibility.We report here the draft genome sequences of  Bradyrhizobium elkanii is known to have the ability to form nodules on soybean (Glycine max) root (B. elkanii strains BLY3-8 and BLY6-1 is restricted by soybean cultivars CNS (Rj3 gene in soybeans (ax) root . Howevervars CNS  and D51 soybeans . In ordeB. elkanii strains BLY3-8 and BLY6-1 was extracted using the ISOPLANT kit  and fragmented to 400 bp using a Covaris S2-A . The libraries for sequencing were prepared by a NEBNext DNA library prep master mix set for Illumina  and paired-end sequenced (2 × 300 bp) on a MiSeq sequencer using a MiSeq reagent kit version 3 . Raw reads were trimmed and de novo assembled using CLC Genomics Workbench 7.5 . Parameters for the trimming were as follows: ambiguous limit, 0; quality limit, 0.01; number of 5′ terminal nucleotides, 10; number of 3′ terminal nucleotides, 14; minimum length, 150 bp. The default settings were used for the de novo assembly parameters .Genomic DNA of B. elkanii strains BLY3-8 and BLY6-1 were assembled into 90 and 79 contigs, respectively. The accumulated lengths of these genomes were 9,198,916 bp  and 9,202,892 bp , respectively. The G+C content of both genomes was 63.8%. The total numbers of coding sequences (CDSs) predicted by the Prokaryotic Genome Annotation Pipeline (PGAP 3.1) were 8,036 and 8,049, respectively.The draft genomes of B. elkanii strains BLY3-8 and BLY6-1 have been deposited at DDBJ/EMBL/GenBank under the accession numbers LWUI00000000 and LXEM00000000, respectively. The versions described in this paper are versions LWUI01000000 and LXEM01000000, respectively.The whole-genome shotgun projects for


ABCB1 c.3435C>T, CYP3A4*1B (g.-392A>G), CYP3A5*3 (g.6986A>G) SNPs and artemether + lumefantrine treatment outcome in 103 uncomplicated malaria patients from Angola. No significant associations with the CYP3A4*1B and CYP3A5*3 were observed, while a significant predominance of the ABCB1 c.3435CC genotype was found among the recurrent infection-free patients (p < 0.01), suggesting a role for this transporter in AL inter-individual performance.Malaria treatment performance is potentially influenced by pharmacogenetic factors. This study reports an association study between the  Artemisinin combination therapy (ACT) has contributed to the remarkable decline by 48% in the malaria mortality rate between 2000 and 2015 [Artemether–lumefantrine (AL) is the most adopted antimalarial by national malaria control programs worldwide. In Angola, it represents the first-line treatment of choice for uncomplicated malaria.Upon AL oral administration, artemether shows an elimination half-life of 1–3 h, CYP3A4 being the main enzyme involved in its conversion towards the  dihydroartemisinin (DHA) metabolite . Only <1CYP3A4 and ABCB1 pharmacogenetic characteristics influencing AL performance.Both lumefantrine and DHA are essentially eliminated through the bile. In the apical biliocanalicular membrane of the hepatocyte, the ABCB1 (MDR1/Pgp) ATP-binding cassette (ABC) transporter is a major biliary efflux pump, particularly for lipophilic substrates, as lumefantrine , 8. SignCYP3A4 and ABCB1 tag SNPs, particularly the promoter located g.-392A>G in the former (CYP3A4*1B) and the synonymous c.3435C>T in the latter, did not yield significant associations [ABCB1 c.3435C>T with altered LUM exposure among HIV positive subjects under Efavirenz based therapy.A previous attempt to correlate lumefantrine pharmacokinetic (PK) parameters with In the present work, we hypothesized that small pharmacokinetic differences might have observable pharmacodynamic consequences, the parasite reaction being a more sensitive parameter of the individual pharmacogenetic influence. Parasite clearance on day 3 post-treatment, recurrent infection prevalence and the 28-day cure rate endpoint of adequate clinical and parasitologic response (ACPR) were herein used as parameters to assess the effect of the patient pharmacogenetic status on AL in vivo anti-parasite performance. To test this hypothesis, a previously performed AL efficacy trial was analysed.Plasmodium falciparum malaria involved in an AL  efficacy trial in the Luanda region, Angola, conducted during the 2011–2013 period [2, D3, D7, D14, D21 and D28. At each time-point, thick blood films were examined for the presence of parasites and a capillary blood sample obtained for PCR analysis.One-hundred and three unrelated patients with microscopy confirmed  uncomplicated 3 period . ClinicaEthical approval was obtained from the Angolan National Public Health Institute/Ministry of Health Ethics Committee. All procedures followed the latest version of the Declaration of Helsinki.® Classic Card, Whatman). DNA extraction was done by phenol–chloroform methods. The ABCB1 c.3435C>T, CYP3A4 g.-392C>G and CYP3A5 g.6986A>G SNPs were analysed by established PCR–RFLP protocols [pfmsp2 PCR amplification of all samples at all time spots [pfmsp2 positive PCR by D3, informative concerning the artemisinin partner performance, in accordance with WHO guidelines; and, (b) pfmsp2 positive PCR during the 28-day follow up (lumefantrine prophylactic effect). The number of clinical failure events (PCR-corrected recrudescence) was too small to warrant their specific analysis. All recrudescences were included in the general recurrence group.Capillary blood sample were collected on filter paper  test and Z statistics were used to determine significant differences between proportions . Five individual associations were herein tested: recurrence vs CYP3A4*1B allele status, recurrence vs ABCB1 c3435C>T SNP status, recurrence vs CYP3A5*3 SNP status, D3 positivity vs CYP3A4*1B SNP status, and D3 positivity vs ABCB1 c.3435C>T SNP status). Accordingly, a Bonferroni-corrected significance threshold at p < 0.01 was considered for these tests. Multivariate Log-linear analysis was performed in order to specifically investigate associations of key three categorical variables: (a) D28 follow-up outcome (recurrence), (b) ABCB1/MDR1 c.3435C>T genotype, and (c) pfmdr1 N86Y status of the initial infection. The multiple testing was Benjamini–Hochberg corrected, assuming a 10% false discovery rate (q).Data on SNPs were analysed using IBM SPSS version 23. Chi square  had positive PCR. During the 28-day follow up, 29/103 patients experienced recurrent parasitaemia, as detected through PCR. Ninety-eight patients were successfully analysed for the CYP3A4 -392A>G SNP. The genotype frequency in this Angolan population was 0.112  for the wild type (g.-392AA), 0.541  minor allele homozygous (g.-392GG) and 0.347  for the heterozygous (g.-392AG). The population was found in Hardy–Weinberg equilibrium for this locus (p > 0.05).The 28-day PCR-corrected cure rate was 91.3%. On DCYP3A4*1B genotype was not found to be significantly associated with either D3 parasite positivity    for the wild type (c.3435CC), 0.079  for the minor allele homozygous (c.3435TT) and 0.158,  for the heterozygous (c.3435CT). The population was found in Hardy–Weinberg equilibrium for this locus (p > 0.05).Concerning the c.3435C>T SNP was also not significantly associated with the D3 parasite PCR positivity   (Table The CYP3A5*3 (c.6986A>G) in 84 samples, as this is the allele is the most investigated as having a robust deleterious effect in the expression of the gene [c.6986AA), 0.048,  for the heterozygous (c.6986AG) and 0.202  for the minor allele homozygous (c.6986GG). The sample population was found in Hardy–Weinberg equilibrium for this locus (p > 0.05).During the completion of the present work, a new report has suggested CYP3A5 as a contributor to lumefantrine metabolism . Followithe gene . GenotypCYP3A4*1B, no significant association was observed between the patient status for carrying a CYP3A5*3 alleles and the parasitological outcome during the 28 day follow up , namely the 2 = 10.84, df = 4, p = 0.0284 vs p (corrected) = 0.0286), and the association between recurrence during follow-up and the ABCB1 c3435C>T status  = 0.0143), after filtering the effect of the presence/absence of the pfmdr1 N86 allele  of 10% for Benjamini–Hochberg multiple test correction, only two associations stood out as near the threshold of significance: the overall interaction between the three analysed variables (GCYP3A and ABCB1 pharmacogenetics and AL pharmacodynamics in vivo endpoints. D3 positivity was not significantly associated with the patient CYP3A4*1B status (Table P. falciparum parasites. Pharmacogenetic driven variable rates of artemether to DHA bioconversion are likely not to be readily visible in terms of the artemisinin effect on the infection. As for the ABCB1 c.3435C>T, the negative observations possibly result from the specific capacity of this transporter to efflux more lypophilic compounds then the final phase II glucuronidated DHA extracted from the liver.The present work focused on finding links between patient CYP3A4*1B and CYP3A4*3 status were not seen to influence the risk of malaria recurrence. The AL post-treatment protective effect is essentially related with the action of lumefantrine, the long half-life partner. In regular conditions, it is expected that the large majority of lumefantrine is eliminated unchanged [CYP3A4*1B and CYP3A5*3 SNPs in modulating AL post-treatment prophylactic action.The nchanged , a resulnchanged . This menchanged . Also, inchanged -and as sABCB1 c.3435TT genotype was found among patients suffering recurrent infections during the 28-day follow up, suggesting a role of the encoded P-glycoprotein. The synonymous c.3435C>T polymorphism has been proposed to be linked with altered rates of protein synthesis, leading to proteins that albeit having the same primary sequence, emerge from the process of translation with different tertiary conformations [ABCB1 c.3435CC genotype among the recurrence-free patients, signalling an increased lumefantrine exposure associated with this genotype, which better shielded the recovering patients from new infections.A significant increase in the frequency of the rmations . The funABCB1 c.3435TT genotype, was suggested to be associated with a significantly decreased D7 lumefantrine levels among patients undergoing malaria treatment with AL. Such an effect in drug exposure can explain the increase risk of these patients towards recurrent infection.A shortcoming of the present study is the unavailability of pharmacokinetic data, namely D7 LUM levels, in order to have a complete pharmacokinetic/pharmacogenetic picture. Nevertheless, it is interesting to note that the present results are in agreement with recent data from Maganda et al. , where tThese data suggest lumefantrine as part of the group of ABCB1 substrates where this genotype is associated with increased drug exposure, probably due to a less efficient efflux. Other examples include tacrolimus , 23, silABCB1 c.3435C>T SNP in the performance of artemether–lumefantrine. The present observations join other recent reports pointing for the importance drug transporter pharmacogenetics in ACT pharmacokinetics and pharmacodynamics [By exploring potential pharmacodynamics/pharmacogenetic associations in anti-malarial therapy, this report shows a non-negligible influence of the host dynamics , 27.


Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read—thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB.Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × 10−4–1 × 10−5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × 105) patient sample.Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h.Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of The online version of this article (doi:10.1186/s13104-017-2649-y) contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis (M. tb)  restriction sites; while a T7 terminator was placed downstream of the Xh0I (5740) and a Xh0I(5740) restriction site. An established purified culture of E. coli BL21 (DE3) was transformed with the recombinant TMKmt gene-carrying pET30a plasmid. A single colony was inoculated into medium ampicillin and kanamycin. Cultures were incubated at 37 °C at 200 rpm. IPTG was introduced for induction. SDS-PAGE was used to monitor the expression. Samplings were isolated at 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 h; and cell pellets isolated by centrifugation. Cell pellets were lysed by sonication, and precipitate after centrifugation was dissolved in urea. Fractions were pooled and refolded followed by 0.22 uM filter sterilization. Proteins were analysed by MALDI-TOF and SDS-PAGE by using standard protocols for molecular weight and purity measurements. The concentration was determined by Bradford assay with Bovine serum albumin (BSA) as a standard  and 2Incubator, USA) for up to 13 days. 10 ml of 2 Marcfland (6 × 108 CFU) standard solution were prepared from each plate using sterile distilled water and subsequently emulsified in 200 ml of Middlebrook 7H9  with tweenR80  supplemented with OADC. The culturing bottles were incubated at 37 °C in incubator shaker . (b) Collection samples for mRNA assay: 0.2 ml aliquots were collected at time intervals of 2.5, 3, 5, 8, 11, 15, 21, 33, 100, 124, 172, and 196 h. The aliquots were collected in triplicates and stored in RNAprotectR —a stabilizing reagent for RNA and kept in a −80 °C freezer  prior to total genomic RNA extraction. (c) Estimating the number of CFUs: Serial dilution, spread-plate technique was used to estimate the number of CFUs that could be recovered from each bottle each day as the broth was incubated throughout. 1 ml from each culture bottle each day was diluted fivefold in Middlebrook 7H9 and 0.1 ml of each dilution was plated on Middlebrook 710 solid media in triplicates. The CFUs were visually counted every after 14 days and recorded and the average was recorded. (d) Extracting total genomic mRNA and converting it into cDNA: The aliquots were thawed from the freezer, centrifuged and the cell pellet harvested from which total RNA was extracted using RNeasy® Mini kit  which was subsequently treated with DNase enzyme using RNase-Free DNase Set . Afterwards the RNA samples were assessed for purity by measuring their absorbance at 260 and 280 nm in Tris EDTA buffer using NanoDrop1000 Spectrophotometer  and only samples with 260/280 ratio of approximately 2 were considered for downward assays. Eventually the RNA was converted to cDNA using ProtoScript® First Strand cDNA Synthesis Kit . (e) Running Real time PCR assays: Conditions for real time PCR assays were optimized in a conventional T100™ Thermocycler  using genomic M. tb DNA with different dilutions employing in-house designed primers for amplification of a 329 bp product length of TMKmt gene (F and R respectively 5-GTTGGTGGAAAAGCTGTCCG-3 and 5-ATTCGATCCGCTGAACCCAG-3)., Additional file ® Imaging System . The conditions were once again optimized on to Rotor Gene Q thermocycler  for real time assay using 2xQuantitect SYBR green PCR mix  instead of Taq2x master mix but keeping the rest of reagents plus their volumes as well as the reaction conditions constant. Details of Optimization of qRT-PCR of TMKmt and M. tb Pol1A are shown in Additional file M. tb PolA 1 gene that code for DNA polymerase 1 was also assayed for its expression using the exact conditions as TMKmt but using in-house designed primers 5′-ACCCAAAGCCTTGCATGAG-3′ and 5′-ACCGGCACTTTCCATCTTC-3′ as forward and reverse respectively targeting a TMKmt amplicon of 382 bp. (f) TMKmt Ag detection enzyme immuno-assays: For detection of TMKmt Ag in frozen centrifugal supernatants of samplings from purified cultures of the Ugandan genotype 1 and H37Rv preserved in SDS, (1) several study aliquotes were prepared by dissolving 1 μl of each sampling in 1000 μl of freshly prepared phosphate buffered saline. (2) 100 µl of resultant serum-diluent was then pipetted into duplicate wells of a sterile 96-well microtiter plate (Nunc) and the plate incubated overnight. (3) The plated wells were then blocked once the following day using 5% BSA in PBS and incubated at 37 °C for 30 min, after which excess solution was discarded and plate left to dry. Blank wells were also made, by adding only PBS rather than sample. (4) TMKmt specific rabbit polyclonals (either PAb-0655 or PAb-0656) were added, and wells incubated at 37 °C for 30 min, after which excess solution was discarded and plate left to dry. The wells-in-use were then washed with PBS three times using an automated plate-washer. (5) 100 µls of goat anti-rabbit IgG horse-radish peroxidase conjugate was added, and the plates incubated at 37 °C for another 1 h. During this incubation, the enzyme–substrate was prepared by adding 1 volume of substrate (TMB) to 1 volume of diluent (hydrogen peroxide) in volumes enough for all thewells in use. (6) 200 µl of freshly prepared substrate was added to each well . (7) The reaction was stopped by adding 100 µl of dilute (1 mol/l) H2SO4. The intensity of the reaction in each well was hence after determined by reading the plate at an optical density (OD) of 450 nm using a single filter of an automated ELISA plate reader .Isolates of the Ugandan genotype 1 used in this study were retrieved from the Mycobacteriology Level III facility at MakCHS, Kampala, Uganda. There were around seventy-seven samples where the genotype of interest (Uganda genotype 1) was represented by more than six strains. Thus six representative strains for the genotype were retrieved, thawed and inoculated onto Middlebrook 7H10  solid media. Two strains that showed acceptable growth fitness patterns (at least showing colonies on the thirteenth day) and no contamination (this was the fitness test) were selected for use in the study. (a) Culture of Isolates: Isolates were thawed and individually streaked onto Middlebrook 7H10 and incubated in a Carbon dioxide incubator at 37 °C  several study aliquotes were prepared by dissolving 1 µl of each sputum-dilution in 1000 µl of freshly prepared phosphate buffered saline. (2) 100 µl of resultant serum-diluent was then pipetted into triplicate wells of a sterile 96-well microtiter plate (Nunc) and the plate incubated overnight. (3) The plated wells were then blocked once the following day using 5% BSA in PBS and incubated at 37 °C for 30 min, after which excess solution was discarded and plate left to dry. Blank wells were also made, by adding only PBS rather than sample. (4) TMKmt specific rabbit polyclonals (either PAb-0655 or PAb-0656) incubated at 37 °C for 30 min, after which excess solution was discarded and plate left to dry. The wells-in-use were then washed with PBS three times using an automated plate-washer. (5) 100 µls of goat anti-rabbit IgG horse-radish peroxidase conjugate was added, and the plates incubated at 37 °C for another 1 h. During this incubation, the enzyme–substrate was prepared by adding 1 volume of substrate (TMB) to 1 volume of diluent (hydrogen peroxide) in volumes enough for all thewells in use. (6) 200 µl of freshly prepared substrate was added to each well . (7) The reaction was stopped by adding 100 µl of dilute (1 mol/l) H2SO4. The intensity of the reaction in each well was hence after determined by reading the plate at an optical density (OD) of 450 nm using a single filter of an automated ELISA plate reader .An intial volume 1 μl of a 1+ AFB patient sample was homogenously diluted (by steering on an automated shaker at 100 rpm) in 10 μls of phosphate buffered saline (PBS). Subsequently, 1 μl of this resultant 1 × 10Additional file 1. This file offers details of recombinant TMKmt cloning, expression and purification in E. coli BL21 (DE).Additional file 2. This file offers details of the expression profile of recombinant TMKmt in E. coli BL21 (DE).Additional file 3. This figures shows optimization curves of qRT-PCR for TMKmt and M.tb Pol1A.Additional file 4. This figures depicts gel electrophoris analysis of PCR amplicons of TMKmt and M.tb Pol1A. Note the 22kDA placement of the TMKmt amplicon relative to protein marker M.Additional file 5. This figure shows TMKmt Ag levels in serial dilutions of 1+ acid fast baccili ladden patient sputum sample detected by PAb-0656. Direct Enzyme Immuno-assay (EIA) were conducted using two custom polyclonal antibodies (PAb-0655 and PAb-0656) in the 1x10-1, 1x10-2, 1x10-3, 1x10-4, 1x10-4, 1x10-5, 1x10-6 dilutions of sputum containing  approximately 104, 103, 102, 10, 1, and 0.1 CFUs/ml of acid fast bacilli are respectively. Note that relative to the blank, TMKmt Ag was detected upto 1x10-4 to 1x10-5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1x105) patient sample by PAb-0655 and PAb-0656, respectively.Additional file 6. This figure shows the GraphPad combined TMKmt antigen expression profile among pure cultures of Uganda genotype 1 detected by PAb-0655 and PAb-0656. In comparision, the cyclic pattern for expression of TMKmt Ag among H37RV (not shown), was different from the Ugandan genotype 1. Specifically, H37RV demonstrated an early but short lived spike in TMKmt Ag levels between 0 and 3 hours post innoculation, with the next spike emerging at 12 hours. This early spike in H37RV TMKmt Ag expression profiles, may explain why this lab strain grows faster than pathogenic strains. Important to note is that, regardless of these differences in cyclic patterns of TMKmt Ag expression, the Ugandan genotype displayed higher amplitudes of TMKmt mRNA expression.Additional file 7. This file offers details of TMKmt Ag expression among the Ugandan genotype 1 and H37RV.Additional file 8. This figure shows TMKmt Ag levels in serial dilutions of 1+ acid fast baccili ladden patient sputum sample detected by PAb-0656 conjugate. Direct Enzyme Immuno-assay (EIA) were conducted using PAb-0656 conjuagte in the 1x10-1, 1x10-2, 1x10-3, 1x10-4, 1x10-5, 1x10-6 dilutions of sputum containing  approximately 104, 103, 102, 10, 1 and 0.1 CFUs/ml of acid fast bacilli are respectively. Note that relative to the blank, TMKmt Ag was detected upto 1x10-5 (containing 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1x105) patient sample by PAb-0656.Additional file 9. This figure shows GraphPad combined TMKmt Ag levels in serial dilutions of 1+ acid fast baccili ladden patient sputum sample detected by PAb-0655 and PAb-0656 conjugates, respectively. Direct Enzyme Immuno-assay (EIA) were conducted using either PAb-0655 or PAb-0656 conjuagte in the 1x10-1, 1x10-2, 1x10-3, 1x10-4, 1x10-5, 1x10-6 dilutions of sputum containing  approximately 104, 103, 102, 10, 1, and 0.1 CFUs/ml of acid fast bacilli are respectively. Note that relative to the blank, TMKmt Ag was detected upto 1x10-4 and 1x10-5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1x105) patient sample by PAb-0655 and PAb-0656, respectively.Additional file 10. This file offers details of raw and adjusted ODs for TMKmt Ag detection in each serial dilutions of a 1+ AFB patient sputum sample.Additional file 11. This file offers details of cross validated by BLAST across the NCBI microbial- databases.


We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked.Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαβ-motif. These toxins contain a toxin signature (K-C These proteins play important roles in cellular signaling processes, such as regulating heart rate and insulin secretionv channels are considered to be ideal pharmacological targets for the development of new therapeutic drugs to treat cancer, autoimmune diseases and cardiovascular, neurological and metabolic disorders. For instance, Kv1.3 constitutes a promising target for treatment of autoimmune diseases, such as multiple sclerosis, as this channel is overexpressed in activated effector memory T cells23Voltage-gated potassium channels , based on the similarity between the primary structures of those toxins56v channels possess a conserved functional core composed of a key basic residue (Lysine or Arginine) associated with a key hydrophobic or aromatic residue  within a 6.6 ± 1-Å distance. Such a functional dyad can be found in a broad range of structurally unrelated peptides from various animals, such as scorpions, cone snails, snakes, and sea anemones8v channels strongly suggest that the functional dyad on its own cannot represent the minimal pharmacophore or prerequisite for Kv1 binding4-X-N)  are key functional residues of α-KTxsLeiurus quinquestriatus hebraeus, to the Shaker potassium channel in Drosophila10v1.3  are predicted to form hydrogen bonds, whereas side chains of Lysine27 directly enter into the pore region to contact the backbone carbonyls of Tyrosine78 on the channel filter v channel in complex with the α-KTx charybdotoxin, though in this complex the location of the Asparagine slightly differs from the NMR-based complex model1214Scorpion toxins, among others, are well reported to interact with Kv blockers are used in clinical settings. For instance, 4-aminopyridine, a Kv1 channel blocker, was marketed as a treatment for multiple sclerosis as it improved the walking speed of patients in phase III clinical trialsv1.5 and Kir3.1/3.4, was approved in Europe as a new antiarrhythmic drug, as it was effective in terminating acute-onset atrial fibrillationv blockers entered clinical trials nowadays, and hence, await results on their efficacy in specific diseases, many K+ channel modulators lack specificity and have significant off-target toxicitiesv blocking compounds in order to treat Kv-related diseases.To date, only few Kv blocking compounds, we focused on another family belonging to the superfamily of cysteine-stabilized αβ (CSαβ) peptides, namely plant defensins. Plant defensins are small, basic, cysteine-rich peptides with antimicrobial activity against a wide range of microorganisms18v channels. Note that introduction of this scorpion toxin signature in the sequence of insect defensins, which does not contain such toxin signature, results in potassium channel blocking activityIn the search for tools to further develop novel Kin silico, based on the presence of the potassium toxin signature sequence. Based on this analysis, we found that the Arabidopsis thaliana defensin AtPDF2.3 sequence contains a partial toxin sequence, and hence, we heterologously expressed this peptide, along with various AtPDF2.3 variants, either bearing a KC, a CXN or a KCXN toxin signature. We tested the ability of all these recombinant (r)AtPDF2.3 variants to block potassium channels. In addition, we investigated a possible link between ion channel inhibitory function of AtPDF2.3 and its variants, and their antifungal activity.In the present study, we analyzed the potential of several plant defensins to interact with potassium channels Arabidopsis thaliana  and even pM  affinities33v1.3-specific peptide isolated from the venom of the scorpion Vaejovis mexicanus smithi. Vm24 inhibits Kv1.3 channels of human lymphocytes with pM affinityThe α-KTx6.1, also known as Pi1, is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pichia pastoris and recombinant (r)AtPDF2.3 was purified using cation exchange and reversed phase chromatography. At least 70 mg/L of culture of purified rAtPDF2.3 was obtained, which was verified by LC-MS.As shown in v1.1–Kv1.6, Kv2.1, Kv4.2, Shaker IR, and hERG) and four cloned voltage-gated sodium channels  (v channels at 5 μM (n ≥ 4) , Shab (Kv2.1), Shal (Kv4.2), and hERG (Kv11.1) subfamilies. These data suggest that rAtPDF2.3 acts as a toxin active on Kv channels, and more specifically, on Kv1.2 and Kv1.6 channels.rAtPDF2.3 was subjected to testing against a wide range of ion channels. The peptide’s activity was investigated on 14 cloned voltage-gated potassium channels (K DmNav1) . rAtPDF2 (n ≥ 4) , whereasv channels and determined IC50 values, i.e. the rAtPDF2.3 concentration resulting in 50% inhibition of the Kv1.2 and Kv1.6 channels. The corresponding IC50 values for Kv1.2 and Kv1.6 are 1.3 ± 0.2 μM and 978 ± 113 nM, respectively  and that rAtPDF2.3 current inhibition is attributed to obstruction of the pore, rather than to altered channel gating. Furthermore, the observation that there is no difference on the percentage-induced block in ND96 or HK leads to the conclusion that channel blockage is independent of the direction of the potassium current flux and is not influenced by the extracellular concentration of potassium ions. Altogether, these experiments imply that current inhibition upon rAtPDF2.3 binding does not result from changes in the voltage dependence of channel gating. The inhibition of Kv1.2 channels occurs rapidly and blocking is reversible because the current recovers quickly and completely upon washout (data not shown). It can thus be assumed that rAtPDF2.3 exerts its Kv channel inhibiting activity by physically blocking the channels, a phenomenon described previously for many Kv channel toxins isolated from scorpions, snakes, cone snails and sea anemones among others3637Next, we determined dose-response curves for rAtPDF2.3’s action against the different Kectively . Kv1.2 cobserved . We furtobserved . rAtPDF2observed . In ND96observed . It can v1.2 and Kv1.6 channels, rAtPDF2.3 variants were produced which contain either only Asparagine36 (rAtPDF2.3[K33A][G36N]), both Lysine33 and Asparagine36 (rAtPDF2.3[G36N]) or neither of these (rAtPDF2.3[K33A]). The native rAtPDF2.3 only contains Lysine33, as indicated in v1.2 and Kv1.6 channels and dose-response curves were constructed  previously identified in scorpion potassium toxinsstructed .v channels, with IC50 values of 611 ± 91 nM and 138 ± 38 nM for Kv1.2 and Kv1.6 channels, respectively, which are 2.1- and 7.1-fold decreased as compared to rAtPDF2.3, respectively. For AtPDF2.3 [K33A][G36N], the IC50 values are 1380 ± 133 nM (Kv1.2) and 366 ± 114 nM (Kv1.6). A reduced activity is observed for rAtPDF2.3[K33A] as compared to rAtPDF2.3, with IC50 values of 2039 ± 383 nM for Kv1.2 and 3500 ± 235 nM for Kv1.6 channels. In conclusion, rAtPDF2.3[G36N] is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A] is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN toxin signature in the plant defensin sequence to block potassium channels.We found that rAtPDF2.3[G36N] has an increased potency as compared to rAtPDF2.3 in blocking KSaccharomyces cerevisiae  and the Asparagine at position 30 (Asparagine30) were found important for channel inhibitory activityF. graminearum, indicating that the toxin signature is not important in determining antifungal activity of this plant defensin. In order to further investigate a potential role for potassium channels in rAtPDF2.3’s antifungal activity, we investigated the effect of rAtPDF2.3 against yeast strains with deletions in genes involved in potassium transport and homeostasis. Note that orthologues of the oocyte Kv channels studied here have not been identified in yeast. Hence, in this study, we focused on all yeast proteins known to be involved in potassium transport and homeostasis in general. Potassium homeostasis is important in yeast, as high intracellular levels are required for many physiological processes, such as protein synthesis, enzyme activation and regulation of intracellular pH40v channels. Hence, potassium transport in yeast seems important for governing AtPDF2.3-specific tolerance. Yet, the mechanisms underlying the observed differences between both peptides in Kv channel blocking activity on one hand, and antifungal tolerance mechanisms on the other hand, remain to be elucidated.In addition, we show that there are no differences in antifungal activity between rAtPDF2.3 and its variants against TRK1 results in a reduced growth rate of this strain, and hence, Trk1p might play an intrinsic role in yeast growth in addition to potassium homeostasis, this protein is suggested to play a role in tolerance towards rAtPDF2.3, since modulators of this protein, including Hal5p, Sat4p and Arl1p, seem to gain tolerance towards rAtPDF2.3 as well. Trk1p is a component of the Trk1p-Trk2p potassium transport system in yeast, which plays a major role in potassium uptake44C. albicans. Similarly, Trk1p was shown to be required for fungicidal activity by lactoferrin 11, bactenecin 16 and virion-associated protein VPR 12 against C. albicans, pointing to Trk1p as a functional effector of these compoundsC. albicans by the human defensins HNP-1, hBD-2 and hBD-3S. cerevisiae. Since HAL5, SAT4 and ARL1 are modulators of the Trk1p-Trk2p transport system, it is not surprising that the corresponding knockout strains were found hypersensitive towards rAtPDF2.3 treatment as well. More specifically, Hal5p activates the Trk1p-Trk2p transport systemHAL5 results in impairment of Trk1p-Trk2p function. Similarly, Sat4p functions as a regulator of the Trk1p-Trk2p transport system and is partially redundant with Hal5pARL1 encodes for a soluble GTPase that was shown to regulate potassium influx via regulation of SAT4 and HAL5ARL1 might result in deregulation of Hal5p and Sat4p, which on its turn would lead to impairment of Trk1p-Trk2p function.Although deletion of QDR2 is involved in mediating tolerance towards rAtPDF2.3, but not rHsAFP1, treatment. Qdr2p functions as a plasma membrane transporter of many mono- and divalent cationsQDR2, in addition to QDR3, confers resistance to cisplatin and bleomycin in yeastQDR2 was found to affect tolerance to oxidative stress, as strains overexpressing and lacking QDR2 exhibited phenotypes when reactive oxygen species-producing agents, such as hydrogen peroxide and menadione, were added to the growth medium. As several plant defensins are reported to induce reactive oxygen species in yeasts  interacting with plant potassium channels to, for instance cause pollen tube burst, as was previously shown for ZmES1-4 from maizein planta.In conclusion, we show that the Plant defensins and scorpion toxins were aligned matching their cysteine residues, using the COBALT alignment toolPichia pastoris strain X33 was used for heterologous production of AtPDF2.3. Botrytis cinerea ), Verticillium dahliae (MUCL19210), Fusarium culmorum , Fusarium oxysporum  and Fusarium graminearum  WT and ∆gcsCandida albicans (SC5314) and Saccharomyces cerevisiae WT (BY4741 and BY4743), ∆ipt1ipt1/∆skn1http://www.euroscarf.de) were used in a yeast growth inhibitory assay and bioscreen assays.P. pastoris was cultured in YPD , BMGY , 1% glycerol, 100 mM K3PO4 pH 6, 4 × 10−5% biotin) or BMMY , 0.5% methanol, 100 mM K3PO4 pH 6, 4 × 10−5% biotin). Plant pathogenic fungi used in the fungal growth inhibitory assay were grown in half strength PDB (1.2% potato dextrose broth). C. albicans and S. cerevisiae WT and knockout strains were grown in minimal medium , 6.7 g/L yeast nitrogen base w/o amino acids , 20 g/L glucose).All culture media were purchased from LabM (UK), unless stated otherwise. For heterologous production, P. pastoris expression system as previously described for recombinant HsAFP1via automated tangential flow filtration  and rAtPDF2.3 was purified by cation exchange chromatography, using 75 mL SP sepharose High Performance resin . For purification of the rAtPDF2.3 variants, a 5 mL SP sepharose High Performance column  was used. Unbound peptides were removed via washing steps with 20 mM sodium phosphate buffers at pH 6.8. The flow rate was maintained at 5 mL/min. Elution of the peptides was carried out by a washing step with 50% (v/v%) elution buffer  for 10 column volumes (CV), followed by a linear gradient to 100% (v/v%) elution buffer in 15 CV, resulting in a peak at approximately 75% (v/v%) elution buffer for rAtPDF2.3, 71% for rAtPDF2.3[K33A], 80% for rAtPDF2.3[G36N] and 69% for AtPDF2.3 [K33A][G36N]. The eluted fraction was further purified by reversed phase chromatography employing a Gemini C18 250 × 10 column  and acetonitrile (ACN) for elution of the bound peptides. The flow rate was maintained at 4.6 mL/min. Elution of the peptides was carried out by a washing step at 0% (v/v%) ACN for 1.2 CV, followed by a linear gradient to 45% (v/v%) ACN in 5.9 CV. rAtPDF2.3 was eluted at 28%, rAtPDF2.3[K33A] at 29%, rAtPDF2.3[K33A][G36N] at 26% and rAtPDF2.3[G36N] at 25%. The eluted fraction was vacuum dried by centrifugal evaporation , re-dissolved in MilliQ water and subjected to a microbicinchoninic acid assay  according to the manufacturer’s instructions, to determine the protein concentration. Bovine serum albumin served as a reference protein. At least 70 mg/L of culture of purified rAtPDF2.3 was obtained. The yields of the rAtPDF2.3 variants were much lower, i.e. in a range of 1.5 mg/L to 45 mg/L of culture. Recombinant HsAFP1 was produced and purified as described previouslyRecombinant AtPDF2.3 and its variants were produced using the pPICZαA transfer vector and the v1.1, rKv1.2, hKv1.3, rKv1.4, rKv1.5, rKv1.6, Shaker IR, hKv3.1, rKv4.3, and hERG) in Xenopus laevis oocytes, the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion). The harvesting of stage V–VI oocytes from an anaesthetized female X. laevis frog was previously described2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4), supplemented with 50 mg/L gentamycin sulfate.For the expression of the voltage-gated potassium channels  or HK (in mM): NaCl, 2; KCl, 96; CaCl2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4). Voltage and current electrodes were filled with 3 M KCl. Resistances of both electrodes were kept between 0.7 and 1.5 MΩ. The elicited currents were filtered at 0.5 kHz and sampled at 2 kHz (for potassium currents) or filtered at 2 kHz and sampled at 20 kHz (for sodium currents) using a four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. Kv1.1–Kv1.6 and Shaker currents were evoked by 250 ms depolarizations to 0 mV followed by a 250 ms pulse to −50 mV, from a holding potential of −90 mV. Current traces of hERG channels were elicited by applying a +40 mV prepulse for 2 s followed by a step to −120 mV for 2 s. Kv2.1 and Kv4.2 currents were elicited by 500 ms pulses to +20 mV from a holding potential of −90 mV. Sodium current traces were, from a holding potential of −90 mV, evoked by 100 ms depolarizations to Vmax . In order to investigate the current–voltage relationship, current traces were evoked by 10 mV depolarization steps from a holding potential of −90 mV. To assess the concentration dependency of the toxin induced inhibitory effects, a concentration–response curve was constructed, in which the percentage of current inhibition was plotted as a function of toxin concentration. Data were fitted with the Hill equation: y = 100/[1 + (IC50/[toxin])h], were y is the amplitude of the toxin-induced effect, IC50 is the toxin concentration at half-maximal efficacy, [toxin] is the toxin concentration, and h is the Hill coefficient. GV curves were calculated from IV relationships as follows: gK = IK/(Em−EK) with EK = (RT/zF)ln[K #0]o/[K #0]i. In these equations gK represents the conductance, IK the potassium current, Em the membrane potential, EK the reversal potential, R the gas constant (8.31 J/K mol), T the temperature, z the charge of the ion (for K+ ions: z = 1), F the Faraday’s constant (96.500 C/mol), [K #0]o and [K #0]i respectively are the extracellular and intracellular K+ ion concentrations. The values of IK or gK were plotted as function of voltage and fitted using the Boltzmann equation: gK/gmax = [1 + exp(Vg−V)/k]−1, were gmax represents maximal gK, Vg is the voltage corresponding to half-maximal conductance, and k is the slope factor. Comparison of two sample means was made using a paired Student’s t test (P < 0.05). All data represent at least three independent experiments (n ≥ 3) and are presented as mean ± standard error.Two-electrode voltage-clamp recordings were performed at room temperature (18–22 °C) using a Geneclamp 500 amplifier  controlled by a pClamp data acquisition system . Whole cell currents from oocytes were recorded 1–4 days after injection. Bath solution composition was ND96 (in mM): NaCl, 96; KCl, 2; CaCl4 spores/mL of the fungus. The IC50 value, which is the concentration required for 50% growth inhibition as compared to control treatment, was determined by microscopy after 48 hours of incubation. The antifungal activity of rAtPDF2.3 and rHsAFP1 against S. cerevisiae and S. cerevisiae knockout strains was analysed according to the standard CLSI protocol M27-A36 cells/mL was suspended in MM for rAtPDF2.3 or MMH (MM supplemented with HEPES pH 7) for rHsAFP1 and added to a two-fold dilution series of the peptides in water. The IC50 value was determined by spectrophotometry (OD490nm) after 24 hours of incubation. Sigmoidal curves were generated with GraphPad Prism , using nonlinear regression. IC50 values were derived from the whole dose-response curves. All data represent at least three independent experiments (n ≥ 3); IC50 values are presented as mean ± standard error. ANOVA followed by Dunnett post hoc test was performed to analyse statistically significant differences between the rAtPDF2.3 or rHsAFP1 IC50 values of the wild types and those of the knockout strains.The antifungal activity of rAtPDF2.3 and its variants against a range of plant pathogenic fungi was analysed following the protocol previously described by Osborn and colleaguesS. cerevisiae and the S. cerevisiae knockout strains that were found hypersensitive towards rAtPDF2.3 . Cultures were grown for 50 hours at 30 °C, shaking, and the OD600nm was measured every 15 min. Growth rates were determined employing the equation Bioscreen assays  were carried out to determine the growth rate of AtPDF2.3 . OvernigHepG2 cells were seeded at 10.000 cells/well in 96-well plates and incubated for 24 hours. Subsequently, cells were treated either with water (untreated) or rAtPDF2.3 (0.01 μM–23.5 μM) for 24 hours after which cell viability was determined using the ‘Cell Proliferation Kit II (XTT) as described previouslyHow to cite this article: Vriens, K. et al. The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels. Sci. Rep.6, 32121; doi: 10.1038/srep32121 (2016).


Here, we demonstrated that multiple H+ sensors, such as ASICs, TRPV1 and proton-sensing G protein coupled receptors (GPCRs) are involved in extracellular acidification-induced intracellular calcium ([Ca2+]i) elevation. By using calcium imaging measures, we observed that both ASIC and TRPV1 channels inhibitors suppressed the [Ca2+]i elevation induced by extracellular acidosis in cultured rat cardiac myocytes. Then, both channels mRNA and proteins were identified by RT-PCR, western blotting and immunofluorescence. ASIC-like and TRPV1-like currents were induced by extracellular acidification, suggesting that functional ASIC and TRPV1 channels jointly mediated extracellular calcium entry. Furthermore, either pre-exhaustion of sarcoplasmic reticulum (SR) Ca2+ with thapsigargin or IP3 receptor blocker 2-APB or PLC inhibitor U73122 significantly attenuated the elevation of [Ca2+]i, indicating that the intracellular Ca2+ stores and the PLC-IP3 signaling also contributed to the acidosis-induced elevation of [Ca2+]i. By using genetic and pharmacological approaches, we identified that ovarian cancer G protein-coupled receptor 1 (OGR1) might be another main component in acidosis-induced release of [Ca2+]i. These results suggest that multiple H+-sensitive receptors are involved in “Ca2+ transients” induced by acidosis in the heart.Acidosis has been known to cause “Ca Several studies have shown that the increase of [Ca2+]i is induced by the influx of extracellular Ca2+ via membrane Ca2+ channels or Na+/Ca2+exchangers32+ also can be resulted from the mobilization of sarcoplasmic reticulum (SR) during acidosis5+-sensitive ion channels or receptors, such as acid-sensing ion channels (ASICs), transient receptor potentialvanilloid-1 (TRPV1) and a group of proton sensing G protein coupled receptors (GPCRs). All of them could be activated by acidosis and mediate “Ca2+ transients”.Acidosis is a noxious stimulus that often comes from inflammation, ischemia or other pathological conditions. It is known to cause “CaC. elegans. Until now, the proton-sensitive members have found to be encoded by four different genes (ASIC1-ASIC4) with six protein subunits cloned, which are ASIC1a (ASIC or BNaC2), ASIC1b (ASIC1β), ASIC2a , ASIC2b (MDEG2), ASIC3 (DRASIC) and ASIC4 (SPASIC)78+ ions. Some of ASIC subunits, such as homomeric ASIC1a2+.ASICs belong to amiloride-sensitive epithelial sodium channel family in vertebrates and the degenerin family of 2+ TRPV1 is a polymorphic sensor to sense various stimuli, such as pain, temperature, tension and acid12Ovarian cancer G protein-coupled receptor 1 (OGR1), T cell death-associated gene 8 (TDAG8), G protein coupled receptor 4 (GPR4) and G2 accumulation (G2A) are all belong to proton-sensing GPCRs family, they share 40–50% homology with different action modes and signaling pathways2+ transients” and above H+-sensitive ion channels or receptors. During the process of diseases that cause acidification, ASICs, TRPV1 and proton-sensing GPCRs would sense external changes of pH value, then induce the opening of ion channels and influx of calcium ion, resulting in the activation of various intracellular signaling cascades. Therefore, cardiac H+ sensors will have an important significance in cardiac diseases.Here, we investigate the relationship between cardiac “Ca2+ imaging measurements in cultured rat cardiac myocytes. As shown in 2+]i) was observed when the pH value of extracellular solution was rapidly lowered from 7.4 to 6.0 or 5.0. This elevation is reversible and could be stably induced when another acidosis solution was given again. The peak of ΔF/F was 1.37 ± 0.08 at pH 6.0 (n = 15 cells) and 2.93 ± 0.22 at pH 5.0  inhibited the increase of [Ca2+]i by 50.07 ± 5.70%  and 46.67 ± 4.25% , respectively. The inhibitory effect was mostly recovered after washout  with 100 μM amiloride, and recovered mostly after washout. Thus, ASIC currents in cardiomyocytes displayed similar electrophysiological property as that in nervous system. Local decrease in extracellular solution to pH 7.0 was sufficient to induce an ASIC-like current, and the amplitudes of ASIC current were increased gradually along with the decrease in pH from 7.4 to 7.0, 6.0, 5.0 and 4.0. Fitting with Hill equations, the curve of pH-current density was “S” shape, and the pH50  was 5.73 ± 0.18  stimulation and CPZ (20 μM) blocked it largely , a neuronal TRPV1-specific inhibitor  to 1.03 ± 0.12  is the inhibitor of SR Ca2+-ATPase that can exhaust the Ca2+ content, so TG was used here to examine the role of SR. The results showed that the extracellular acidification-induced [Ca2+]i elevation in the absence of extracellular Ca2+ was almost totally abolished by TG (3 μM) with the inhibition ratio of 90.59 ± 1.46%  and U73122, the inhibitor of PLC in cytoplasm were measured here. As shown in 2+ elevation evoked by extracellular acidification in the absence of extracellular Ca2+, the inhibitory ratios were 49.80 ± 4.85% (n = 15 cell) and 53.93 ± 5.14% , respectively. To rule out the influence of DMSO on responses of cardiomyocytes to acidification, we measured the acidic solution (pH 5.0)-triggered Ca2+ signals with incubation of 0.1% DMSO, and no significant decay of Ca2+ signals was observed after DMSO exposure , and Cu2+, the inhibitor of the protonation of extracellular histidines residues in OGR1, could inhibit this elevation to 0.46 ± 0.05  of Ca2+ transient induced by pH 5.0 solution  in rat ventricular cardiomyocytes, only OGR1 was considered to be the primary protein that transferred extracellular proton signals to Gq proteinssolution . Taken t2+]i in cultured rat cardiac myocytes; We then identified that ASICs and TRPV1 were expressed in the cardiac myocytes and jointly mediated extracellular calcium entry; Thirdly, another sensor of extracellular protons OGR1 was also found in cardiomyocytes, it mediated the IP3R-gated mobilization of intracellular Ca2+ in SR via OGR1-PLC-IP3-IP3R signaling pathway.In the present study, we first found that extracellular acidification can induce an elevation of [Ca2+]i followed by various functional changes in cardiomyocytes2+]i in a pH-dependent manner. Since H+-sensitive ion channels or receptors may be one of the mediators for the influx of extracellular Ca2+, the effects of ASICs and TRPV1 were investigated. The antagonists of ASIC and TRPV1 channels inhibited in part the augmentation of [Ca2+]i, respectively. Meanwhile, blockade of both ASIC and TRPV1 channels inhibited the elevation of [Ca2+]i more significantly, and the inhibition ratio was similar to the effect of EGTA, where extracellular Ca2+ was absent. Therefore, ASIC and TRPV1 channels contributed prominently to the acid-evoked Ca2+ influx.Acidosis is a detrimental condition accompanied with some cardiac disease, for example, myocardial ischemia. The outcome of acidosis is the increase of i that triggered by extracellular acidification, suggesting the possible contributions of intracellular Ca2+ stores in acidosis-induced elevation of [Ca2+]i. Under absence of extracellular Ca2+ conditions, when the Ca2+ contents in SR were exhausted by using TG, the elevation of [Ca2+]i was almost totally abolished, demonstrating that acidosis elevated [Ca2+]i via prompting the release of Ca2+ from SR in cardiomyocytes. Although it is well accepted that SR can be mobilized by ryanodine receptor activation3 receptor has attracted more and more attentions recently, especially for their functions in cardiomyocytes. The activity of IP3 receptors can be directly potentiated by intracellular IP3, triggering the release of Ca2+ that elicits excitation-contraction coupling or cardiac hypertrophy, and predisposes ventricle to arrhythmia272829303 receptor with 2-APB significantly attenuated the acidification-induced [Ca2+]i elevation in cardiomyocytes, which was also observed in the specific inhibitor of PLC, U73122-treated groups, thus, IP3 receptors should mediate the extracellular acidification-induced [Ca2+]i elevation via IP3-PLC signaling. However, the possible contributions of RyRs to the [Ca2+]i elevation could not be completely ruled out. In fact, there is a clear potential for cross-talking between RyRs and IP3R-mediated Ca2+ signaling pathways in heart muscle2+ stores to acidification-induced [Ca2+]i elevation would be further investigated in cardiomyocytes.In the present study, the depletion of extracellular Ca3-generated PLC is mediated by Gq protein that are usually coupled with GPCRs33192+ mobilization from SR by acidification. Using specific antibody for OGR1, we also examined the existence of OGR1 in ventricular cardiomyocytes, and calcium imaging found that OGR1 had a relative high sensitivity to protons, when extracellular pH was lowered from 7.6 to 7.0, it has already been activated. Furthermore, the possible involvement of OGR1 in the proton-induced activation of PLC and the following mobilization of intracellular Ca2+ were also tested by application of Cu2+. Pre-incubation of Cu2+ significantly attenuated the elevation of [Ca2+]i via binding to essential histidine residues in OGR12+ was properly non-specific, the specificity of Cu2+ for proton sensing GPCRs other than OGR1 has not been reported. Hence, we concluded that OGR1 mediated the IP3R-gated mobilization of intracellular Ca2+ in SR via sensing extracellular protons.The activation of IP2+ transients” in cultured rat cardiac myocytes. As H+-sensitive ion channels, ASICs and TRPV1 can be activated by acidosis solution and mediate extracellular Ca2+ entry. On the other hand, OGR1 mediates the mobilization of intracellular Ca2+ from SR via OGR1-PLC-IP3-IP3R signaling pathway  and approved by the Committee of Animal Care of Huazhong University of Science and Technology.In these studies, all experimental protocols were conducted in accordance with Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health . Every 3–5 minutes, the digest was sucked into the prepared Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1:1)  with 10% fetal bovine serum , and 100 U/ml penicillin – streptomycin to terminate digesting. About repeating 5–6 times, the shivers could be digested completely. The dissociated cells were collected by centrifugation at 118 × g for 6 minutes and suspended again in DMEM/F-12. Bromodeoxyuridine  at a final concentration of 0.1 mM was added during the first 36 hours to prevent proliferation of cardiac fibroblasts. Myocyte purity was about 95% 48 hours after plating. The cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere incubator. Experiments were performed on days of 3–5.Firstly, neonatal Sprague-Dawley rats (1–2 days old) were euthanized by decapitation, ventricular tissues were excised and washed three times in phosphate-buffered saline (PBS), and then were cut into shivers with the size less than 1 mm−1 intraperitoneally) before decapitation, and then the heart was removed rapidly and retrogradely perfused with normal Tyrode solution containing (mM): NaCl 135, KCl 5.4, MgCl2 1, CaCl2 1.8, NaH2PO4 0.33, HEPES 10, Glucose 10 and pH adjusted to 7.2 with NaOH, and then with nominally Ca2+-free Tyrode solution for 5 minutes. Subsequently, Ca2+-free Tyrode solution with 0.25 mg/ml collagenase , 0.15 mg/ml protease  and bovine serum albumin  was perfused through the heart for 8–9 minutes. The perfusate was oxygenated with 100% oxygen and kept constant at 36–37 °C. The digested ventricular myocardium were excised and stored in normal Tyrode solution at room temperature for later experiments.Rat ventricular myocytes were prepared by enzymatic dissociationTM FirstStrand cDNA Synthesis system for RT-PCR kits . Methods were similar to our previous report with slightly modified36Total RNA of cultured ventricular myocytes were isolated and cDNA was synthesized with the RevertAid3VO4, 1 PMSF, and with 1% Nonidet P-40, and protease inhibitor cocktail . The lysates were centrifuged at 12000 × g for 15 minutes, and the supernatant was recovered. After denatured, equal amounts of lysate proteins were separated on 10% SDS/PAGE gels, followed by transferred to nitrocellulose membranes . After blocking, the proteins were probed with the appropriate primary antibodies against ASIC1, ASIC2a, ASIC3, TRPV1  and OGR1 . Membrane-bound primary antibodies were detected using proper secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique37Cultured cardiomyocytes were lysed on ice for 30 min inlyses buffer containing (in mM) 50 Tris - HCl (pH 7.4), 1 EDTA, 100 NaCl, 20 NaF, 3 NaVentricular myocytes were fixed with 4% paraformaldehyde in 0.01 M PBS, pH 7.4 for 30 minutes and then rinsed three times with PBS for 10 minutes. Followed by permeabilized with PBS/0.3% TritonX-100 for 30 minutes, cells were blocked with 2% goat serum and 1% BSA in PBS for 1 hour, and then incubated with 1:50 ASIC1, ASIC2a, ASIC3, TRPV1 or OGR1 antibody in PBS/0.3% TritonX - 100/1% BSA/2% goat serum overnight at 4 °C. After rinsed three times in PBS, cells were incubated with 1:100 goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated  IgG in PBS containing 0.3% TritonX-100, 2% goat serum and 1% BSA for 1 hour at room temperature. After washed three times in PBS, cells were mounted on glassslides with 50% glycerin and imaged using a confocal laser scanning microscope . For double-label immunofluorescence, a mouse monoclonal antibody Troponin I (C-4)  was used to mark myocardium with goat anti-mouse Rodamine conjugated  IgG, or Hoechst33258 was used as nucleus marker.2·6H2O, 5 EGTA, 2 MgATP, 10 HEPES, pH 7.4 with Tris-OH. Cardiac myocytes were voltage-clamped at −80 mV throughout the experiments. A multibarrel perfusion system was used to achieve a rapid exchange of extracellular Tyrode solution (see above), pH adjusted to 7.4, 7.0 and 6.0 with Tris-OH. MES was used instead of HEPES to buffer bath solution pH ranging from 6.0 to 5.0 and 4.0. All experiments were performed at room temperature (22–25 °C).The ASIC current was induced by rapidly lowering pH for 6 seconds and TRPV1 was for 10 seconds.For cultured rat ventricular myocytes, the beating cells during 3–5 days cultured were selected; for acute isolating cells, quiescent, rod-shape cardiomyocytes showing clear striations were selected to perform patch-clamp recording after 15–20 minutes rest at room temperature. The whole-cell patch-clamp techniques were performed in a voltage-clamp mode with HEKA EPC-10  as our previous describe36382·6H2O, 2 mM CaCl2, 10 mM Glucose, 10 mM HEPES, pH 7.4 with Tris-OH) and incubated with 1 μM Fura-2/AM for 20 minutes at 37 °C. Followed by three washes and additional incubation in standard extracellular solutions for 30 minutes, the coverslips were then transferred to a chamber fixed on the movable stage of an inverted microscope . Fura-2/AM loaded cells were illuminated at 340 nm for 150 milliseconds and 380 nm for 50 milliseconds at 1-second intervals using a TILL Polychrome monochromator . Fura-2 fluorescence emission was imaged at 510 nm by an intensified cooled charge coupled device  through an IX-70 fluorescence oil immersion lens  and a 460 nm long-pass barrier filter. Paired F340/F380 fluorescence ratio images were acquired every second for [Ca2+]i. Ratio images (340/380 nm) were analyzed by TILLVISION 4.0 software. The amplitude of [Ca2+]i transient (∆F) represented the difference between baseline concentration (F) and the transient peak response to the stimulation, and [Ca2+]i response amplitude was defined as the normalized variations of [Ca2+]i (∆F/F)37Cultured cardiac myocytes grown on glass coverslips were washed three times with standard extracellular solutions : y = a∙ xb/(cb + xb); a, maximum current density; b, Hill coefficient; c, pH50.Data are expressed as the mean ± s.e.m. Comparisons were made using Student’s How to cite this article: Hu, Y.-L. et al. Multiple H+ sensors mediate the extracellular acidification-induced [Ca2+]i elevation in cultured rat ventricular cardiomyocytes. Sci. Rep.7, 44951; doi: 10.1038/srep44951 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.