Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1  gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade. Piriformospora indica colonizes the roots of many plant species and promotes their growth. The lack of host specificity suggests that the plant response to this endopyhte is based on general signalling processes. In a genetic screen for Arabidopsis plants, which do not show a P. indica-induced growth response, we isolated a mutant in the OXI1  gene. Previously, this protein kinase has been shown to play a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that deletion of PDK1 also abolishes the growth response to P. indica. PDK1 is activated by phosphatidic acid (PA). P. indica triggers PA synthesis and mutants impaired in PA synthesis do not show growth promotion in response to fungal infection. Since defense processes are repressed by P. indica, we propose that a pathway consisting of the PLD-PDK1-OXI1 cascade mediates the P. indica-induced growth response.Like many root-colonizing microbes, the primitive Basidiomycete fungus  Piriformospora indica, a cultivable basidiomycete of Sebacinales, colonizes the roots of many plant species including Arabidopsis P. indica is found worldwide in association with roots P. indica also confers resistance against abiotic P. indica indicates that the beneficial interaction may be based on general recognition and signalling pathways. Little is yet understood about the molecular steps leading to P. indica-induced growth promotion. Plant growth can be induced by a fungal exudate component The endophytic fungus In mammals, the phospholipid-binding 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1) sustains and regulates the balance between growth, cell division and apoptosis Arabidopsis roots 2O2Hyaloperonospora arabidopsidis (previously known as Peronospora parasitica) infection and root hair growth OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) is a serine/threonine kinase necessary for oxidative burst-mediated signalling in P. indica. By positional cloning, we have identified OXI1 as the responsible gene for the growth phenotype induced by P. indica. Since OXI1 is an AGC protein kinase that can be activated by H2O2 and PDK1, we also tested whether mutants in PDK1.1 and PDK1.2 are defective in the P. indica-induced growth phenotype. We found that pdk1.1 pdk1.2 double knock out mutants do not respond to P. indica. The fungus stimulates PA, but not H2O2 synthesis in Arabidopsis plants. PA is produced by several pathways including by PLD. When PA synthesis was reduced by inactivation of phospholipase D (PLD)α1 or PLDδ, the P. indica-induced growth promotion was compromised. These results suggest that P. indica stimulates growth by PA-mediated activation of PDK1, which subsequently activates OXI1.In this work, we report on the results of a genetic screen for Arabidopsis mutants, which do not respond to P. indica are taller than the uncolonized controls Piriformospora indica-insensitive12 (pii12), was smaller in the presence of the fungus  uncovered that P. indica does not induce H2O2 accumulation 2O2 accumulation in the roots .Previously, it was shown that oxi1 plants to respond to P. indica might be caused by their shorter root hairs P. indica translation elongation factor1 (Pitef1) were comparable in oxi1 and wild-type roots  and share a conserved PDK1 binding site, the FxxF motif, at their C-terminal ends OXI1 mRNA level, the AGC2-2 mRNA level is not regulated by ROS (https://www.genevestigator.com). agc2-2 plants did not show any visible phenotype, produced the same amount of seeds, and – in contrast to oxi1agc2-2 plants were not shorter than those of wild-type plants  is the closest homolog of OXI1 , which are present in the Arabidopsis genome  romotion . pldα1 wvels cf. , and themulation . These rP. indica and Arabidopsis the OXI1 pathway constitutes a protein kinase signalling pathway that confers growth stimulation . P. indica was cultured as described previously A. thaliana seedlings were transferred to nylon disks (mesh size 70 µm) and placed on top of a modified PNM culture medium 2, 0.01 µM FeSO4, 70 µM H3BO3, 14 µM MnCl2, 0.5 µM CuSO4, 1 µM ZnSO4, 0,2 µM Na2MoO4, 0.01 µM CoCl2, 10.5 g L−1 agar, pH 5.6), in 90 mm Petri dishes. Fungal plugs of 5 mm in diameter were placed at a distance of 1 cm from the roots. Control seedlings remain untreated. Plates were incubated at 22°C under continuous illumination from the side (80 µmol m−2 sec−1).Wild-type rhd2  Salk_043276, oxi1 (At3g25250) Gabi_355H08, agc2-2 (At4g13000) Salk_083220, pdk1.1a (At5g04510) Salk_113251, pdk1.1b (At5g04510) Salk_007800, pdk1.2 (At3g10540) Sail_450_B01, pldα1-1  Salk_122059, pldε (At1g55180) Koncz68434. pdk1.1 pdk1.2 was generated by crosses between pdk1.1 and pdk1.2.The following homozygote T-DNA insertion lines were used: P. indica. Arabidopsis seedlings, grown for 14 days on MS media, were transferred to vermiculite (rather than soil), because this allowed to harvest the intact roots including the lateral roots. The growth response of the plants to P. indica on soil and on vermiculite is comparable (data not shown). The vermiculite was mixed with the fungus  which was dissolved in PNM medium. 70 ml of liquid PNM medium or inoculated PNM medium was used per plant. The fungal mycelium was obtained from two weeks old liquid cultures after the medium was removed and the mycelium was washed with an excess of distilled water. Cultivation occurred in pots in a temperature-controlled growth chamber at 22°C under short-day conditions (light intensity: 80 µmol m−2 sec−1). The sizes of the plants were monitored weekly and after six weeks the fresh weights of the shoots were determined and the roots harvested for RNA or DNA extraction.6 week-old adult plants were used for interaction studies with −2 sec−1) from above. Fresh weights were determined (data not shown) and RNA was extracted of the root material.12-day-old seedlings were directly transferred from MS medium to a plate with a fungal lawn. The fungal lawn was obtained by placing a fungal plug on Kaefer medium and the fungus was allowed to grow for 14 days at 24°C in the dark, before the seedlings were transferred to the plate. Control seedlings were transferred to Kaefer medium without the fungus. The plates were incubated for 7 days at 22°C under continuous illumination . RT-PCR was conducted with the primer pairs given in Figure S6 in P. indica was monitored with a primer pair for the translation elongation factor 1  was used as housekeeping genes for Arabiopsis roots.RNA was isolated from the roots with an RNA isolation kit . For quantitative RT-PCR, RNA from Arabidopsis roots grown in the absence or presence of  Pitef1; . The colGAPC2 and UBQ5 message levels. Fold induction values were calculated with the ΔΔCP equation of Pfaffl (2001) Semiquantitative analysis was performed after 27 PCR cycles: the products were analysed on 2% agarose gels, stained with ethidium bromide, and visualized bands were quantified with the ImageQuant 5.0 . Real-time quantitative RT-PCR was performed using the iCycler iQ real-time PCR detection system and iCycler software version 2.2 . For the amplification of the PCR products, iQ SYBR Supermix (Bio-Rad) was used according to the manufactureŕs instructions in a final volume of 23 µl. The iCycler was programmed to 95°C 2 min, 35× , 72°C 10 min followed by a melting curve programme (55–95°C in increasing steps of 0.5°C). All reactions were repeated twice. The mRNA levels for each cDNA probe were normalized with respect to the 2O2 was determined by an assay coupled to the peroxidase 4/insoluble PVP (5%). The supernatant was clarified by centrifugation, adjusted to pH 5.6 with 5 M K2CO3 solution and incubated with 1U ascorbate oxidase for 10 min to oxidize the ascorbate. The reaction in 0.1 M phosphate buffer (pH 6.5), 3.3 mM 3-(dimethylamino) benzoic acid, 0.07 mM 3-methyl-2-benzothiazoline hydrazone and 0.3 U peroxidase was started by adding the oxidized extracts and followed by absorbance change at 590 nm and 25°C. NBT staining has been described previously H43−. Samples (3 seedlings each) were treated by adding 100 µL water with or without elicitor for the times and concentrations indicated. Treatments were stopped by adding 50 µL 50% perchloric acid (w/v) and shaking the samples vigorously for 5 min. Liquid was then removed and replaced by 375 µL of CHCl3/MeOH/HCl [50∶100∶1 (v/v)] followed by 100 µL 0.9 % NaCl (w/v), to extract the lipids while shaking (10 min). A two-phase system was induced by the addition of 375 µL of CHCl3 and 200 µL of 0.9% (w/v) NaCl. The remainder of the extraction was performed as described before 3/MeOH/25%NH4OH/H2O [90∶70∶4∶16 (v/v)], essentially as described in Arabidopsis seedlings (5-days-old) were labeled overnight in 400 µL buffer  containing 10 µCi of carrier-free POt-Test (p≤0.05) in Excel.All data were analysed with one-side, unpaired students OXI1 , At3g25250, NP_189162.1; AGC2-2 , At4g13000, NP_193036.1; PDK1.1 , At5g04510, NP_568138.1; PDK1.2 , At3g10540, NP_187665.2; RHD2 , At5g51060, NP_199919.1; IRE (other names: INCOMPLETE ROOT HAIR ELONGATION), At5g62310, NP_201037.1; PLDα1 , At3g15730, NP_188194.1; PLDδ , At4g35790, NP_849501.1; PLDα3 , At5g25370, NP_197919.1; PLDε , At1g55180, NP_175914.1http://www.ncbi.nlm.nih.gov/ and http://www.arabidopsis.org/Information from Text S1Supporting information.(DOC)Click here for additional data file.


The X-ray diffractometry (XRD) results show the phases of ZnO, anatase TiO2 and Zn2Ti3O8 coexisted when the zinc titanate powders were calcined at 600 °C for 1 h. When calcined at 900 °C for 1 h, the XRD results reveal the existence of ZnO, Zn2TiO4, rutile TiO2 and ZnTiO3. Scanning electron microscope (SEM) observations show extensive large agglomeration in the samples. Transmission electron microscope (TEM) and electron diffraction (ED) examination results indicate that ZnTiO3 crystallites formed with a size of about 5 nm on the matrix of plate-like ZnO when calcined at 700 °C for 1 h. The calcination samples have acceptable absorbance at a wavelength of 400 nm, indicating that the zinc titanate precursor powders calcined at 700 °C for 1 h can be used as an UVA-attenuating agent.Synthesis of Zn UVA radiation is a major culprit in photoaging and skin cancers. Moreover, UVB, which primarily reaches the top-most layer of skin, is thought to be responsible for acute photodamage, including sunburn and some non-melanoma skin cancers . Therefo–280 nm, 2, are extensively used as agents to attenuate (scatter and/or absorb) UV radiation, and have many attractive characteristics, such as a long history of topical use, broad spectrum absorption, high photostability and low irritancy /[Ti4+] was 1.0. An aqueous solution of Zn(NO3)2 was added to TiCl4 solution under an air atmosphere. The pH of the mixture was then raised to 7.0 by using NH4OH aqueous solution and stirring the resulting solution for 2 h at room temperature. Subsequently, the solution was kept in an autoclave at 150 °C for 1 h. After cooling, the precipitates obtained were filtered, and washed thoroughly three times with a large amount of deionized water and ethanol  to remove Cl−. The final precipitates were dried at −55 °C in a vacuum and the white zinc titanate precursor powders were thus obtained.The Zn2.2.2O3. The calcination temperature was determined from the DTA result.Differential thermal analysis  was conducted on 50 mg zinc titanate precursor powders at a heating rate of 10 °C/min in air with a reference material of Al/IIIV, Tokyo, Japan) with Cu Kα radiation and Ni filter, operated at 30 kV, 20 mA and a scanning rate of 0.25°/min.The crystalline phase was identified using an X-ray diffractometer  and transmission electron microscope . The crystal structure of the post-calcined powders was determined by selected area electron diffraction (SAED) analysis. The TEM samples were prepared by dispersing the post-calcined powders in an ultrasonic bath and then collected on a copper grid.3.3.1.2- into N2 and H2 . 3 with the ZA of [ıı̄ı].3.4.4.2Ti3O8 powders prepared by a hydrothermal method without a dispersant agent or mineralizer for use in UVA-attenuating applications have been investigated using DTA, XRD, SEM, TEM, ED and UV/VIS. The results are summarized as follows:2 and Zn2Ti3O8 coexisted in the sample. However, when calcined at 900 °C for 1 h, the XRD result reveals the existence of Zn2TiO4, rutile TiO2, and ZnO.When the zinc titanate precursor powders were prepared at pH = 7 and calcined at 600 °C for 1 h, the XRD results show that the phases of ZnO, anatase TiOThe SEM results reveal significant agglomeration in both the freeze-dried and post-calcined samples.2Ti3O8 crystallites with size of about 5 nm on larger ZnO particles with a length of 200 nm and width of 100 nm. The microstructure ZnTiO3 shows a somewhat belt-shaped morphology, with a length of 200 nm and width of 50 nm for precipitates calcined at 900 °C for 1 h.The TEM and ED examination indicates the existence of near spherical ZnThe calcined samples have an acceptable transmittance when the wavelength is 400 nm. This result indicates that zinc titanate precursor powders calcined at 700 °C for 1 h can be used as an UVA-attenuating agent.Zn


Individuals with schizophrenia show increased smoking rates which may be due to a beneficial effect of nicotine on cognition and information processing. Decreased amplitude of the P50 and N100 auditory event-related potentials (ERPs) is observed in patients. Both measures show normalization following administration of nicotine. Recent studies identified an association between deficits in auditory evoked gamma oscillations and impaired information processing in schizophrenia, and there is evidence that nicotine normalizes gamma oscillations. Although the role of nicotine receptor subtypes in augmentation of ERPs has received some attention, less is known about how these receptor subtypes regulate the effect of nicotine on evoked gamma activity.We examined the effects of nicotine, the α7 nicotine receptor antagonist methyllycaconitine (MLA) the α4β4/α4β2 nicotine receptor antagonist dihydro-beta-erythroidine (DHβE), and the α4β2 agonist AZD3480 on P20 and N40 amplitude as well as baseline and event-related gamma oscillations in mice, using electrodes in hippocampal CA3. Nicotine increased P20 amplitude, while DHβE blocked nicotine-induced enhancements in P20 amplitude. Conversely, MLA did not alter P20 amplitude either when presented alone or with nicotine. Administration of the α4β2 specific agonist AZD3480 did not alter any aspect of P20 response, suggesting that DHβE blocks the effects of nicotine through a non-α4β2 receptor specific mechanism. Nicotine and AZD3480 reduced N40 amplitude, which was blocked by both DHβE and MLA. Finally, nicotine significantly increased event-related gamma, as did AZD3480, while DHβE but not MLA blocked the effect of nicotine on event-related gamma.These results support findings showing that nicotine-induced augmentation of P20 amplitude occurs via a DHβE sensitive mechanism, but suggests that this does not occur through activation of α4β2 receptors. Event-related gamma is strongly influenced by activation of α4β2, but not α7, receptor subtypes, while disruption of N40 amplitude requires the activation of multiple receptor subtypes. Individuals with schizophrenia display changes in auditory event-related potentials (ERPs) including decreased amplitude of the P50 and N100 components and disrupted gating of the P50 In addition to changes in the ERP, nicotine has been shown to enhance power in the gamma frequency range of the EEG Recent studies have attempted to identify the specific nicotinic acetylcholinergic receptor subtypes responsible for regulating the effects of nicotine on ERPs, and more recently, evoked gamma activity. At present the mechanism by which nicotine enhances P20 amplitude is not entirely clear, although both α7 and α4β2 nicotinic receptors have been implicated ad libitum. Experiments were conducted at the University of Pennsylvania during the light phase between 9∶00 AM and 3∶00 PM. Mice were allowed at least one week to acclimate to the housing facility before starting any procedure. All protocols were performed in accordance with University Laboratory Animal Resources guidelines and were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania (803887).Thirteen male C57BL/6J mice were used for experiment 1 (nicotine antagonist study) and 9 additional mice were used for experiment 2 (AZD3480 study). All mice were obtained at 8 weeks of age from Jackson Laboratories , and were assessed at 10 to 12 weeks of age. Mice were housed four to five per cage until electrode implantation and single-housed thereafter in a light- and temperature-controlled Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility. Water and standard rodent chow were available Nicotine hydrogen tartrate salt (1.0 mg/kg), methyllycaconitine (MLA –10.0 mg/kg), and dihydro-beta-erythroidine (DHΒE - 2.0 mg/kg)  were dissolved in 0.09% saline. Nicotine was administered intraperitoneally (I.P) at a volume of 0.1 ml. MLA and DHΒE were administered subcutaneously, also at a volume of 0.1 ml. All drug doses are expressed as drug base and were selected based on previous research on nicotine’s effects on ERPs Unipolar recording electrodes were unilaterally placed in the CA3 hippocampal region under isoflurane anesthesia,  and referenced to the surface of the ipsilateral frontal hemisphere . Since the recording and reference electrodes are located far apart from one another, it is likely that activity recorded using this configuration extends far beyond the localized field generated within the CA3 region, and, therefore, as in human EEG recordings, reflects brain activity across a widespread area. For this reason, histological verification of electrode placement was not performed in the current study, although this has been reported elsewhere using the same surgical procedures For experiment 1, five days of ERP testing was conducted over a two week period with a minimum of 24 hours washout period between drug exposures and three recording sessions per test day. The first recording session of each test day was an acclimation trial followed by a baseline saline (vehicle) trial. The third testing session consisted of the injection of the test compound(s). On the first testing day, animals were acclimated to all handling and testing procedures and received nicotine (1 mg/kg). On test day two, animals received MLA (10 mg/kg), on test day three a combination treatment of MLA (10 mg/kg) followed 5 minutes later by nicotine (1 mg/kg). On the fourth day of testing, animals received an injection of DHβE (2 mg/kg) and on the fifth and final testing day animal received a combination treatment of DHβE (2 mg/kg) followed 5 minutes later by nicotine (1 mg/kg). For experiment 2  and were delivered through speakers attached to the cage top. Recordings were performed in 8 standard mouse cages, all of which were located within a Faraday cage. 50 pairs of white noise clicks  were presented with a 9-s interstimulus interval against 70 dB of background noise. Waveforms for the ERP and gamma analyses were filtered between 1 and 500 Hz and baseline corrected at stimulus onset. Individual sweeps were rejected for movement artifact based on a criterion of 2 times the root mean squared amplitude per mouse. Average waves were created from 50-ms pre-stimulus to 200-ms post-stimulus. The P20 component was selected for each ERP by determining the maximum positive deflection between 10 and 30 ms (S1) and between 510 and 530 ms (S2). The N40 was selected by determining the maximum negative deflection between 25 to 60 ms (S1) and between 525 and 560 ms (S2) post-stimulus. Since the latency of the maximum deflection can differ greatly between animals, this method of quantification will produce values for P20 and N40 that are oftentimes higher than what appears in the grand average waveform, which tends to smooth out maximal differences. Sensory gating was analyzed by comparing the amplitude of S1 to the amplitude of S2, with successful sensory gating being defined as a statistically significant difference between S1 relative to S2, with S1 being greater than S2, and sensory gating failure being defined as a lack of statistically significant difference between S1 and S2. This was done separately for P20 and N40.Event-related gamma (30 to 80 Hz) data were processed using EEGLAB . Epochs were extracted from between −199 and 399 msec relative to tone onset. Power was calculated using Morlet wavelets in 116 logarithmically spaced frequency bins between 4 and 120 Hz, with wavelet cycle numbers ranging from 2 to 10 p≤0.05 unless otherwise noted.Both the antagonist and AZD3480 experiments used a repeated within-subjects design in which all animals were exposed to each level of DRUG and each level of STIM (S1 and S2 components). Separate analyses were conducted for the P20, N40 and for event-related gamma The P20 and N40 data were analyzed using a 2 factor repeated measures ANOVA (DRUG by STIM), while gamma data were analyzed using a single factor repeated measures ANOVA (DRUG). In both experiments, repeated measures analyses of variance (ANOVAs) were performed on baseline data for each ERP component to rule out possible confounding effects of time and repeated testing. In the case of a significant main effect or interaction, Fisher’s LSD post-hoc tests were conducted. All tests were two-tailed. Results are significant at Nicotine significantly increased amplitude of the P20 component and reduced amplitude of the N40, consistent with previous studies  [10], , indicating that there were no significant lasting effects of any drug treatment. The mean amplitude and standard error of the mean (in parentheses) for each drug condition for S1 was; saline: 42.27 (6.84); nicotine: 67.98 (7.04); MLA: 30.65 (7.42); DHβE: 53.69 (7.01); MLA+nicotine: 78.96 (14.55) and DHβE+nicotine: 28.83 (3.55). Amplitude of the P20 component was significantly affected by drug treatment [F = 6.33]. There was also a significant main effect for STIM [F = 27.2], suggesting that significant sensory gating occurred, and for the STIM×DRUG interaction [F = 11.38], suggesting that drug effects on P20 amplitude differed for the first and second stimuli. Post hoc testing on responses following S1 revealed that nicotine alone significantly increased P20 amplitude while neither MLA nor DHβE alone had a significant effect on P20 amplitude. The effect of Nicotine on P20 amplitude was blocked by pre-treatment with DHβE but not by pre-treatment with the α7 antagonist MLA. There were no significant treatment effects on P20 amplitude following S2  = 0.06, p>0.05], indicating that there were no significant lasting effects of AZD3480 treatment across the duration of testing. Mean values for P20 amplitude were; Saline: 23.37 (3.7); 1 mg/kg AZD3480; 25.24 (3.8) and 10 mg/kg AZD3480∶30.31 (9.2). No significant effects were observed for either DRUG or for the STIM x DRUG interaction following AZD3480 treatment, although there was a significant main effect for STIM [F = 56.6]. Comparison of S1 and S2: Post-hoc tests showed increased S1 amplitude relative to S2 in all treatment conditions, suggesting that AZD3480 did not affect P20 gating.Decreases in N40 amplitude following nicotine are not blocked by pretreatment with MLA or DhβE.  A repeated measures ANOVA on data from the pre-drug trial of each session for the N40 component revealed no significant differences over time , indicating that the baseline N40 at each session was not affected by repeated testing or previous drug exposures. Amplitude of the N40 component was significantly affected by drug treatment [F = 3.20] and by STIM [F = 7.2]. There was no significant STIM×DRUG interaction, suggesting that drug effects on N40 amplitude did not differ for the first and second stimuli. Post hoc testing for the main effect of drug treatments revealed that nicotine significantly decreased N40 amplitude. A significant reduction in N40 amplitude was observed following MLA by itself. In contrast, DHβE alone did not alter N40 amplitude and neither antagonist blocked nicotine-induced decrement of N40 amplitude. The mean amplitude and standard error of the mean (in parentheses) for each drug condition for S1 was; saline: 95.87 (17.6); nicotine: 47.47 (18.9); MLA: 62.08 (11.9); DHβE: 95.46 (30.6); MLA+nicotine: 81.89 (31.2) and DHβE+nicotine: 83.84 (26.0). Comparison of S1 versus S2: A significant effect was observed for STIM [F = 7.17], with N40 amplitude being significantly greater following S1 relative to S2. A lack of a significant STIM x DRUG interaction suggests that the difference between S1 and S2 was consistent across treatment conditions  = 1.88, p>0.05], indicating that the baseline N40 at each session was not affected by repeated AZD3480 treatment. Mean values for N40 amplitude following drug treatment were; Saline: 53.02 (9.9); 1 mg/kg AZD3480; 57.9 (9.9) and 10 mg/kg AZD3480∶15.04 (10.9). A significant effect was seen for DRUG [F = 8.02], STIM [F = 13.66], and the STIM x DRUG interaction [F = 24.0]. Post hoc tests revealed significantly reduced S1 amplitude following treatment with the high (10 mg/kg) dose α4β2 agonist, relative to saline vehicle  = 13.66], as well as a significant interaction between STIM x DRUG [F = 24.0]. Post hoc tests comparing S1 and S2 response as a function of drug condition revealed significant differences within the vehicle (p = 0.0001) and low dose (p = 0.0001) groups. In contrast, no difference was observed between S1 and S2 in the high dose group, suggesting disrupted gating. Further, significant differences in N40 amplitude between the high dose and vehicle groups was only seen for the S1 component (p = 0.0001), demonstrating that reduced gating was due solely to reduced S1 response  = 0.11, p<0.05], indicating that the evoked gamma activity was not affected by repeated testing or previous drug exposure. Event-related gamma was significantly affected by drug treatment . Post hoc tests showed enhanced gamma power following nicotine (p = 0.037) and following MLA + nicotine (p = 0.0002). No change was observed following DHβE treatment alone, while pretreatment with DHβE blocked the effect of nicotine on gamma power. MLA treatment by itself had no effect on gamma. Mean values for evoked-gamma were: Saline: 1.31 (0.28); Nicotine; 1.82 (0.29); MLA: 0.83 (0.33); DHβE: 1.7 (0.45); MLA+Nicotine: 2.39 (0.31) and DHβE+Nicotine: 1.41 (0.57). This pattern of results suggests that the effects of nicotine on event-related gamma are due to activation of the a4b2 receptor.The a4b2 agonist AZD3480 significantly enhanced evoked-gamma oscillations. A significant effect of AZD3480 was observed on event-related gamma [F = 3.7]. Post hoc tests showed a significant increase in gamma power following low dose (p = 0.016), but not high dose (igh dose . Mean vaOne limitation of this study was the within-subject design. Minimum washout periods of 24 hours were provided between each drug treatment and the half-lives in rodents of the drugs used in this study did not exceed 30 minutes Consistent with previous reports Similar to previous reports, nicotine significantly decreased N40 amplitude Gamma activity has been associated with perceptual and cognitive processes as well as positive and negative symptoms in schizophrenia


Streptomyces sviceus which can circumvent the limitations of fungal laccases. Ssl1 belongs to the family of small laccases that contains only few characterized enzymes. After removal of the twin-arginine signal peptide Ssl1 was readily expressed in E. coli. Ssl1 is a small laccase with 32.5 kDa, consists of only two cupredoxin-like domains, and forms trimers in solution. Ssl1 oxidizes 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and phenolic substrates like 2,6-dimethoxy phenol, guaiacol, and syringaldazine. The kcat value for ABTS oxidation was at least 20 times higher than for other substrates. The optimal pH for oxidation reactions is substrate dependent: for phenolic substrates the highest activities were detected at alkaline conditions , while the highest reaction rates with ABTS were observed at pH 4.0. Though originating from a mesophilic organism, Ssl demonstrates remarkable stability at elevated temperatures  and in a wide pH range (pH 5.0 to 11.0). Notably, the enzyme retained 80% residual activity after 5 days of incubation at pH 11. Detergents and organic co-solvents do not affect Ssl1 stability. The described robustness makes Ssl1 a potential candidate for industrial applications, preferably in processes that require alkaline reaction conditions.Fungal laccases are well investigated enzymes with high potential in diverse applications like bleaching of waste waters and textiles, cellulose delignification, and organic synthesis. However, they are limited to acidic reaction conditions and require eukaryotic expression systems. This raises a demand for novel laccases without these constraints. We have taken advantage of the laccase engineering database LccED derived from genome mining to identify and clone the laccase Ssl1 from  They catalyze the one-electron oxidation of substrate molecules and transfer thereby abstracted electrons to molecular oxygen which is reduced to water. The electrons are channeled through highly conserved copper binding residues from the substrate oxidizing T1 copper site to the T2/T3 trinuclear copper cluster where oxygen is bound and reduced to water by four electrons Although significant progress has been achieved in enzyme engineering, the discovery and characterization of novel enzymes from diverse (micro)organisms still plays an essential role for the development of biocatalytic processes. Especially in the case of laccases it has been demonstrated that very few positions can be mutated without loss of activity Trametes sp. or Pleurotus sp. Many of those fungal laccases exhibit high redox potentials and therefore possess high activities towards their substrates. However, owing to pH preference and stability The most exhaustively investigated laccases originate from white-rot fungi such as B. subtilisE. coliCorynebacterium glutamicumThe physiological functions of most characterized bacterial laccases remain unknown. The few described functions include spore pigmentation as found for the laccase CotA from Thermus thermophilus shows extreme stability at high temperatures with a half-life of thermal inactivation at 80°C of more than 14 h Bacillus halodurans and Streptomyces coelicolor exhibit maximum activities towards syringaldazine or 2,6-dimethoxyphenol at pH values of 7.5 or 9.4 By now, the characteristics and biotechnological potential of these enzymes are poorly investigated and still few reports on bacterial laccases have been published. Nevertheless, these reports demonstrate the thermal robustness and more alkaline activity profiles of bacterial laccases compared to fungal enzymes. Exemplarily, the laccase from Similar to other multicopper oxidases, laccases commonly consist of three cupredoxin-like domains with the T1 copper coordinated by two histidines and a cysteine residue in domain 3 and the trinuclear T2/T3 cluster at the interface of domain 1 and 3 coordinated by eight histidines Streptomyces sviceus. Ssl1 demonstrated moderate thermostability, alkaline pH-activity profile, and stability in a wide pH range up to pH 11 and in presence of organic solvents. Thereby its catalytic properties were distinct from other laccases.Here, we describe the cloning, expression and characterization of the small two-domain Ssl1 laccase from E. coli DH5α . E. coli BL21(DE3), E. coli BL21(DE3) pLys, E. coli Rosetta(DE3)  and E. coli BL21-CodonPlus (DE3)-RP  served as expression hosts. Genomic DNA of Streptomyces sviceus (DMS 924) was purchased from the DSMZ .All reagents were of analytical grade or higher and purchased from commercial sources. Enzymes for molecular cloning, nucleotide ladders and protein ladders were obtained by Fermentas . Molecular cloning and plasmid propagation were carried out in ssl1 gene (SSEG_02446) was amplified by PCR with the primers CTTgctagcATGCATCATCATCATCATCATGCCCCGGGCGGCGAG and GGCaagcttTCAGTGGTGGTGTTCGGCCCGC  using genomic DNA of S. sviceus as template. NheI and HindIII endonuclease recognition sites are shown in lowercase, the sequence of the hexahistidine tag is underlined. The genomic sequence of ss1l was truncated at the 5′ end in order to remove a natural signal sequence of the twin arginine translocation pathway. The PCR product was purified and cloned into the pET22H plasmid NheI and HindIII restriction endonucleases. The sequence of the ssl1 insert in the resulting pET22-ssl1 plasmid was verified by sequencing (Eurofins MWG Operon).The E. coli expression strains were transformed with pET22ssl1 and grown in 200 mL medium containing ampicillin (100 µg ml−1) and, when required, chloramphenicol (34 µg ml−1). Cultures were grown at 30°C or 37°C and 140 rpm. Expression conditions were optimized with regard to E. coli expression strain (BL21(DE3), BL21(DE3) pLys, Rosetta(DE3), BL21-CodonPlus (DE3)-RP), medium , induction OD600 , inducer concentration , expression temperature  and expression duration (3 to 24 h).E. coli BL21-CodonPlus (DE3)-RP grown at 30°C and 140 rpm in TB medium, when expression was induced at an OD600 of 1.0 with 40 µM IPTG. 2 mM copper(II) sulfate were added to the culture medium upon induction, and the expression was carried out for 8 h at 25°C and 140 rpm.Highest volumetric activities were obtained in After expression, cultures were harvested by centrifugation at 11000 g at 4°C for 20 min. The cell pellets were resuspended in potassium phosphate buffer  containing 0.1 mM PMSF and 0.3 mM copper(II) sulfate. Cell disruption was done by sonication with a Branson sonifier SLPe  with at least 1 min on ice between the cycles. Cell debris was removed by centrifugation at 31000 g for 30 min at 4°C. The soluble fraction was incubated for 20 min at 65°C and precipitate was removed by centrifugation at 48000 g for 30 min at 4°C. The resulting supernatant was filtered through a cellulose acetate membrane with 0.45 µm pores. For purification of Ssl1 by immobilized metal affinity chromatography (IMAC), the filtrate was loaded onto a Talon  packed and preequilibrated gravity flow column (7 mL bed volume). The column was washed with 5 column volumes (CV) equilibration buffer  and,thereafter, with 5 CV washing buffer . Ssl1 was eluted in 1 CV elution buffer . The eluate was concentrated with Vivaspin 15 columns  and imidazole and sodium chloride were removed by use of PD miditrap G-25 desalting columns . Purified Ssl1 was stored at -20°C without loss of activity until use. The purity of Ssl1 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% gel.The absorption spectrum of Ssl1 was determined in the range of 300 to 700 nm with a Lamdba 35 spectrophotometer . The solution contained 125 µM Ssl1 in 50 mM MOPS buffer at pH 7.5. Ssl1 concentrations were determined by the Bradford method with BSA as standard, calculation of molar concentrations of Ssl1 were based on the computationally determined and experimentally verified molar mass of 32.5 kDa.The copper content of Ssl1 was determined by atom absorption spectroscopy with an AAnalyst 100  at 324.8 nm with a slit width of 0.7 nm.−1 flow with 50 mM potassium phosphate buffer at pH 7.5. The injection volume was 100 µL with a Ssl1 concentration of 7.5 mg mL−1. Refraction index and scattering intensity were measured on-line by an Optilab rEX differential refractometer and a miniDAWN TREOS triple-angle light scattering detector . Data were integrated and analyzed by the Astra software package.The molar mass of Ssl1 in native form was determined by multi-angle static light scattering. Therefore, Ssl1 was applied to an equilibrated Superdex 200 10/300 GL size exclusion chromatography (SEC) column mounted on an ÄKTA purifier FPLC system . The SEC was conducted at 0.5 mL min−1 cm−1), the reaction mixture contained 0.5 mM 2,6-DMP. The oxidation of ABTS (0.5 mM) was followed at 420 nm (ε = 36 mM−1 cm−1), oxidation of guaiacol (2 mM) to the dimeric product tetraguaiacol was followed at 470 nm (ε = 26 mM−1 cm−1) and oxidation of SGZ  was followed at 525 nm (ε = 65 mM−1 cm−1). Ssl1 activity towards tyrosine was tested at pH 4.0, 7.0 and 9.0. The reaction contained 0.1 mM tyrosine and 2 µM Ssl1, oxidation was monitored at 280 nm (ε = 4.4 mM−1 cm−1). Activity assays were performed in triplicate. For pH activity profiles, activities at optimal pH were set as 100%.The activity of Ssl1 towards the substrates 2,6-dimethoxyphenol , 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), guaiacol, and syringaldazine (SGZ) was tested in 50 mM McIlvaine’s buffer (pH 3–6), 50 mM potassium phosphate buffer (pH 7–8), 50 mM glycine-sodium hydroxide buffer (pH 9–10) and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS)-sodium hydroxide buffer (pH 11) at room temperature. The oxidation of 2,6-DMP was followed spectrophotometrically at 468 nm (ε = 49.6 mMa(t) = a0 exp (-t ln2/T1/2)) by OriginPro 8.5 . Data were collected as triplicate.For testing thermal stability, Ssl1 (75 µM in 50 mM glycine-sodium hydroxide buffer with pH 9.0) was incubated at 50°C, 60°C, 70°C and 80°C for 0 to 240 min. After incubation, samples were immediately chilled on ice. Residual activities were measured with 2,6-dimethoxyphenol as described above and the half-time of activity loss was determined by non-linear regression to the equation of exponential decay  was incubated at pH 3, 4, 5, 6 (in 50 mM McIlvaine’s buffer), 7, 8 (in 50 mM potassium phosphate buffer), 9, 10 (in 50 mM glycine-sodium hydroxide buffer) and 11 (in 50 mM CAPS-sodium hydroxide buffer) at room temperature and residual activities were determined with 2,6-dimethoxyphenol.n-hexane), samples were agitated with 600 rpm in a thermomixer for the same period. Activities were measured with 2,6-dimethoxyphenol as described above. Stability towards organic solvents was accessed by incubation with solvent for 20 h at ambient temperature and subsequent activity measurement with 2,6-dimethoxyphenol.Relative activities of Ssl1 in presence of organic solvents were determined after 10 min incubation in glycine-sodium hydroxide buffer  containing solvent in indicated concentrations at ambient temperature. In case of water immiscible solvents (isooctane and −1 cm−1), 420 nm  and 525 nm . Initial rates were determined in triplicate and resulting data were fitted by non-linear regression to the hyperbolic equation (v = maxv [S]/(mK+[S])) in OriginPro 8.5.Kinetic constants of Ssl1 were determined with the substrates 2,6-DMP, ABTS and SGZ. Reactions contained 3.5 µM Ssl1 and 0 to 6 mM 2,6-DMP in 50 mM glycine-NaOH buffer (pH 9.0), 2.3 µM Ssl1 and 0 to 10 mM ABTS in 50 mM McIlvaine buffer (pH 4.0), or 22 nM Ssl1, 0 to 40 µM SGZ and 2% dimethyl sulfoxide in 50 mM potassium phosphate buffer (pH 8.0). The reactions were conducted at room temperature and followed spectrophotometrically at 468 nm  S. sviceus contains two laccases of different type, one belonging to SUBfamiliy K of SLAC homologues from S. coelicolor and the second belonging to SUBfamily J with homologues of CueO from E. coli. Here we describe the cloning, expression and characterization of the laccase Ssl1 (S. sviceus laccase 1) from SUBfamiliy K. To this subfamily belong also the previously described EpoA laccase from Streptomyces griseus, SLAC laccase from S. coelicolor, and SilA laccase from S. ipomoea. An alignment of the Ssl1 amino acid sequence with these two-domain laccases  a 6-hexahistidine tag was introduced. The resulting 882 bp PCR fragment was cloned into pET22H As indicated by a Conserved Domain Search E. coli BL21-CodonPlus (DE3)-RP as expression host (800–1000 U L−1). This expression strain contains a plasmid encoding rare tRNAs for arginine and proline that are frequently found in genes of GC-rich genomes such as in Streptomyces strains. In case of Ssl1 there are three prolines  and four arginines  that are encoded by the CCC or AGG codon which are transcribed by two of the CodonPlus-RP tRNAs. Since ssl1 contains further codons that are rarely used in E. coli, codon optimization of the ssl1 nucleotide sequence can be expected to increase expression efficiency and volumetric activities.Highest volumetric activities of Ssl1 were obtained with 600 reached 1.0 when expression was induced by only 40 µM IPTG with 2 mM copper sulfate as supplement. After 16 h at 25°C the cells were harvested and lysed. The soluble fraction was incubated for 20 min at 65°C which lead to precipitation of most E. coli proteins while the activity of Ssl1 remained stable. Precipitated proteins were removed and the lysate was applied to a single IMAC purification step which resulted in homogeneously pure Ssl1 as judged by SDS-PAGE (−1 (with ABTS as substrate).For optimal Ssl1 expression, cultures were grown in TB medium at 30°C until ODSDS-PAGE . TypicalE. coli was observed repeatedly As determined by atomic absorption spectroscopy Ssl1 contained 2.5 copper ions per molecule. According to the four canonical copper binding sites of laccase, we expected four copper equivalents instead of 2.5. The used expression and purification strategy resulted in partially copper depleted enzyme. Partial copper depletion of bacterial laccases when heterologously expressed in In SDS-PAGE Ssl1 migrated at 33 kDa corresponding to the theoretically determined molecular weight of 32.5 kDa. When incubated in loading buffer containing SDS but without reducing agent, Ssl1 migrated at approximately 100 kDa (not shown). Accordingly, in multiangle static light scattering experiments the molecular weight of active Ssl1 was determined to be 98.3 kDa. This molecular weight corresponds to a homotrimeric oligomerization state of active Ssl1. Other two-domain laccases were found to form either homotrimers EpoA,  or homod−1 cm−1).Purified Ssl1 solutions are characterized by a deep blue color. Accordingly, the absorption spectrum of Ssl1 in the range of 300 to 700 nm  showed tSsl1 was able to oxidize syringaldazine but did not accept tyrosine as substrate. In combination with the typical UV-vis spectrum and conservation of the canonical copper binding residues, Ssl1 could be classified as laccase, not as tyrosinase. Ssl1 oxidized a range of other typical laccase substrates like 2,6-DMP and guaiacol. ABTS was also oxidized by this enzyme. Thereby the activity-pH dependence was bell-shaped for all substrates . MaximalThe bell-shaped activity profiles with phenolic substrates can be ascribed mainly to two antagonistic effects: (a) the redox potential of phenolic substrates decreases with increasing pH, which results in a larger redox potential difference of substrate and T1 copper and thus in higher activity; (b) hydroxide ions bind at the trinuclear cluster and inhibit the oxygen reduction which reduces activities at high pH −1 mM−1) than for syringaldazine (3.66 s−1 mM−1) and 2,6-DMP (0.361 s−1 mM−1). Observed Km values were in the micromolar range, turn over numbers (kcat) strongly depended on the substrate and ranged from 443 min−1 for ABTS to 19.3 min−1 and 3.47 min−1 for 2,6-DMP and syringaldazine, respectively , the catalytic features of these four two-domain laccases differ substantially. It is notable that EpoA could not oxidize 2,6-DMP, guaiacol and syringaldazine. SLAC and SilA accept these substrates but show catalytic constants that differ from the constants measured for Ssl1. For 2,6-DMP oxidation by Ssl1 the Ktima see . In a fuSsl1 showed remarkable stability with regard to pH, temperature and presence of organic solvents. In a broad pH range from 5 to 10, Ssl1 retained 40 to 60% residual activity after 5 days of incubation . At moreE. coli host proteins, while Ssl1 activity remained unaltered. Thermal stability is not only beneficial for this simple and efficient purification step, but is also an ideal prerequisite for directed evolution experiments since stable enzymes can tolerate more destabilizing mutations and thereby allow screening within a larger mutational space as was shown e.g. for P450 monooxygenases Ssl1 also showed moderate stability at elevated temperatures, the half-times of residual activities were 226±12 min at 50°C, 88±9 min at 60°C, 29±4 min at 70°C and 10±0.4 min at 80°C. This thermal robustness was utilized in the purification process by heat precipitation of most n-hexane and isooctane did not alter either activity or stability of the enzyme (Further, stability of Ssl1 in the presence of several organic solvents was examined. Presence of a second phase of the water immiscible organic solvents e enzyme . With 40S. sviceus is a small two-domain laccase with unusual properties. It can be easily expressed in E. coli and combines stability in a wide pH range, at elevated temperatures and in presence of organic solvents with an alkaline activity profile. This makes Ssl1 a suitable candidate for industrial biocatalysis, especially in processes that cannot be accessed by other laccases due to the requirement of high pH values or organic co-solvents.Ssl1 from


Stability is a crucial ecosystem feature gaining particular importance in face of increasing anthropogenic stressors. Biodiversity is considered to be a driving biotic force maintaining stability, and in this study we investigate how different indices of biodiversity affect the stability of communities in varied abiotic (composition of available resources) and biotic (invasion) contexts.Pseudomonas fluorescens genotypes and grew the communities in different resource environments or in the presence of model invasive species. Biodiversity significantly stabilized community productivity across treatments in both experiments. Path analyses revealed that different aspects of diversity determined stability: genotypic richness stabilized community productivity across resource environments, whereas functional diversity determined stability when subjected to invasion.We set up microbial microcosms to study the effects of genotypic diversity on the reliability of community productivity, defined as the inverse of the coefficient of variation of across-treatment productivity, in different environmental contexts. We established a bacterial diversity gradient ranging from 1 to 8 Biodiversity increases the stability of microbial communities against both biotic and abiotic environmental perturbations. Depending on stressor type, varying aspects of biodiversity contribute to the stability of ecosystem functions. The results suggest that both genetic and functional diversity need to be preserved to ensure buffering of communities against abiotic and biotic stresses. Diversity was expressed as genotypic richness and functional diversity  as described previously 600 of 0.5. All genotypes have a similar body size, and a comparable OD to CFU/mL ratio, and OD600 is a reasonable proxy for the biomass of the inoculated bacteria. We set up 21 different bacterial communities of different composition by randomly assembling these strains, establishing diversity levels of 1 (each of the eight monocultures once), 2 (eight different communities), 4 (four different communities) and 8 genotypes (one community replicated four times)  . Each ge2HPO4 7.01 g l−1, KH2PO4 6.8 g l−1, MgSO4 *7H2O 1.19 g l−1, (NH4)2SO4 1.2 g l−1, CaCl2*7H2O 8.8*10−2 g l−1, FeSO4 *7H2O 7.0 *10−3 g l−1, (NH4)6Mo7O24 * 4 H2O 2.0*10−4 g l−1, Na-EDTA 2.5*10−3 g l−1. ZnSO4 *7H2O 1.11 g l−1, MnSO4×H2O 1.54*10−3 g l−1, CuSO4*5H2O 3.90*10−4 g l−1, Co(NO3)2×6 H2O 2.50*10−4 g l−1, Na2B4O7×10 H2O 1.80*10−4 g l−1, NiCl2*6H2O 1.30*10−3 g l−1;−1). Each bacterial community was grown in each of the resource treatments .We set up a resource richness gradient by combining the carbon sources glucose, mannose, fructose, sucrose and citrate in order to cover a gradient of 1 (each of the five single carbon sources), 2 (four combinations), 3 (four combinations) and 5  carbon sources . We grewP. fluorescens communities as described above, either alone or subjected to the invasion by Serratia liquefaciens MG1 or Pseudomonas putida IsoF, as described previously P. fluorescens strains, and the functional similarity between invaders and resident bacteria was expected to foster competitive interactions In order to test the effect of invasion on community performance, we selected the four resource treatments with three carbon sources (600 = 0.05). After 24 h, we measured the optical density (OD600) as proxy for total community productivity.In both experiments, bacteria were grown in 384-well microtiter plates  at 25°C with agitation. All microcosms contained the same biomass at the beginning of the experiment (start OD600) after 24 h at 25°C of each genotype on each of the five resources as phenotypic parameters. Functional diversity is a well established biodiversity index reflecting in our case the potential of mixed communities to use various substrates compared to monocultures. An index of 0 indicates the absence of complementarity in resource use, and higher values show an increased metabolic potential of the community. Stability (reliability) of community productivity was calculated as the inverse coefficient of variation (CV−1) in the varying treatments Functional diversity of the community was calculated according to Petchey and Gaston R2 = 0.88, p<0.0001; F- and p-values for genotypic richness and functional diversity given in text refer to those where these variables were fitted first, allowing to compare their relative importance. In order to prevent the usage of pseudo-replicates , if necessary. We used sequential GLMs (type I sum of squares) to test in a hierarchical way the effects of genotypic richness and functional diversity per se directly influenced stability of community productivity, or if productivity was indirectly stabilized by increasing functional diversity. In our path analyses arrows represent causal relationships or processes, while rectangles represent manipulated (genotypic richness) or measured variables . We hypothesized that genotypic richness (exogenous variable) influences stability (endogenous variable) either directly or indirectly through increasing bacterial functional diversity (endogenous variable). The adequacy of the model was determined via 2χ tests and AIC  and low AIC values indicate that the model cannot be rejected as a potential explanation of the observed covariance structure As a complement to the GLM approach used to investigate treatment effects and to account for the composition of bacterial communities, path analysis was employed to investigate if bacterial diversity effects on stability of community productivity were due to genotypic richness or functional diversity. Path analysis allows testing the strength of direct and indirect treatment effects by involving multiple causal pathways 1,19F = 6.90, p = 0.0166; 1,19F = 8.42, p = 0.0092; 1,19F = 5.40, p = 0.0314; 1,19F = 17.82, p = 0.0005; 1,10F = 6.63, p = 0.0276), but not in the varied resource experiment (p>0.3). In contrast, the increase in stability with genotypic richness was no longer significant in these analyses . Data on invader performance will be presented in a separate study .Genotypic richness significantly stabilized community productivity in the varied resource experiment Click here for additional data file.


In the spring of 2013, a novel avian-origin influenza A (H7N9) virus in Eastern China emerged causing human infections. Concerns that a new influenza pandemic could occur were raised. The potential effect of chemical agents and physical conditions on inactivation of the novel avian influenza H7N9 virus had not been assessed.7.7 EID50 were exposed to physical conditions  or treated with commercial chemical agents  respectively. After these treatments, the viruses were inoculated in SPF embryonated chicken eggs, the allantoic fluid was collected after 72–96 hours culture at 35°C and tested by haemagglutination assay.To determine the inactivation effectiveness of the novel avian influenza A (H7N9) virus under various physical conditions and chemical treatments, two H7N9 viruses A/Anhui/1/2013 and A/Shanghai/1/2013 were treated by varied temperatures, ultraviolet light, varied pHs and different disinfectants. The viruses with10Both of the tested viruses could tolerate conditions under 56°C for 15 minutes or 60°C for 5 minutes, but their infectivity was completely lost under 56°C for 30 minutes, 65°C for 10 minutes, 70°C, 75°C and 100°C for 1 minute. It was also observed that the H7N9 viruses lost their infectivity totally after exposure of ultraviolet light irradiation for 30 minutes or longer time. Additionally, the viruses were completely inactivated at pH less than 2 for 0.5 hour or pH 3 for 24 hours, however, viruses remained infectious under pH treatment of 4–12 for 24 hours. The viruses were totally disinfected when treated with Sodium Hypochlorite, Virkon®-S and Ethanol at recommended concentrations after only 5 minutes.The novel avian influenza A (H7N9) virus can be inactivated under some physical conditions or with chemical treatments, but they present high tolerance to moderately acidic or higher alkali conditions. The results provided the essential information for public health intervention of novel H7N9 avian influenza outbreak. AIV A (H7N9)) with genomic features of low pathogenic virus causing severe infections in humans was identified in China [On March 31, 2013, a novel reassortant avian influenza A (H7N9) virus (7.7 egg infectious dose (107.7 EID50/50 μl) viruses were treated at 56°C, 65°C, 70°C, 75°C and 100°C. As shown in Table To evaluate the effectiveness of heat treatments in inactivating the novel avian influenza H7N9 virus, 50 μl virus stock solutions containing 107.7 EID50 viruses were spread on a Petri dish and then treated with UV irradiation in a Biosafety Cabinet for varied times at room temperature. The results showed that the viruses were inactivated after exposed to UV irradiation for 30 min or longer  virus or the decontamination methods at laboratory or field conditions. The results indicated that the novel avian influenza H7N9 viruses can be effectively inactivated by temperature treatment under 56°C for 30 min, 65°C for 10 min, 70°C, 75°C and 100°C for 1 min. But, it should be noted that the H7N9 viruses still could survive after treatment under 56°C for 15 min or 65°C for 5 min. Similar results were demonstrated with highly pathogenic avian influenza (HPAI) H5N1 virus, which was inactivated after exposure at 56°C for 30 min . Low patN9 virus AIV inactivation through UV light irradiation has a potential sterilizing application in the laboratories. The present study showed that UV light irradiation on the novel H7N9 virus is effective under the UV light exposure for 30 min or longer within 75 cm distance. However, the virus can survive if exposed to UV for shorter than 20 min. The result suggested that enough UV exposure time is necessary for avoiding cross-contamination on lab-works. In addition, other disinfection measures should be implemented after lab works since only microbes on the surface of material and in the air can be killed by UV light . UV irraOrthomyxoviridae viruses are considered to be sensitive to acid pH values, although their retention of infectivity is dependent on degree of acidity and virus strain . Our stuIn general, on the basis of their resistance to chemical agents, viruses can be divided into three categories  according to the presence/absence of lipids on the virus particle and size of virus. AIVs belong to category A, which can be inactivated easily by all major classes of disinfectants if used properly . CommercThe results indicated that the novel avian influenza H7N9 viruses can be completely inactivated using high temperature (e.g. 56°C or above), UV light irradiation, and commercial disinfectants . But the virus presents a high tolerance to moderately acidic or higher alkali conditions. The present results would provide essential information for public health intervention of novel avian influenza H7N9 outbreaks.50/ml, were evaluated according to the Reed and Muench method  [In March 2013, three urban residents of Shanghai or Anhui, China, were identified to be infected with AIVs A (H7N9) [h, 1938) . The vir7.7 EID50 were added into thin-walled 0.2 ml polypropylene thermocycler tubes  and placed in a precision tube-holding heating block of the thermocycler with heated lid  at different temperatures, including 56, 65, 70 and 75°C for 5, 10, 15, 30, 45 and 60 min and additionally, 70°C, 75°C and 100°C for 1 and 2 min. The treated viruses were placed in an ice bath immediately to stop the heat treatment and 450 μl PBS was added for inoculation of SPF embryonated chicken eggs.The avian influenza H7N9 viruses were subjected to the conditions of various temperatures. The efficacy of heat treatments inactivating H7N9 viruses was determined. The tested protocol was modified from Swayne and Beck . For tes7.7 EID50 was spread on Petri dish  surface using 10 μl tip  and exposed to UV light (distance of 75 cm and wavelength of 250-270 nm) in Biosafety Cabinet  at room temperature. The cover of the Petri dish was removed when samples were exposed to the UV light, 450 μl PBS was added to the Petri dish after UV exposure for 10, 20, 30, 45 and 60 min respectively. And then the recovery of the virus was tested immediately by inoculation of SPF embryonated chicken eggs.50 μl virus stock solution containing 107.7 EID50 were mixed thoroughly with 450 μl PBS with pH 1, 2, 3, 4, 6, 8, 10, 11 and 12, adjusted by using sodium hydroxide or hydrochloric acid, and treated for 0.5, 1 and 24 h at room temperature respectively. And then the recovery of the virus was tested immediately by inoculation of SPF embryonated chicken eggs.To determine the tolerance of the novel virus on the acidic and alkali conditions, the viruses AH1 and SH1 were exposed to different pH values (1–12) for different time intervals at room temperature, as described in previous study with some modifications . 50 μl v7.7 EID50 were mixed thoroughly with 450 μl of each working disinfectant, 4.5 ml PBS was added to the mixture to stop the disinfection after 5, 10, and 30 min treatment at room temperature respectively. And then the recovery of the virus was tested immediately by inoculation of SPF embryonated chicken eggs.Commercial disinfectants, including Sodium Hypochlorite, Virkon®-S  and ethanol, were tested in this study. The tested protocol was modified from Suarez et al. . BrieflyAll treated viruses were inoculated into 9-day-old SPF embryonated chicken eggs to determine their survival. Each of the virus suspension exposed to physical conditions  was made 10-fold dilution with PBS, 100-fold dilution for the untreated virus stock solution as positive control, 10-fold dilution for each disinfectant control. Each diluted samples was filtered through 0.22 μm filter  before inoculation into six 9-day-old SPF embryonated chicken eggs for 72-96 h. Embryo mortality observed within 24 h post inoculation was considered nonspecific and discarded. Allantoic fluid (AF) was collected from each egg and checked for HA as the sign of viral growth in the eggs.In view of the biosafety issues involved in handling avian influenza H7N9 viruses, all the experiments were conducted in a biosafety level 3 facility for this study.AIV A (H7N9): A novel reassortant avian-origin influenza H7N9 virus; AIV: Avian influenza virus; UV: Ultraviolet light; EID50: 50% egg infectious dose; AH1: A/Anhui/1/2013(H7N9); SH1: A/Shanghai/1/2013(H7N9); HA: Haemagglutination assay; HPAI: Highly pathogenic avian influenza; LPAI: Low pathogenic avian influenza; SPF: Specific pathogen-free; AF: Allantoic fluid.The authors declare no competing interests. The contents of this article are solely the responsibility of the authors and do not necessarily represent the views of China CDC and other organizations.YLS designed the research and draft the paper; SMZ and JFG performed the research; SMZ and RBG joined into the designation of the study, analysed the data and draft the paper; LBD,YZ, JD and HB helped with titration of the virus; JFZ and KQ helped to draft the manuscript. All authors read and approved the final manuscript.


The duration of FiO2 1.0 tended to be shorter in the CPAP group than in the PPV group (CPAP vs. PPV: 65 (33–105) vs. 100 (40–280) s; p = 0.05), SpO2 >95% occurred more often in PPV group (53% vs. 69%) and lasted longer (70(40–95) vs. 120(50–202) s). In CPAP group, minute volume increased from 134 (76–265) mL/kg/min 1 minute before to 240 (157–370) mL/kg/min (p<0.01) 1 minute after start FiO2 1.0 and remained stable at 2 minutes (252 (135–376) mL/kg/min; ns). The rate of rise to maximum tidal volume increased (from 13.8 (8.0–22.4) mL/kg/s to 18.2 (11.0–27.5) mL/kg/s; p<0.0001) to 18.8 (11.8–27.8) mL/kg/s; ns). In the PPV group respiratory rate increased from 0(0–4) to 9(0–20) at 1 minute (p<0.001) to 23 (0–34) breaths per minute at 2 minutes (p<0.01).Results are given as median (IQR) or percentages where appropriate. Suitable recordings were made in 50 infants (GA 27 (26–29) weeks), 17 received CPAP and 33 PPV. SpOIn preterm infants at birth, a rapid increase in oxygenation, resulting from a transient increase to 100% oxygen might improve respiratory drive, but increases the risk for hyperoxia. However, hypoxia inhibits breathing movements in the fetus 2 sensitivity of infants changes in days-weeks after birth Since SpO2 1.0 if needed and then titrating down in preterm infants at birth. An oxygen saturation (SpO2) ≤70% at 5 minutes was used to increase FiO22 reduction was advocated once the infant was stabilized.From 2008 until 2010, the local guidelines of the Royal Women's Hospital  and the Leiden University Medical Center  recommended starting in air and switching to FiO2 and respiratory drive in preterm infants right after birth in the delivery room after switching from air to FiO2 1.0.Our aim was to investigate the change in SpO2 1.0 when: 1) cardiac massage was needed, 2) positive pressure ventilation (PPV) was administered for 1 minute and heart rate (HR) was <100 beats per minute (bpm) or 3) SpO2 <70% at 5 minutes. FiO2 was then titrated down as quickly as possible (when SpO2 >90%). Recordings were only made when the research team was available.The local institutional review boards (IRBs) of the Leiden University Medical Center  and Royal Women's Hospital  approved physiological- and video recordings at birth in the delivery room when respiratory support was necessary for research purposes. Written parental consent to use the recordings for research was obtained after birth. A retrospective study was performed in both hospitals with data collected between 2008 and 2010. During the period of data collection local guidelines recommended that support was started with air and switched to FiO2O) in preterm infants during apnea or HR<100 bpm. In breathing infants and HR>100 bpm, CPAP (5–6 cmH2O) is given. Changing pressures was left to the discretion of the caregiver.Respiratory support was delivered with a T-piece resuscitator  and face mask. Local resuscitation guidelines recommended to start PPV , a Masimo SET pulse oximeter , an oxylog  and Spectra program  for physiological recordings has been described in detail in previous publications 2 1.0 were identified and divided into two groups. This was based on the type of respiratory support they received around the time point FiO2 1.0 was started: group 1) infants were breathing on continuous positive airway pressure (CPAP) and group 2) received positive pressure ventilation (PPV).All recordings of infants born at <32 weeks gestation between 2008 and 2010 were reviewed. Using video and respiratory function monitoring other interventions were identified performed during the analyzed period. Infants receiving FiO2 was increased to 1.0, for what reason(s) , duration and the downward titration rate of FiO2 1.0. Furthermore we noted the increase in SpO2 duration of SpO2 >95%. We used SpO2 >95% as an indication for increased risk for hyperoxia.In all infants we recorded when FiO2 1.0 on respiratory drive was investigated. To measure the change in respiratory effort, we analyzed the respiratory rate (RR), expired tidal volume (Vte), minute volume (MV) and the rate of rise to maximum tidal volume (mL/kg/second) during inspiration from 1 minute before until two minutes after starting FiO2 1.0 (which served as a control period). To measure the maximum rate of tidal volume increase we used spontaneous breaths without mask leak (Vti = Vte).In group 1 (CPAP group) the effect of FiO2 1.0. Breaths in between and coinciding with inflations were identified according to previous described methods In group 2 (PPV group) the tidal volumes and rate of rise will be influenced by the positive pressure ventilation given and we only analyzed RR of the spontaneous breaths from 1 minute before until two minutes after starting FiO2 can influence flow and volume measurements 2 1.0, both inspired and expired tidal volumes were corrected by −6% when using cold dry gas and by −10% when heated gas was used.As changing of FiOData are presented as mean (± SD) or median (IQR) where appropriate. Differences were analyzed with a paired samples t-test for parametric data or a Wilcoxon signed rank test for non-parametric data where appropriate using . A two-sided p-value <0.05 was considered statistically significant.Data from 80 recorded infants were reviewed, 30 were excluded (no respiratory support (n = 10), no supplemental oxygen (n = 7), low quality recordings (n = 12) and 1 infant was born dead). Thus, 50 infants with GA 27 (26–29) weeks were analyzed ; during 2O, PPV-group; PIP 21.6 (20.3–24.9) cmH2O and PEEP 4.2 (3.5–4.7) cmH2O) and no readjustments of mask position were observed. After the study window 1 infant of the CPAP-group was intubated, but reason was unclear. In the PPV-group, 5 infants were intubated after the study window for apnea and low SpO2 despite FiO2 1.0. Cardiac massage was not provided to any infant.The infants in the CPAP-group did not receive PPV during or after the study window. In both groups ventilation pressures were not increased during the study window. (pressures given: CPAP-group CPAP level 5.3 (4.6–5.9) cmH2 1.0 was started 300 (225–315) seconds after birth and was given for 65 (33–105) seconds. FiO2 was weaned in 20 (5–60) seconds to 21 (21–21) %. In all infants oxygen was started for low SpO2 and HR was >100 bpm.In the CPAP-group FiO2 1.0 was started 180 (120–270) seconds after birth and was given for 100 (40–280) seconds. FiO2 was weaned in 25 (10–47) seconds to 21 (21–30)%.In the PPV-group FiO2 occurred in the first minute after starting oxygen  infants of in group 1 and in 23/33 (69%) infants of group 2. The starting point and duration of SpO2 >95% are depicted in SpO2 to 1.0 increased RR from 30 (18–41) 1 min before to 35 (24–45) breaths per minute (ns) 1 minute after to 39 (31–44) breaths per minute (p<0.05) in the 2nd minute.In the CPAP-group, increasing FiOst minute after increasing FiO2 to 1.0 and remained stable in the 2nd minute (Vte: from 4.9 (2.3–8.8) mL/kg to 6.7 (3.6–10.4) mL/kg (p<0.001) to 6.5 (3.7–10.2) mL/kg (ns); MV: from 134 (76–265) mL/kg/min to 240 (157–370) mL/kg/min (p<0.01) to 252 (135–376) mL/kg/min (ns)).Vte and MV increased significantly in 12 to 1.0 and remained stable at 18.8 (11.8–27.8) mL/kg/s (ns) in the 2nd minute.The rate of rise to maximum tidal volume increased from 13.8 (8.0–22.4) mL/kg/s in the minute before to 18.2 (11.0–27.5) mL/kg/s (p<0.0001) in the minute after increasing FiO2 1.0 and at that moment SpO2 was 87% (11) and HR 147 (19) bpm.PPV was given in 23 apneic infants and in 10 infants for poor respiratory drive. Apneic infants started breathing 80 (50–180)s after FiOnd minute after switching to FiO2 1.0.RR increased from 0 (0–4) 1 min before to 9 (0–20) breaths per minute (p<0.001) 1 minute after to 23 (0–34) breaths per minute (p<0.01) in the 22 1.0 on SpO2 and respiratory drive in preterm infants at birth. Most infants with SpO2 near the 10th percentile had a good HR but FiO2 was increased to 1.0 because of low SpO2. After increasing FiO2 to 1.0, respiratory drive improved simultaneously with a rapid increase in SpO2. However, SpO2 >95% occurred in the majority of infants, especially in the infants who received PPV, which probably reflects the difficulty of simultaneously performing PPV and titrating oxygen. These observations suggest that targeting a higher percentile as currently recommended in international guidelines th–50th percentile) might improve respiratory drive. A more stepwise increase in FiO2 and more diligence in reducing FiO2, for example when SpO2 >85%, could reduce the risk of hyperoxia.We investigated the influence of switching from air to FiO2 was increased and SpO2 improved. Antenatally, hypoxia suppresses fetal breathing movements 2 may be involved 2 1.0 was only required for a short time and could be rapidly weaned allowing most infants to remain stable with little extra oxygen. Although our weaning rate was fast, studies comparing high versus moderate FiO2 levels in preterm infants found similar levels of FiO2 at 10 minutes We observed that preterm infants started to breathe more vigorously, as indicated by an increased rate and effort, once FiO2 to 1.0 in hypoxic lambs just after birth improved blood oxygenation, but had no effect on lung compliance and pulmonary blood flow 2 1.0 is achieved by increasing the partial pressure gradient for oxygen diffusion compensating the ventilation perfusion mismatch due to low FRC Experimental studies have shown that pulmonary vascular resistance at birth is related to ventilation onset and oxygen had little impact 2 and remained stable in the minute thereafter. Also, RR increase cannot be explained by improving compliance. Alternatively, increased pressures could have elevated Vte, but these remained unchanged. Increasing FiO2 increases gas density which can influence measurements 2 1.0 and when considering the rate of rise is also increased it is more likely to be the infant's own effort.The reported Vte increased in infants on CPAP could be explained by improved lung compliance. However, volume increase occurred right after increasing FiO2 of <1.0, most infants starting with low FiO2 levels needed an increased FiO2 (0.45–0.6) to reach target SpO2 levels 2 below target. Therefore, starting in air may not be the right approach. Although it is unclear how detrimental a short period of FiO2 1.0 is at birth (1–2 minutes), more vigilance in preventing SpO2 levels >95% is needed Although studies showed it is feasible to support preterm infants at birth with a FiO2 >95% occurred more often and lasted longer, increasing the chances of hyperoxia. These observations may indicate the algorithm was difficult to follow. This will become even more difficult if separate SpO2 targets for each minute after birth are defined and may lead to a change in focus away from adequate ventilation. Adding an extra person to the resuscitation team could be helpful.In line with our recent report 2 1.0 complicates comparing with the respiratory drive of infants receiving air. Also, the observational nature of this study prevented us to have a control group of infants needing no support. However, we were interested in the effect of FiO2 1.0 and measurements before starting oxygen served as a control period.The retrospective nature of the study and the relative small sample size precludes any hard conclusion regarding respiratory drive and oxygenation. Recording respiratory parameters at birth is challenging, similar studies do not include large number of infants 2 1.0 increased the risk for hyperoxia. No hard conclusions can be drawn, but our observations might suggest that respiratory drive increased after supplemental oxygen was given and oxygenation improved. The role of SpO2 levels in stimulating respiratory drive at birth merits further investigation.We observed that during respiratory support of preterm infants switching from air to FiO


Methanobacterium thermoautotrophicum ΔH, at 2.2 Å. Unlike typical Sac10b family proteins, Mth10b is an acidic protein and binds to neither DNA nor RNA. The overall structure of Mth10b displays high similarity to its homologs, but three pairs of conserved positively charged residues located at the presumed DNA-binding surface are substituted by non-charged residues in Mth10b. Through amino acids interchanges, the DNA-binding ability of Mth10b was restored successfully, whereas the DNA-binding ability of Sso10b, a typical Sac10b family member, was weakened greatly. Based on these results, we propose a model describing the molecular mechanism underlying the interactions of typical Sac10b family proteins with DNA that explains all the characteristics of the interactions between typical Sac10b family members and DNA.The Sac10b protein family is regarded as a family of DNA-binding proteins that is highly conserved and widely distributed within the archaea. Sac10b family members are typically small basic dimeric proteins that bind to DNA with cooperativity and no sequence specificity and are capable of constraining DNA negative supercoils, protecting DNA from Dnase I digestion, and do not compact DNA obviously. However, a detailed understanding of the structural basis of the interaction of Sac10b family proteins with DNA is still lacking. Here, we determined the crystal structure of Mth10b, an atypical member of the Sac10b family from  Sulfolobus acidocaldariusS. shibataeS. solfataricusMethanocaldococcus jannaschiiArchaeoglobus fulgidusin vitro.The Sac10b protein family, also named Alba, is generally regarded as a family of DNA-binding proteins that are highly conserved and widely distributed within the archaea in vivo, resulting in a several-fold reduction in its nucleic acid-binding affinity et al. first proposed a model for the interaction of Sso10b with the DNA duplex S. solfataricusS. shibataeM. jannaschiiA. fulgidusA. pernix K1P. horikoshii OT3The crystal structure of Sso10b was solved in 2002 M. thermoautotrophicum ΔHRecently, we identified an atypical member of the Sac10b family, Mth10b from 1-α1-β2-α2-β3-β4. As shown in 1, β2 and β4 are parallel, while strand β3 is antiparallel to the other strands. This structure model covers 86 of the total 91 residues, and no electron density was observed for the four N-terminal residues or for C-terminal Asp91. The monomer structure is asymmetric with approximate dimensions of 47×27×21 Å. Two adjacent asymmetric Mth10b monomers interact with each other side-by-side along the long dimension of the monomer, forming a dimer with a crystallographic two-fold axis perpendicular to the long dimension of the dimer  from M. jannaschii, Afu10b (1NFJ) from A. fulgidus, Ape10b2 (2H9U) from A. K1, and Pho10b (2Z7C) from P. horikoshii OT3, gave rmsd values of 0.624 Å, 0.803 Å, 0.526 Å, 0.687 Å, 1.442 Å and 0.822 Å, respectively. The main differences among these structures occur at the tips of the outstretched β-hairpin arms  and purified to homogeneity. Protein purity was higher than 95% as confirmed by 15% SDS-PAGE  was immobilized on a SA sensor chip. Increasing concentrations of purified proteins were injected and the interactions between these proteins and the DNA duplex were measured in different running buffers.D) values of 2.1×10−6 M, 2.6×10−7 M and 1.6×10−5 M for N11K, I39R and T77R, respectively. Slight cooperativity was observed in these three mutants  without NaCl was used, no interaction was observed between wild type Mth10b and the DNA duplex, agreeing well with EMSA results  mutants . These rD values of 3.0×10−5 M, 6.5×10−6 M and 7.0×10−7 M, respectively , while measurements for the other three mutants of Mth10b, N11K, I39R and the triple mutant N11KI39RT77R, gave Kectively . Relativectively . MeasureD value of the triple mutant N11KI39RT77R  cells were used for plasmid cloning and protein expression, respectively. Enzymes and reagents for DNA manipulations were purchased from TAKARA. Plasmid Miniprep Kits and Gel Extraction Kits were obtained from OMEGA. Yeast extract and Tryptone were purchased from OXOID. Isopropyl β-D-thiogalactoside (IPTG) was obtained from MERCK. All chemicals were of analytical grade for biochemical use. All apparatus and chromatography materials were purchased from GE.The plasmid pET11a from Novagen was used to make the vector-DNA construct. Expression plasmids pET11a-4)2SO4, 0.1 M HEPES, pH 7.5. A 2.2 Å dataset was collected on beamline BL17U at the Shanghai Synchrotron Research Facility (SSRF) and processed with HKL2000 Purified Mth10b in a buffer containing 10 mM HEPES, pH 7.2, 150 mM NaCl was concentrated to ∼10 mg/ml for the initial screening of crystals. Crystals were grown by the hanging drop vapor diffusion method at 4°C for about 3 days, using an equal volume (0.5 µl) of protein and crystallization buffer over a 0.5 ml reservoir composed of 0.1 M NaCl, 1.6 M  and purified as described previously All expression plasmids of Mth10b variants and Sso10b variants were constructed from the parental plasmids pET11a-mth10b gene (MTH1483) was amplified from the plasmid pET11a-mth10b by PCR with the forward primer 5′- gTgATgTCAgAggAgAATgTAgTATAC -3′ and the reverse primer 5′- gCTTTAAggATCACTTCACTggTC -3′. The forward primer was labeled with 5′-biotin. The PCR product was purified from a 1% agarose gel using a Gel Extraction Kit (OMEGA). The extracted DNA duplex was diluted about 10-fold in basic running buffer  and applied to the streptavidin-coupled sensor chip (SA chip) surface at 10 µL/min to generate about 75 response units (76.4 RU). Measurements were performed in basic running buffer containing different concentrations of NaCl at 25°C. Protein samples were prepared by serial dilution with running buffer from the stock solution and applied to the sensor surface at a flow rate of 30 µL/min for 1–2 min. Postinjection disassociation was monitored in running buffer at the same rate for 2 min. Surface regeneration between injections was achieved using a 1-min injection of basic running buffer containing 0.5 M NaCl. All experiments were performed in triplicate and any background signal from the reference flow cell without immobilized ligand was substracted from all data sets. Sensorgrams, RU versus time, at different concentrations, for the binding of each protein to DNA were obtained, and the RU in the steady-state region was determined by linear averaging over a selected time span. When there was no obvious cooperativity, binding parameters  and the apparent KD) were obtained by data analysis with a simple 1∶1 Langmuir interaction model using BIAevaluation software. When there was obvious cooperativity, Rmax was estimated from the binding curves directly, and the apparent KD was estimated from the concentration of the protein required to bind with 50% saturation. The saturation binding stoichiometries of tested proteins were calculated according to Rmax values and the amount of DNA fixed on the chip.DNA-protein interactions were analyzed by SPR using a BIAcore 3000 system (GE). To create a non-specific DNA duplex for SPR experiments, the N-terminal 110 bp fragment of the In order to test whether or not the DNA was protected from nuclease attack by the tested proteins, a simple experiment was designed. Approximately 400 ng plasmid pAS22 (3789 bp) was incubated with 10 µg recombinant protein in binding buffer (20 mM Tris/pH 7.5) in a total volume of 15 µl at room temperature for 20 min. After addition of Dnase I (TAKARA), the incubation was continued at room temperature for another 20 min. SDS was then added to a final concentration of 1% (W/V) to disable the nuclease and disassemble the DNA-protein complex. Samples were resolved by electrophoresis in 1.5% agarose gels in 1×TAE buffer at constant voltage. After electrophoresis, the gels were stained with ethidium bromide and visualized under UV light. Several controls were performed parallely.


Here we show the utility of EMD Millipore's 3L and 50L CellReady single use bioreactors for the cultivation of adherent mammalian cells on microcarriers. Cytodex3® and Solohill® collagen microcarriers were first tested in a mixing study to assess feasibility. We evaluated the normalized mixing speed required in the 3L and 50L to achieve a suspension of the microcarriers and enable growth of the cells.The Mobius Working with this assumption, mixing studies where performed using the Cytodex3® microcarriers in 3L Mobius® CellReady and Solohill® Collagen coated in 50L single use bioreactor to determine the slowest agitation speed or the just suspended mixing power inputs (P/V)js, required to fully suspend the microcarriers so that the beads are equally distributed in the bioreactor.Manufacturer specifications show CytodexMicrocarrier distribution was assessed by sampling the bioreactor at varying depths. Then the dry weight of the microcarrier was used to determine the% relative sample weight to the target weight.js to be ~0.6W/m3 in both the 3L and 50L single use bioreactors Data show the (P/V)3® in 3L bioreactor and 15g/L Solohill® Collagen microcarriers in 50L bioreactor100% distribution corresponds to the theoretical concentration of microcarriers, which is 3g/L Cytodex® CellReady single use bioreactor. The conditions that showed the best performance could then scaled to the 50L Mobius® bioreactor.Initial cell culture runs were performed with MDCK and Human Mesenchymal Stem Cells (hMSCs) to evaluate the bioreactor agitation to support cell growth in the 3L Mobius3® microcarriers grew to a peak cell density of ~1e6cells/mL using a power input of 0.6W/m3 with a 2L working volume after 3 days.1. Cultured MDCK cells on Cytodex® microcarriers grew to a maximum total cell number of 6e6 cells using power input of 0.6-0.8W/m3 with a 2.4L working volume after 12 days.2. Cultured hMSCs on Solohill3.1. Data from the mixing experiments demonstrate the just suspended mixing power input was determined to be ~0.6W/m® CellReady bioreactor with total number of hMSCs reaching 4e8 and 9e9 cells after 12 days at a agitation power input of 0.6-0.8W/m32. Cell growth experiments with hMSCs demonstrate comparable cell growth in the 3L and 50L Mobius3 may provide the best performance for cell growth with peak cell densities ~1.0e6 cells/mL after 3 days3. Initial cell growth experiments with adherent MDCK cells demonstrate an agitation power/volume input of 0.6W/m4. Comparable MDCK cell growth is observed:® CellReady Bioreactor 3LMobius® CellReady Bioreactor 50LMobiusRocking Bioreactor 20L


TCCO2) with regard to arterial carbon dioxide partial pressure (PaCO2) in severe obese patients undergoing laparoscopic bariatric surgery. Twenty-one patients with BMI>35 kg/m2 were enrolled in our study. Their PaCO2, end-tidal carbon dioxide partial pressure (PetCO2), as well as PTCCO2 values were measured at before pneumoperitoneum and 30 min, 60 min, 120 min after pneumoperitoneum respectively. Then the differences between each pair of values (PetCO2–PaCO2) and. (PTCCO2–PaCO2) were calculated. Bland–Altman method, correlation and regression analysis, as well as exact probability method and two way contingency table were employed for the data analysis. 21 adults  were finally included in this study. One patient was eliminated due to the use of vaso-excitor material phenylephrine during anesthesia induction. Eighty-four sample sets were obtained. The average PaCO2–PTCCO2 difference was 0.9±1.3 mmHg (mean±SD). And the average PaCO2–PetCO2 difference was 10.3±2.3 mmHg (mean±SD). The linear regression equation of PaCO2–PetCO2 is PetCO2 = 11.58+0.57×PaCO2 , whereas the one of PaCO2–PTCCO2 is PTCCO2 = 0.60+0.97×PaCO2 (r2 = 0.89). The LOA (limits of agreement) of 95% average PaCO2–PetCO2 difference is 10.3±4.6 mmHg (mean±1.96 SD), while the LOA of 95% average PaCO2–PTCCO2 difference is 0.9±2.6 mmHg (mean±1.96 SD). In conclusion, transcutaneous carbon dioxide monitoring provides a better estimate of PaCO2 than PetCO2 in severe obese patients undergoing laparoscopic bariatric surgery.To investigate the correlation and accuracy of transcutaneous carbon dioxide partial pressure (P The end-tidal carbon dioxide partial pressure (PetCO2) measurement has been widely used for the continuous noninvasive monitoring of carbon dioxide in patients with tracheal intubation during general anesthesia, However, many factors may possibly affect the accuracy of PetCO2 monitoring, such as mismatch of ventilation to blood flow (V/Q ratio), chronic obstructive pulmonary disease, obstructive sleep apnea syndrome, surgery postures, smoking, ect. Recently, noninvasive transcutaneous carbon dioxide partial pressure (PTCCO2) monitoring has been used in infants and in adult patients with good accuracy Currently the “gold standard” technique for the measurement of arterial carbon dioxide partial pressure , fentanyl (2–4 μg.kg −1), and rocuronium (0.6 mg.kg−1) by the same anesthetist. After tracheal intubation, patients were ventilated with 100% oxygen (2 L/min) under the mode of intermittent positive pressure ventilation (IPPV), with a tidal volume of 6–10 ml/kg and an I:E ratio of 1∶2. The ventilatory frequency and tidal volume were adjusted to maintain normocarbia . The PetCO2 was monitored by side stream spirpometry . PTCCO2 was monitored with a TCM-4 device . One of the authors calibrated, placed, and maintained the monitor. Before placement, the electrode was cleaned, a new membrane applied, and calibration done according to the manufacturer’s recommendations. The working temperature of the electrode was set at 44°C and the electrode was placed on the chest. The area where the electrode was placed was swabbed with alcohol in order to to facilitate adhesion of the disk to the skin. Re-calibration was required once the position of the electrode was changed. The electrode was removed, adjusted, and replaced in a different location on the chest every 2 h to avoid thermal injury.Anesthesia was induced with propofol , remifentanil (0.1–0.2 μg kg−1.min−1), and atracurium (0.6 mg. kg−1.h−1) to keep the variation of blood pressure and HR within 20% of baseline values. Those patients whose blood pressure drop was more than 20% baseline value or who needed a vasoconstrictor to maintain hemodynamics stable were excluded from the study. yet the data from them before hypotension occurred could still be used for the analysis. The patient’s body temperature was continuously monitored nasopharyngeally and maintained at 36°C to 37°C. The room temperature was maintained at 23°C to 25°C. Pneumoperitoneum was established and intraperitoneal CO2 infusion pressure was maintained at 12–14 mmHg during the surgery.An AS/5 monitor  was employed to monitor the patients’ electrocardiography, pulse oxymetry, and noninvasive blood pressure. Before anesthesia, patients’ heart rate (HR) and arterial blood pressure were both recorded as baseline. A 20-G or 22-G arterial catheter was inserted into the left radial artery under local anesthesia for ABG sampling. PaCO2 and PTCCO2 or PaCO2 and PetCO2 using Bland–Altman method. Pearson correlation coefficient and linear regression analysis were used to establish the relationship. The exact probability method and two way contingency table were employed to compare the difference of 5 mmHg or less and 3 mmHg or less between PaCO2 and the other two noninvasive variables. A P value of 0.05 or less was considered statistically significant.Data were presented as mean±SD. Statistical analysis was performed by SPSS version 17.0.We assessed the agreement between P2, 42.1(5.4) kg/m2) were recruited into this study. All patients underwent laparoscopic bariatric surgery. The PaCO2, PetCO2, and PTCCO2 values were recorded at 4 time points. Eight-four samples were finally obtained. The mean values of these variables at different time points are presented in TCCO2 was correlated with PaCO2 at each time point . PetCO2 was correlated with PaCO2 at each time point . The PaCO2 values were ranging from 42.2 to 58.4 mmHg. The average PaCO2–PetCO2 difference was 10.3±2.3 mmHg and the average PaCO2–PTCCO2 difference was 0.9±1.3 mmHg. In those samples, both PetCO2 and PTCCO2 were closely correlated with PaCO2. The linear regression equation between PetCO2 and PaCO2 was PetCO2 = 11.58+0.57×PaCO2, r2 = 0.64, P<0.01. Only one PetCO2–PaCO2 difference  was 5 mmHg or less while all values of PTCCO2–PaCO2 difference  were 5 mmHg or less (P<0.01). According to Bland-Altman analysis, the 95% limits of agreement (LOA) of the average PaCO2–PetCO2 difference was 10.3±4.6 mmHg yr; weight from 86 to 160 kg, 119.3(22.1)kg; BMI from 35.3 to 51.1 kg/m4, P<0.01; and PTC9, P<0.01. In all etCO2) measurement has been widely used in the anesthetic management. However, quite a number of factors may possibly affect the accuracy of PetCO2 measurement, including alveolar ventilation volume, V/Q ratio, chronic obstructive pulmonary disease, etc. Due to the effects of weight loss, remission of diabetes mellitus and improvement of health-related quality, bariatric surgery gets rapid development in clinical practice 2 pneumoperitoneum results in the further decrease of FRC and greater degree of intrapulmonary shunt, which diminishes the accuracy of PetCO2. Nevertheless, PTCCO2 monitoring uses heated electrodes to improve local perfusion , which facilitates the absorption of carbon dioxide into the heated electrodes via skin diffusion. The carbon dioxide inside the electrodes changes the internal pH value, which results in PTCCO2 signals. Reid CW et al aCO2–PetCO2 difference increased along with PaCO2 levels. In the patients undergoing bariatric surgery the PetCO2 is usually greater than 40 mmHg, and PaCO2 may be underestimated, while the PTCCO2 is still accurately reflect the PaCO2 levels. Especially after the extubation, PetCO2 monitor becomes unavailable. And it is highly likely that hypoxia and hypercapnia happens. PTCCO2 monitoring is particularly valuable. In our study, the average PaCO2–PetCO2 difference was 10.3±2.3 mmHg whereas the average PaCO2–PTCCO2 difference was 0.9±1.3 mmHg. Those findings are unanimous with the past study.The end-tidal carbon dioxide .Previous studies reported that the COTCCO2 was more accurate in patients with a BMI greater than 40 kg/m2 undergoing transabdominal bariatric surgery. Maniscalco M et al 2) with chronic obstructive pulmonary disease (COPD), obstructive sleep apnea syndrome (OSAS), hypopnea syndrome (OHS) and respiratory failure (RF), PTCCO2 still accurately reflected the PaCO2,compared with the blood gas analysis. Our findings indicated that PTCCO2 was more accurate in reflecting the real levels of PaCO2 than PetCO2 in patients with BMI>35 kg/m2 undergoing laparoscopic bariatric surgery. And only one PetCO2 readings was 5 mmHg or less from PaCO2 while all values of PTCCO2 were 5 mmHg or less.Griffin J et al TCCO2 monitoring depends on monitor and a series of patient factors. Although TC-CO2 monitoring more accurately reflected PaCO2 in most of the patients in the previous studies, several technical factors may affect the accuracy of monitor, including trapped air bubbles, improper placement, damaged membranes, and inappropriate calibration techniques. Patient-related factors may also affect the accuracy, such as variations in skin thickness, the presence of edema, tissue hypoperfusion, or the use of vasoconstricting drugs and oxygen deficiency acidosis. Nishiyama T et al TCCO2 and PTCO2 more precisely predict the PaCO2 and PaO2 when the electrodes were put on the chest, compared to the placement at the upper arm and forearm. Nishiyama T etc The successful PTCCO2 and PTCO2 could accurately estimate PaCO2 and PaO2 respectively. Sorensen LC et al Electrode heating temperature can significantly affect the accuracy of the measurement results. Nishiyama T et al TCCO2 monitoring. Lower environmental temperature resulted in skin vascular contraction, reduced blood flow, In this study temperature in the operating room always maintained above 23°C. Meanwhile, patients’ exposure was reduced to the lowest possible level. Currently there are still disputes about the accuracy of PTCCO2 monitoring with the use of vasopressors, and past studies suggested vasopressors affected accuracy of PTCCO2 monitoring TCCO2 monitoring. Most of general anesthetics have the effect of vasodilation. Propofol administration produced venodilation and peripheral vasodilation in humans Skin tissue perfusion also affected the accuracy of the PetCO2 may underestimate PaCO2 because of inadequate washout of the anatomical dead space by a small tidal volume and the relatively slow response time of infrared CO2 analyzers. Especially during use of high frequency jet ventilation (HFJV) aCO2 in situations where continuous and precise control of CO2 levels in perioperative with HFJV TCCO2 monitoring especially useful during use of HFJV in obese patients which can avoid them in high risks of hypercapnia.We all know that during jet ventilation (JV), PTCCO2 can not substitute the PetCO2 monitoring completely as PetCO2 monitoring has many unique advantages, including the judgment of successful intubation, the warning of breathing circuit disconnected and indicator of pulmonary embolism, etc. In addition, the PetCO2 wave pattern had more clinical significance. The PTCCO2 monitoring was limited by many factors, including longer priming time, adjuscting before use, periodically electrode replacement, no CO2 waveform and the risk of skin burn injury However, PTCCO2 can estimate the PaCO2 more accurately than PetCO2 in obese patients under laparoscopic bariatric surgery. Moreover, the application of PTCCO2 monitoring might improve the quality of the anesthesia management.In conclusion, our study demonstrated that P