In this study, we evaluated the effect of an H2S slow-releasing compound GYY4137 (GYY) on a mouse PD model induced by acute injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). GYY was intraperitoneally (i.p.) injected once daily into male C57BL/6J mice 3 days before and 2 weeks after MPTP  administration. Saline was given as a control. Behavioral tests  showed that 50 mg/kg GYY significantly ameliorated MPTP-caused motor impairments. At lower doses (12.5 and 25 mg/kg) GYY exhibited a less obvious effect. Consistent with this, immunohistochemistry and western blot analysis demonstrated that 50 mg/kg GYY attenuated the loss of tyrosine hydroxylase (TH) positive neurons in the substantia nigra and the decrease of TH expression in the striatum of MPTP-treated mice. Moreover, at this regimen GYY relieved the nitrative stress, as indicated by the decreases in nitric oxide (NO) generation and neuronal NO synthase (nNOS) upregulation elicited by MPTP in the striatum. The suppression of GYY on nNOS expression was verified in vitro, and the results further revealed that Akt activation may participate in the inhibition by GYY on nNOS upregulation. More important, GYY reduced the nitrated modification of α-synuclein, a PD-related protein, in MPTP-induced mice. Overall, our findings suggest that GYY attenuated dopaminergic neuron degeneration and reduced α-synuclein nitration in the midbrain, thus exerting neuroprotection in MPTP-induced mouse model of PD.The neuromodulator hydrogen sulfide (H Its etiology remains elusive. Several pathogenic factors such as oxidative stress, mitochondrial dysfunction, protein misfolding and neuroinflammation, have been reported to be involved. In addition, higher level expressions of NO and its synthases were detected in the brains of PD patients and animal models . Inhibit2S or NaHS has been shown to exhibit neuroprotection in neurotoxins-induced rodent models of PD  that does not accurately mimic the biological process of H2S release in vivo. These limit our understanding to the role of H2S in PD pathogenesis. As such, in this study we evaluated the effect of GYY, which slowly releases low but consistent amounts of H2S within several hours in aqueous solution at physiologic pH and temperature  were purchased from the SLRC Laboratory . The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University. All mice were housed in a SPF animal facility with a temperature at 21–25°C and a controlled light–dark cycle.Parkinson’s disease animal models were established by intraperitoneally (i.p.) injecting the mice with MPTP  four times at 2-h intervals. Saline was given as controls. To evaluate the effect of GYY , the mice were administered with GYY at individual doses  once daily for 3 days prior to MPTP treatment, and continued throughout the experimental period. The animals were sacrificed 2 weeks after MPTP administration for biochemical studies.Behavioral assessments were performed at 2 weeks after MPTP injection. The data were analyzed by independent observers blinded to the treatments.Rotarod test was performed to estimate motor balance and coordination. Before test, each mouse was trained three times on the rod with an accelerating speed at 5–20 rpm/min. During the test session, the rotational speed remained constant at 18 rpm/min. A trial was stopped if the mouse fell down or the latency to fall reached 5 min. Each mouse was subjected to three trials with an interval of 30 min during the test session, and the average value of the latency to stay on the rod was calculated and included for further analysis.The mice were placed on a batten and tempted with fodder to cross a wooded balance beam . The test was stopped and repeated again if the mouse fell off. The time that a mouse successfully transversed the balance beam was recorded. The test was repeated three times at 5 min intervals, and the mean value of the walking time was presented as the data for analysis. A longer latency to cross the beam indicated a poorer motor coordination.∗ 40 cm wire grid with 3 ∗ 3 cm grid squares (iron wire at a diameter of 4 mm) in a quiet room with dim lighting, and allowed to walk for 30 s. The number of hindlimb slips was counted by an independent experimenter when the paw completely failed to hold a rung. The animal was put on the grid twice for habituation before test without pre-training. Each trial was repeated three times and the average of foot slips was used for analysis.Grid walking test was applied to evaluate the sensorimotor coordination of hindlimbs in mice. The mice were placed on a 50 2O2 in 0.01 M PBS for 30 min. Next, the sections were incubated in 5% BSA with 0.1% Triton X-100 in PBS for 30 min, followed by incubation with mouse monoclonal anti-TH  at 4°C overnight. Subsequently, the sections were washed in PBS, incubated with goat anti-mouse secondary antibody at room temperature for 1 h and stained using a DAB kit . The sections were observed and photographed using the Zeiss microscope .Following the behavioral tests, the animals were deeply anesthetized and perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde (PFA). Mouse brains were immediately removed from the skull and post-fixed in PFA solution overnight. After that, brains were dehydrated in a serial of alcohol solution and dimethylbenzene was applied to make the brains transparent. Brains were then embedded in paraffin wax. Next, 4 μm thickness sections were cut through SN compacta (SNpc), and every fourth slice was reserved. For staining, sections were deparaffinized and rehydrated, followed by epitope retrieval by boiling the sections in 0.01 M citrate buffer (pH 6.0) for 10–15 min and cooling at room temperature for 20–30 min. After that, sections were thoroughly washed in 0.01 M PBS three times. Endogenous peroxidase activity was blocked with 3% H+ levels were determined as previously described . An aliquot (20 μl) of each supernatant was eluted through a C18 reverse phase column  attached to the HPLC system  at a flow rate of 1.0 ml/min. The mobile phase consists of 15% acetonitrile, 50 mM potassium phosphate adjusted to pH 3.2 with ultrapure 18 M phosphoric acid. The detection limit is 3 ng/ml and this method provides good reproducibility.MPTP/MPPg for 30 min at 4°C. Next, an aliquote (50 μl) of the resulting supernatant was mixed with the dilution buffer at an equal volume, followed by addition with 5 μl nicotinamide adenine dinucleotide phosphate (NADPH), 10 μl flavin adenine dinucleotide (FAD) and 5 μl nitrate reductase and incubation at 37°C for 15 min. After that, 10 μl lactate dehydrogenase (LDH) was added and incubated for another 5 min. Subsequently, 50 μl Griess reagent I (0.1% naphthylethylene diaminedihydrochloride in 1% sulfanilamide) and 50 μl Griess reagent II (2.5% H3PO4) were added and incubated for 10 min. The optical density of the mixture was measured at 540 nm using a microplate reader . The results were calculated by a standard curve with sodium nitrite, and expressed as μmol per gram protein. The protein concentrations of striatal homogenates were determined using the BCA protein assay kit .The striatal NO content was evaluated by measuring the accumulation of nitrite and nitrate using the kit from Jiancheng Bioengineering Institute . Briefly, striatum tissues were homogenized (1:10 m/v) in lysis buffer and centrifuged at 15,000 Whole striatum tissues were homogenized with lysis buffer and centrifuged at 13,200 rpm for 20 min at 4°C. Protein concentrations of the homogenates were determined using the BCA protein assay kit. Equal amounts of total proteins were loaded in 8–12% sodium dodecyl sulfate polyacrylamide gels and then transferred onto PVDF membranes. Membranes were incubated with specific antibodies: anti-TH , anti-nNOS , anti-nitrated α-syn , anti-α-syn , anti-p-Akt , anti-Akt , and anti-β-actin  at 4°C overnight. Blots were then briefly washed with PBS followed by incubation with the corresponding secondary antibodies  for 1 h at room temperature. Protein densities were detected using an enhanced chemiluminescence kit and analyzed by ImageJ software.2/95% air at 37°C. For experimentation, cells were seeded into a 12-well plate and cultured overnight before treatment.PC12 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (China), and cultured in RPMI1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The cultures were maintained in a incubator with 5% COTukey’s post hoc analysis. Statistical significance was set at P < 0.05.All data were presented as mean ± SEM of at least three independent experiments. All statistical analyses were performed with GraphPad Prism 5.0 software. The differences among groups were analyzed using one-way analysis of variance (ANOVA) followed by Figure 1. Compared to saline-treated group (control), the latency to fall off the rod significantly decreased in MPTP-injected mice . Co-treatment with GYY at 25 and 50 mg/kg prolonged the time of staying on the rod compared to MPTP-only group. Balance beam walking test also showed that 50 mg/kg GYY co-treatment improved the MPTP-induced motor deficits, as indicated by a shorter time period to cross the beam compared to MPTP only group . Similarly, in grid walking test we found that MPTP injection increased the number of foot slips in mice, implying that the sensorimotor function was also impaired by MPTP. This was markedly attenuated by 50 mg/kg GYY co-treatment . And the treatment with GYY alone at 50 mg/kg, the maximal tested dose in this study, did not have any impact on the motor behavior.To study the ability of GYY to affect MPTP-induced impairment in motor function, we assessed the animals’ motor coordination using rotarod, balance beam walking, and grid walking test at 2 weeks after MPTP injection. The results are shown in + via monoamine oxidase (MAO) in the nigrostriatal system, the effect of GYY on striatal MPP+ levels was determined in a parallel study. In this part, 10 male C57BL/6J mice (10 weeks old) were applied and randomly divided into two groups: MPTP and GYY + MPTP group (n = 5 per group). For MPTP group, the mice received four injections with MPTP . For GYY + MPTP group, the mice were treated with GYY  once daily for 3 days followed by MPTP administration at 1 h after the last injection with GYY. No significant changes were observed in the striatal MPP+ level in MPTP-treated mice with and without GYY  pretreatment , which was measured 90 min after the last MPTP injection. Measurement of serum MPP+ levels revealed no significant difference between the two tested groups either . This indicates that the observed effects of GYY on MPTP-induced neurotoxicity are not caused by the reduction of MPTP conversion into MPP+.As MPTP-caused neurotoxicity to dopaminergic neurons is dependent on the metabolism of MPTP to its active metabolite MPPFigures 3A,B). Treatment with GYY alone did not have any impact on TH-positive staining in the SN. Western blot analysis showed that 50 mg/kg GYY co-treatment prevented the reduction of striatal TH protein level caused by MPTP . These results indicate that co-treatment with GYY at a suitable dose was able to prevent dopaminergic neuron losses in the SN of MPTP-treated mice.The dopaminergic neuron degeneration was examined by quantifying the number of TH immunoreactive neurons in the SNpc using immunohistochemistry study. The result showed that MPTP caused an approximate 40% decrease in the number of dopaminergic neurons in the SN compared to saline, and this was attenuated in the mice co-treated with 50 mg/kg GYY . This increase of NO generation was abated in the mice co-treated with GYY at the dose of 25 and 50 mg/kg, respectively. Likewise, co-treatment with GYY at this regimen inhibited the MPTP-induced upregulation of nNOS expression in the striatum . Treatment with GYY alone did not modulate NO or nNOS expression level. The result was verified by an in vitro study, which demonstrated that pre-treatment with 100 μM GYY prevented MPP+  elicited increase of nNOS expression in PC12 cells .Overproduction of NO and its associated nitrative stress participate in PD pathology . In lineFigure 4D).Excessive NO generation often results in peroxynitrite formation and tyrosine nitration in proteins. Human α-syn contains four tyrosine residues  that can be nitrated , which m+ was found to reduce the phosphorylation of Akt Ser 473 in a time-dependent manner, which was detected early at 0.25 h and remained to be dramatically declined at 1 h after treatment in PC12 cells . Pretreatment with 100 μM GYY almost reversed the decline of Akt phosphorylation caused by MPP+ exposure . However, co-treatment with Akt inhibitor perifosine (5 μM) was able to attenuate the GYY-mediated inhibition on nNOS expression in MPP+-treated cells . These results indicate that Akt signaling may be activated and thus contribute to the inhibition on nNOS expression by GYY.Last, we studied the signaling pathway that underlies the inhibition by GYY on nNOS expression. Treatment with 100 μM MPP2S slow-releasing compound attenuated dopaminergic neuron losses in the SNpc caused by MPTP. Last, GYY mitigated MPTP-induced nitrative stress and α-syn nitration in vivo. In addition, GYY preventive treatment did not affect MPTP metabolism, as serum and striatal MPP+ levels in GYY + MPTP group did not differ from those in MPTP group. To our knowledge, this is the first study demonstrating the neuroprotection of an H2S slow-releasing compound in the MPTP-induced mouse model of PD.In this study we demonstrate the potential of GYY as a neuroprotectant against dopaminergic neuron losses in a mouse model of PD based on the following findings. First, intraperitoneal injection with GYY at an appropriate dose (50 mg/kg) alleviated MPTP-induced motor impairments in mice. Second, this H2S in PD was subsequently confirmed by other studies. Two independent groups demonstrated that systemic administration with NaHS or inhaled H2S prevented neurodegeneration and movement dysfunction in the MPTP-induced mouse model of PD  of Soochow University. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University.XH: study design, data acquisition, data analysis and manuscript preparation. YY and YS: experiments design, sample collect and data analysis. BY, YW, and JZ: sample acquisition, data collection, statistical analysis. C-FL and XZ: study design and manuscript revision. L-FH determined the study theme, designed the experiments, ensured all the data and approved the final version to be published. All authors have contributed to this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins.Jeske et al. show that LOTUS domains present in the germline proteins Oskar, TDRD5, and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. In vivo, the localization of  Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS  domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of  RNA helicases are key enzymes involved in almost all RNA metabolic processes, such as RNA synthesis, processing, translation, and decay . DEAD-boDrosophila oogenesis, Vasa localizes to two functionally distinct compartments in the egg chamber: the germ plasm in oocytes and the nuage in nurse cells (Drosophila germ plasm (or pole plasm) is assembled at the posterior tip of the oocyte and specifies the Drosophila germ cell precursors called pole cells, which form at the posterior pole during early embryogenesis  domain  domain .Drosophila, the TDRD5 and TDRD7 orthologs Tejas and Tapas are jointly required for localization of Vasa to the nuage and play a role in piRNA-mediated retrotransposon silencing  is conserved in bacteria, fungi, animals, and plants and was originally suggested to bind to RNA . In animilencing . Moreoveilencing . This obilencing , raised Drosophila revealed that Vasa recruitment to the nuage and pole plasm depends on its interaction with eLOTUS domains. Our analysis identified the eLOTUS domain as a novel DEAD-box RNA helicase regulator and sheds light on the function of LOTUS domain proteins in animals.Here, we address this question directly and show that the LOTUS domains of Oskar, Tejas, and Tapas physically interact with Vasa. We further demonstrate that the LOTUS domain stimulates the Vasa DEAD-box helicase activity and that this function is conserved from insects to humans. We present the crystal structure of the Oskar LOTUS domain in complex with the C-terminal RecA-like domain of Vasa and show that the LOTUS domain occupies a novel binding surface on a DEAD-box helicase. Based on our finding that Vasa binding requires a particular C-terminal extension present in the LOTUS domains of Oskar, TDRD5, and TDRD7 but not in any of the LOTUS domains of MARF1, we divided the LOTUS domains into two subclasses; namely, extended LOTUS (eLOTUS) and minimal LOTUS (mLOTUS) domains. Finally, our mutational analysis in Drosophila Schneider 2 R+ (S2R+) cells, which do not express Oskar and Vasa endogenously. When Short Oskar and Vasa were expressed as C-terminal fusions to either GFP or mCherry, transfected GFP-Oskar localized in speckles within the nucleus, while mCherry-Vasa was distributed ubiquitously in the cytoplasm and nucleus  or in speckles (Tapas). Upon cotransfection, Vasa was no longer distributed uniformly within the cytoplasm and nucleus but was recruited to sites of Tejas and Tapas localization . The direct interaction of the LOTUS domains of Tejas and Tapas with Vasa was also observed in GST pull-down assays performed with purified proteins  of ∼10 µM for the LOTUS–Vasa-CTD complex -rich extension that is predicted to be disordered A. To ide complex C. We prease core . This suOskar binding to Vasa was mapped previously not to the Vasa-CTD but to regions for which we detected no interaction with Oskar. In one study, Oskar was shown to bind to a stretch residing in the Vasa-NTD (amino acids 200–252) . HoweverWe asked whether the binding of the LOTUS domain of Oskar to Vasa might modulate Vasa's DEAD-box helicase activity. Studies of several different DEAD-box proteins have defined the individual enzymatic steps of the helicase cycle . For simplicity, the experiments shown contained only ssRNA, as we did not observe a difference in ATPase activity whether ssRNA or dsRNA was present. Incubating ATP with high Vasa concentrations (20 µM) revealed a low level of ATP hydrolysis activity . The crystal structure consists of one LOTUS dimer and two molecules of Vasa-CTD, each one bound to a LOTUS monomer on the side opposite to the dimerization interface   .In the complex, the LOTUS domain is contacted mainly via the α2 helix of the Vasa-CTD D. ConverVasa interacts with the LOTUS domains of Oskar, Tejas, and Tapas but not the LOTUS domain protein MARF1 . The difSupplemental Fig. S2).In order to validate the Vasa–eLOTUS interface and design mutant transgenes for in vivo analysis of the Vasa interaction with LOTUS domain proteins (see below), we aimed to identify point mutations that lie in the interface of the complex A. All ofSupplemental Fig. S3A). The residue 162 bears a small side chain, which is either an alanine in the eLOTUS domains of Oskar and various TDRD7 proteins or a serine in TDRD5 proteins. The residue 228 is small and hydrophobic and either a leucine in the eLOTUS domain of Oskar or an isoleucine in the eLOTUS domains of TDRD5 and TDRD7 proteins. Together, this suggests that the eLOTUS–Vasa interaction tolerates little variation of the LOTUS interface residues.In Oskar, we identified one mutation each in the α2 helix (A162E) and α5 helix (L288E) of the eLOTUS domain that prevents its interaction with Vasa in GST pull-down assays B. The reSupplemental Fig. S3B). Belle, the DEAD-box protein with the highest sequence similarity to Vasa, also contains an aromatic residue at the position equivalent to residue 508 in Vasa . However, Belle does not interact with Oskar, suggesting that a single aromatic residue is not sufficient for LOTUS domain binding, which is consistent with our mutational analysis of Vasa . We conclude that LOTUS domains bind specifically to the Vasa helicase.In Vasa, we identified two point mutations in the α2 helix of the CTD (F504E and F508E), each of which prevents the Vasa–eLOTUS interaction in GST pull-down assays C. F504 aSupplemental Fig. S5A), strongly suggesting that the mutations do not interfere with the proper folding of Vasa. However, the activity of the Vasa mutant proteins was not stimulated by the eLOTUS domain of Oskar . We generated transgenes that encode GFP fused to either wild-type or mutant Vasa, resulting in GFP-Vasa-WT and GFP-Vasa-F504E proteins, respectively .We also tested the Vasa and eLOTUS orthologs of the silk moth ase core B. MoreovS domain C,D. Finabyx Vasa E, they dSupplemental Fig. S3A).Taken together, our functional assays demonstrate that the eLOTUS domain is a widely conserved positive regulator of Vasa. This is remarkable considering the low sequence identity among eLOTUS domains  and TDRD7 (Tapas) contain a single eLOTUS domain, some TDRD5 and TDRD7 proteins from other animals harbor mLOTUS domains in addition to their N-terminal eLOTUS domain. Whether the mLOTUS domains from MARF1, TDRD5, and TDRD7 have related activities or are functionally distinct remains to be determined.Our study provides molecular insight into the function of animal LOTUS domain proteins, factors involved in diverse germline functions. We showed that the DEAD-box helicase Vasa interacts with the LOTUS domains of Oskar, TDRD5/Tejas, and TDRD7/Tapas but not with MARF1. In Drosophila eLOTUS domain proteins Oskar, Tejas, and Tapas have been considered to be scaffolding proteins whose function is to recruit Vasa and other germline factors to germ plasm or the nuage. While LOTUS domains were originally predicted to be RNA-binding domains , whereas Vasa stimulation by Oskar in the pole plasm likely has a distinct role. Vasa was suggested to activate translation of mRNAs in the egg chamber through recruitment of eIF5B . We conclude that Vasa and eIF5B do not physically interact and that the recruitment of eIF5B by Vasa might be mediated through RNA or other proteins. It is equally plausible that Vasa's role in translation might be that of a DEAD-box RNA helicase involved in remodeling RNA–protein complexes. Given its importance in germline biology, the mechanism by which Vasa promotes translation of mRNAs merits thorough re-examination.The  domains , we were domains . Our preof eIF5B , which cof eIF5B . Vasa haof eIF5B . A Vasa Bombyx Vasa associates with the Piwi proteins Siwi and Ago3  with primers introducing the required restriction sites. The sequences of the eLOTUS domain  of Bombyx TDRD7 and human TDRD5 and TDRD7 were amplified from synthetic gene fragments (gBlocks purchased from IDT) codon-optimized for E. coli using primers introducing the required restriction sites. Sequences of the eIF5B constructs were amplified from an eIF5B plasmid obtained from Paul Lasko. All constructs were verified by sequencing. Proteins were expressed in and purified from E. coli (Rosetta 2) cells. The Bombyx mori Vasa protein sample comprised residues 135–564 and was a kind gift from Leo Nesme and Teresa Carlomagno. Details of the generation of recombinant proteins used in this study are provided in the Supplemental Material.For crystallization, Oskar 139–240 and Vasa 463–623 were mixed in a 2:1 molar ratio to obtain a 35 mg/mL protein mixture in crystallization buffer . Equal volumes of protein complex and reservoir solution  were mixed and subjected to the hanging drop vapor diffusion method. Crystals appeared the same day at 18°C and were flash-frozen 1 wk later, and diffraction data were collected at the ID23-1 beam line of the European Synchrotron Radiation Facility . The structure was solved by molecular replacement using PHASER . Search The crystal structure of the Oskar LOTUS–Vasa-CTD complex has been deposited in the Protein Data Bank under the accession number 5NT7.GST pull-down assays were performed as described  with pro32P] ATP  in a volume of 20 µL in the presence or absence of 10 µM RNA oligo of the sequence (AGCACCGUAAAGC)2. The protein concentrations used are indicated in the figure legends. Per time point, 4 µL of the reaction was transferred into 50 µL of 5 mM EDTA (pH 7) to stop the reaction. The mixture was subjected to phenol/chloroform extraction, and 2 µL of the aqueous phase were analyzed by TLC using polyethyleneimine-cellulose (Merck) and 1 M formic acid and 0.5 M LiCl as solvent. The thin layer plates were analyzed by phosphorimaging, ATP hydrolysis was quantified using Fiji, and the data were fitted using SigmaPlot.Proteins were incubated at 23°C with 0.1 µL (corresponding to 16.6 nM) of 10 µCi/µL [γ-w1118) or vasaPD/vasaD1 background. Details of the generation of the transgenic fly lines are provided in the Supplemental Material. Hatching assays were performed as described  (AttB vectors containing the EGFP fusion of Vasa with or without the F504E mutation were used for ΦC31 integrase-mediated transgenesis. Transgenes were studied in the wild-type (escribed . For imm(1:2000) , rabbit Drosophila cDNA (MARF1 and Tapas cDNAs obtained from the DGRC) and cloned into the blunt-end EcoRV site of either the pAc5.1B-EGFP or pAc5.1B-mCherry vector. Vasa E400Q and Short Oskar R266E constructs were obtained by site-directed mutagenesis of the respective templates. Oskar, Tejas, and Tapas constructs lacking the LOTUS domain were generated by amplification of the vector omitting the LOTUS sequence using phosphorylated primers and religation of the PCR product. The correct sequence of all constructs was verified by sequencing.The pAc5.1B-EGFP plasmid was a kind gift from Elisa Izaurralde. The pAc5.1B-mCherry plasmid was generated by replacing the EGFP with the mCherry sequence using the KpnI/HindIII sites. The sequence of Short Oskar (amino acids 139–606), full-length Vasa, Vasa truncations , MARF1, Tejas, Tapas, and Belle were amplified from Drosophila medium + (L)-glutamine (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum Gold (PAA) and 100 µ/mL penicillin–streptomycin (Thermo Scientific). Cells (1.5 mL) were seeded in glass-bottomed six-well plates and cotransfected with 0.2 µg of each of the respective EGFP and mCherry plasmids using the Effectene transfection reagent (Qiagen) according to the instruction manual. After 2 d, images of the live cells were taken with a 63× oil objective and a Zeiss LSM 780 confocal microscope and processed with Fiji.S2R+ cells (obtained from the DGRC) were grown in Schneider's


Brachylagus idahoensis), during the summer and winter by quantifying energetic costs associated with resting above and below ground. We used indirect calorimetry to determine the relationship between energy expenditure and ambient temperature over a range of temperatures that pygmy rabbits experience in their natural habitat. We also measured the temperature of above- and below-ground rest sites used by pygmy rabbits in eastern Idaho, USA, during summer and winter and estimated the seasonal thermoregulatory costs of resting in the two microsites. Although pygmy rabbits demonstrated seasonal physiological acclimatization, the burrow was an important thermal refuge, especially in winter. Thermoregulatory costs were lower inside the burrow than in above-ground rest sites for more than 50% of the winter season. In contrast, thermal heterogeneity provided by above-ground rest sites during summer reduced the role of burrows as a thermal refuge during all but the hottest periods of the afternoon. Our findings contribute to an understanding of the ecology of small mammals in seasonal environments and demonstrate the importance of burrows as thermal refuge for pygmy rabbits.Small mammals in habitats with strong seasonal variation in the thermal environment often exhibit physiological and behavioral adaptations for coping with thermal extremes and reducing thermoregulatory costs. Burrows are especially important for providing thermal refuge when above-ground temperatures require high regulatory costs  or exceed the physiological tolerances of an organism. Our objective was to explore the role of burrows as thermal refuges for a small endotherm, the pygmy rabbit ( Parus atricapillus;Mustela nivalis;Tamiasciurus hudsonicus;In mid- and high-latitudes, the thermal environment can vary substantially across spatial and temporal scales, such that ambient conditions can be energetically challenging for animals. For example, black-capped chickadees (Tb) through changes in posture, fur or feather orientation, and blood flow at the periphery (Tuc) and on the cool end by the lower critical temperature (Tlc). As ambient temperature increases above the Tuc, resting metabolic rate (RMR) increases due to evaporative cooling , and as temperature decreases below the Tlc, RMR increases to maintain Tb (T) if the animal is inactive. The energetic costs of thermoregulation over the range of temperatures animals experience in their environment can be estimated using data on the TNZ, the RMRT, and the relationship between RMR and temperatures below the Tlc and above the Tuc  is the range of ambient temperatures over which an animal can maintain body temperature , uses the refuge of a burrow to address thermal challenges in a strongly seasonal environment. Pygmy rabbits are endemic to the arid sagebrush habitats in the Intermountain West  measure the relationship between temperature and oxygen consumption during summer and winter using indirect calorimetry; (2) measure the thermal environment within burrows and at above-ground rest sites near burrow systems known to be used by pygmy rabbits during summer and winter; and (3) combine these datasets to estimate the approximate thermoregulatory costs of resting in burrows and above-ground microsites during both seasons. We hypothesized that the relationship between energy expenditure and temperature would vary from summer to winter, reflecting seasonal physiological acclimatization  to prevailing thermal conditions, such that thermoregulatory costs would be higher in summer than winter at temperatures below the TNZ. Because burrow use by pygmy rabbits is poorly understood and can be influenced by a variety of factors , we did not articulate specific hypotheses about how burrow use should differ with thermal conditions. Instead, we sought to quantify the costs associated with thermoregulating inside the burrow relative to above-ground rest sites in both seasons as a first step towards evaluating the costs and benefits of burrow use. However, we did expect that the burrow would serve as a thermal refuge for a greater proportion of time during winter than summer because temperatures can remain well below freezing for extended periods during the winter. We also expected that the thermoregulatory costs associated with resting in the burrow would be lower than above-ground rest sites overnight in winter and during mid-day in summer. Elucidating the role of burrows as thermal refuges for pygmy rabbits contributes to an understanding of the species’ thermal ecology and the functional role of habitat in mitigating physiologically stressful conditions.We evaluated RMR as rates of oxygen consumption by adult pygmy rabbits captured in east-central and south-central Idaho and in southwestern Montana, USA . We maintained the animals in captivity at the Small Mammal Research Facility at Washington State University. Animals were housed individually in 1.8 × 1.2 m mesh pens lined with pine shavings inside of a barn with a roof and partial walls. This arrangement exposed the rabbits to ambient temperatures but protected them from direct solar radiation, wind, and precipitation. Cages had corrugated pipe and nest boxes for enrichment and refuge. Food  and water were available ad libitum. Daily maximum and minimum ambient temperatures were recorded in the facility using a digital thermometer  during winter  and summer  trial periods. Mean daytime ambient high temperatures in the facility during winter were 7.8 °C  and mean lows were 1.1 °C . During summer, mean daytime ambient highs were 35.5 °C  and mean lows were 8.4 °C .2/min) during winter  and summer  across a range of temperatures typical of natural habitats. During winter, we evaluated 3). Because body heat can influence the internal temperature of the metabolic chamber, we measured temperature inside the chamber using two iButtons®  positioned on diagonally opposed corners. For all temperatures ≥0 °C, the metabolic chamber was placed inside a temperature controlled environmental cabinet. For the −5 °C winter temperature trials, the chamber was placed inside of a small freezer. We used a wireless infra-red camera  to monitor the activity and welfare of animals.We measured rates of oxygen consumption . Flow rate into the chamber and oxygen concentrations were averaged over 5 s intervals, converted to digital signal by an A–D converter , and recorded to a laptop using Warthog LabHelper software (http://www.warthog.ucr.edu). We collected baseline concentrations of room air for 3–5 min at the start of the trial and approximately every 40 min thereafter to correct for drift in the oxygen analyzer. We used Warthog LabAnalyst software to fit a regression to baseline oxygen concentrations and corrected oxygen concentrations accordingly. The 2; FiO2 is the fractional oxygen concentration in dry, CO2-free air (0.2095); and FeO2 is the fractional oxygen concentration of excurrent air scrubbed of water vapor and CO2. Data were visually inspected, and mean values of oxygen consumption were obtained when C; mL O2 h−1 °C−1; multiply by 0.00558 for watts/°C) for each animal at the coldest trial temperature in summer and winter according to the Irving–Scholander model, Tb for pygmy rabbits  and Washington State University (Protocol #04398-011), and they were in accordance with guidelines for the use of wild mammals in research published by the American Society of Mammalogists  is the dominant shrub species, with black sagebrush (A. nova), three-tip sagebrush (A. tripartita), green rabbitbrush (Chrysothamnus viscidiflorus), and rubber rabbitbrush (Ericameria nauseosa) occurring less frequently. The matrix between clusters of sagebrush supports a highly variable mix of low-growing shrubs, forbs, and bare ground in the lowest elevations, and more continuous grass and forb cover at higher elevations.We evaluated the thermal environment in above-ground microsites and burrows available to pygmy rabbits in sagebrush steppe habitat in the Lemhi Valley of east-central Idaho, USA. The valley is high-desert shrub-steppe  and receives on average <25 cm precipitation annually , most ofTe) to characterize the thermal environment of above-ground sites available to pygmy rabbits , and estimated summer RMRT. We tested for a seasonal difference in thermal conductance using a mixed-effects model with fixed effects for season and mass and a random effect for individual identity. Analyses were conducted using the “nlme” and “lme4” packages in R  using measurements of mean Te and mean burrow temperature. We calculated the proportion of time in each season that the burrow could serve as a thermal refuge for a resting pygmy rabbit. In winter, this was defined as the amount of time that mean Te < Tlc, but the burrow was warmer than mean Te and therefore had lower associated thermoregulatory costs. In summer, it was calculated as the amount of time that Te > 35 °C .We measured p = 0.017) on the slope of the Tlc. During summer, the slope was −0.21 ± 0.04 mL O2 min−1 °C−1 (p < 0.001), but in winter the slope was −0.11 ± 0.02 mL O2 min−1 °C−1  versus summer and winter data combined). Summer RMRT was 4.78 ± 0.51 mL O2/min for an animal of average size. We did not detect the Tuc in either summer or winter , but we did observe differing postures between high and low trial temperatures. During warmer trials , animals extended their bodies and assumed a sprawled posture, presumably to maximize contact with the chamber floor. This contrasted with the typical spherical posture during cooler trials. The mean values of thermal conductance for summer- and winter-acclimatized animals was 15.14 ± 1.35 mL O2 h−1 °C−1 and 12.28 ± 0.91 mL O2 h−1 °C−1, respectively . There was no significant difference between summer and winter thermal conductance , nor was there a significant effect of mass on thermal conductance .Season had a significant effect (Te) of above-ground rest sites was more extreme and variable than the burrow thermal environment during both seasons, but burrows provided more stable microclimates during winter than summer based on visual inspection. Notably, mean temperatures both above ground and in burrows remained below the estimated Tlc (25.2 °C) of pygmy rabbits for nearly the entire duration of winter monitoring. During winter, mean hourly Te ranged from −18.0 to 23.1 °C, and burrow temperatures ranged from −4.3 to 1.7 °C . Mean hourly Te in summer ranged from 1.5 to 49.8 °C . Accounting for the lowest mean temperature measured by a single Te sensor in a given hour, the number of days in which Te exceeded 35 °C (the estimated Tuc) dropped to six days, and Te remained above 35 °C for 2.5 h per day . High temperatures occurred during mid-day (1000–1700 h), likely as a result of solar radiation. During these periods, burrow temperatures averaged 17.7 °C (sd = 1.5) and were 22.5 °C cooler than Te, suggesting that burrow use could reduce the energy and water costs of thermoregulation when Te is considerably greater than Tuc.The burrow satisfied our definition of thermal refuge for a greater proportion of time during winter than summer. Burrows were thermal refuges on 30 of the 31 days that we monitored temperature during the winter, for an average of 13.4 h per day . Burrows were generally warmer than Te because the burrow temperatures often were below Tlc  for a 462 g eutherian mammal  among species of Otomyinae across a mesic-arid gradient, and suggested that these properties reflected similar refuge strategies rather than prevailing environmental conditions. The ability of pygmy rabbits to use burrows may contribute to seasonal constancy of Tlc and RMRT by allowing the animal to use behavioral means of increasing thermal resistance rather than physiological acclimatization alone. Exploring relationships between the use of thermal refuges and seasonal morphological and physiological acclimatization in pygmy rabbits remains a fruitful area for future research.Although we could not quantify a shift in a result . Althougquirrels , black-cquirrels , greenfiOur data showed that burrows provide important thermal refuge for pygmy rabbits during winter by reducing the energetic costs of thermoregulation relative to above-ground microsites. Winter temperatures at our study site rarely warmed to the TNZ of pygmy rabbits in either microhabitat, but burrows provided buffered microclimates and imposed lower thermoregulatory costs than above-ground rest sites for more than 50% of the monitoring period. Our observations of the role of the burrow as thermal refuge are consistent with the behavior of pygmy rabbits during winter. Te at the study site was typically above estimated Tuc for several hours daily, Te at some above-ground microsites was considerably lower and within the TNZ throughout the day because of the shade provided by dense overhead canopy. Shaded microsites and cool soil would negate the need to seek thermal refuge inside a burrow for an animal at rest. During the brief periods when above-ground temperatures exceeded the TNZ, the cost of cooling in the sheltered locations was predicted to be lower than the cost of warming inside the burrow at the same time because the difference in temperature between the burrow and Tlc was greater than that between the Tuc and Te. Piute ground squirrels (Spermophilus mollis) relied on burrows less in sagebrush steppe than in grassland habitats because the structural complexity of sagebrush offered more thermal heterogeneity and suitable microclimates: instead of using burrows for cooling, ground squirrels stretched out on the ground in the shade  during the winter, such that exposure to wind radically elevated metabolic rate at low temperatures. Thus, the thermoregulatory cost savings of using a burrow as a thermal refuge during the winter is potentially larger than our model predicts. Even given the uncertainty associated with the propagation of model error on thermoregulatory costs in each of the microhabitats, we believe that our results represent the relative utility of the burrow as a thermal refuge when above-ground climatic conditions are inhospitable. During the summer, the lowest Te values on our study site occurred during the night and early morning and were colder than the burrow and below the Tlc. Pygmy rabbits are active through the night and crepuscular periods during the summer . Furthermore, our estimates of energy expenditure do not address evaporative water loss, which is likely also an important factor in the thermal physiology and overall fitness of this species. Nonetheless, because pygmy rabbits demonstrated a high capacity for behavioral thermoregulation  and long-tailed weasels (Mustela frenata), are capable of retrieving a pygmy rabbit from a burrow system (Burrow use also is influenced by other factors besides behavioral thermoregulation including reproduction and predator avoidance . Additiow system , and vigMicrohabitat selection and its influence on physiology can have important ramifications for individual fitness . Our res10.7717/peerj.4511/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4511/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.4511/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.4511/supp-4Supplemental Information 4This dataset is required to perform the bootstrapping procedure to estimate standard errors of resting metabolic rate.Click here for additional data file.


Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called  By comparing S2] type . The anaS2] type . Considea°) and PDI-M(a) variants, we have investigated the contribution of each catalytic domain to the PDI-M redox properties using the same assays i.e., RNase A oxidative refolding, DTNB and insulin reduction, but also by testing activities typical of Trx i.e., the capacity to activate the chloroplastic NADP-MDH, a well-characterized redox-regulated chloroplastic protein catalyzing the conversion of oxaloacetate to malate, and to sustain the peroxidase activity of a chloroplastic Prx belonging to the 2-Cys Prx subgroup by regenerating the reduced active form of the protein.In subsequent experiments, we focused our effort on the atypical PDI-M. Using PDI-M(a°) presented an efficiency comparable to the one of PDI-M whereas PDI-M(a) exhibited only 50 to 60% of this activity after 15 minutes  was more efficient than PDI-M(a°) (a) was even slightly more active than PDI-M whereas PDI-M(a°) was less active  . In thisDI-M(a°) . This was active . Altogeta°) domain was as efficient as PDI-M whereas PDI-M(a) was only very poorly able to promote DTNB reduction (a), which was in fact the most efficient for insulin reduction when DTT was used as a reductant, obviously indicates that the a domain is not or only poorly reduced by NTR and that the catalytic disulfides of PDI-M are asymmetrically targeted by NTR. On the other hand, insulin reduction by PDI-M(a°) was less efficient than by PDI-M (a°) and PDI-M had comparable efficiency for DTNB reduction, these results indicate that mutating the active site cysteines of the a domain may somehow affect the redox properties of the a° module (possibly at the level of the 3D structure or of substrate recognition (here insulin)) which incidentally suggests that these two adjacent domains are connected.The properties of PDI-M domains have been further examined using the DTNB assay and AtNTRB as an electron donor. The PDI-M(eduction . To checeduction . The absby PDI-M . Since PArabidopsis 2-Cys Prx (At2-Cys Prx). As shown in a) was almost inefficient, only ca 10% activity was retained compared to PDI-M. The PDI-M(a°) retained about 50% activity. Such a decrease may again indicate that having mutated the active site cysteines of the a module impacted the a° module reactivity.Finally, we have taken advantage of the NADPH-coupled NTR reduction system to examine the reduction of Prx by PDI-M. Indeed, it was shown recently that an ER-targeted human 2-Cys Prx (PrxIV) can accept electrons from two PDI isoforms, P5 and ERp46 , 38. Altin vitro and also possibly in vivo depending on the substrates, on their subcellular localizations and/or on the cellular and subcellular redox potentials. In connection with these elements and with the variety of substrates, there are multiple PDI with variable module arrangements which provide the necessary flexibility in the PDI activity pattern  cluster was totally unexpected because this has to our knowledge never been observed for a PDI member. It appears a bit less surprising when taking into account that proteins sharing the same thioredoxin fold, i.e. some thioredoxins or glutaredoxins, including the ER-targeted GRX6 isoform of S. cerevisiae, are able to do this APWCGHC . We initrequired . Besides do this –51. Howen the ER .a°-a-b domain organisation quite different from the a-b-b’-a’ of the classical and well-characterized PDI-L. In particular, the organisation of the catalytic domains in tandem raises the question of their respective contribution to protein reactivity and of their possible connectivity. The latter point is in fact one of the unsolved questions for most PDI, although such cooperativity has been proposed based on the U shape arrangement of the four Trx domains of the yeast PDI1p, very similar to DsbC or DsbG [The second aim of the study was the biochemical characterization of a poplar PDI-M isoform possessing an  or DsbG . We have or DsbG .a modules of PDI-L1a possess the usual Glu and Lys residues, whereas both domains of PDI-M contain the Glu but an Ala and a His in the a° and a modules respectively , fully oxidized (16%) and partially oxidized forms of the a or a’ modules (18/15%) [i.e., without producing H2O2, should clearly decrease reactive oxygen species formation in the ER [These dual and opposite oxidase/reductase activities observed (18/15%) . These r(18/15%) , 56–58. n the ER .a’ module  of a human regular PDI-L, which presents the highest redox potential (ca -150 mV), possesses a preponderant oxidase activity toward reduced and scrambled RNase A [in vivo redox state [a domains might be important for the formation of native disulfides in proteins entering the secretory pathway by ensuring either oxidase or isomerase reactions.It is documented that the  RNase A . Also, i RNase A . With thox state , these ea domains in PDI-M. The a module, having the cysteine pairs with the lowest redox potential (-170 mV) is the most efficient for reduction reactions  , is more efficient for oxidation reaction than the a module, promoting the recovery of RNase A activity similar to PDI-M levels. Accordingly, the a° module of orthologs  is also the major contributor in the oxidative refolding of reduced or scrambled RNase A [a° module can efficiently oxidize NTR contrary to the a module. The incapacity of the a module to oxidize NTR or RNase A may relate to steric hindrance or unfavourable protein/protein interactions rather than to thermodynamic factors, the redox potentials of both domains being still rather close. The structure of the E. coli NTR-Trx complex indicated that Trx interacts with NTR mostly via hydrophobic residues  and via R73 [a domains of PDI-L1a and PDI-M, it is interesting to note that two of these five residues differ in the a domain of PDI-M compared to the three other a domains analyzed where they are all strictly similar (not shown). Hence, it is tempting to attribute the inability of the a domain of PDI-M to interact with NTR to these changes. Another possible explanation could rely on steric factors. In PDI-L1a, if it adopts a regular structure, the two a domains should be found at the extremity of the U-shape structure and thus be very likely accessible to NTR. On the contrary, the fact that the two a domains in PDI-M are adjacent and that the a domain is sandwiched between the a° and b domains may be problematic for the accessibility of NTR which is an homodimer of two identical 35 kDa subunits.Although experiments have not been achieved with poplar PDI-L1a, we have observed such a duality among the DH) Figs  and 7. O RNase A , 60. In  via R73 . By compa° module of PDI-M is reduced by AtNTRB whereas the a module is not or very poorly, we have observed some sort of cross-talk or interference between both domains. Indeed, PDI-M(a°) is as efficient as PDI-M in the assay with the small DTNB molecule (a module is needed for a maximal efficiency of the a° module with some substrates. Alternatively, introducing cysteine substitutions in the a module may have generated subtle conformational changes which hamper recognition of large substrates by the a° module. These substitutions may have more impact in PDI-M because the two catalytic domains are adjacent.In the course of these experiments and taking advantage of the fact that the molecule  whereas molecule . Thus, i


Positive impact of SiC nanoparticles on pinning grain boundaries and inhibiting grain growth was not obvious when deformation temperature was below 623 K, but was remarkable when the temperature was above 623 K. By comparing compressive stress-strain curves of AZ91 alloy and nano-SiCp/AZ91 composites, the addition of nanoparticles could improve the deformation ability of a matrix alloy under high-temperature conditions. There was no essential difference of deformation mechanism between AZ91 alloy and the composite, but hot deformation activation energy of the composite was significantly lower than that of the AZ91 alloy. The AZ91 alloy and the composite had the same workability region of 600 K–673 K and 0.001–1 s−1, while instability region for the composite was reduced compared with that of AZ91 alloy at high temperature.The hot deformation behavior and processing characteristics of AZ91 alloy and nano-SiCp/AZ91 composite were compared at temperature ranges of 523 K–673 K and strain rates of 0.001–1 s With high demand for energy and fuel conservation in the transportation sector, there have been wide range of research interests focused on magnesium and magnesium-based alloys ,2. Howev2O3 composite had been investigated by Prasad et al. .4XC optical microscope (OM) and MIRA 3XMU scanning electron microscope (SEM) were used for microstructure observation after hot compression. The specimens to be analyzed were cut parallel to compression direction using wire electrical discharge machining. The specimens were ground, polished and then etched [5 mL acetic acid + 6 g picric acid + 10 mL H−1 to 0.001 s−1. With the increase of deformation temperature from 523 K to 673 K, DRX extent also increases under the same strain rate. There are a high amount of twins existing in the AZ91 at a deformation temperature of 523 K and strain rates of 1 s−1 and 0.1 s−1. The number of enabled slip systems is few for magnesium with HCP structure, and larger critical shear stress is required for the activation of basal slip system in low temperature and high speed [−1. The microstructure is made up of fine recrystallized grains and a few coarse grains. The volume fraction of dynamic recrystallization is 98% at a deformation temperature of 673 K and a strain rate of 0.01 s−1. Further decreasing the strain rate to 0.001 s−1, fine recrystallized grains grow up and the deformed structure tends to coarsen. gh speed . Thus, s−1, volume fraction of DRX reaches 80% in the matrix alloy. Among the four deformation temperatures, microstructure deformed at 623 K is relatively uniform under different strain rates. As deformation temperature further increases to 673 K, complete recrystallization and grain coarsening take place. The grain size is small around SiC nanoparticles while the grains are coarse away from SiC nanoparticles within the rectangular red areas “A” and “B” at 673 K as shown in With respect to nano-SiCp/AZ91 composite, as shown in −1, as shown in −1 at 623 K, original grains in either AZ91 or composite are completely replaced by recrystallized grains, and recrystallized grain coarsening occurs. The average grain size of AZ91 alloy is 25.4 μm while that of the composite is 20.1 μm at deformation condition of 623 K/0.001 s−1, indicating the addition of SiC nanoparticles can refine the matrix alloy. Accordingly, the effect of SiC nanoparticles on the DRX changes with deformation temperature. When the deformation temperature is below 623 K, the positive impact of SiC nanoparticles on the DRX is not obvious but remarkable when the temperature is above 623 K. As mentioned earlier, during low-temperature compression, slip systems of AZ91 and nano-SiCp/AZ91 composite are limited and difficult to activate. So basal slip accompanied by twinning (giving c+a strain) is the main way for incoordinate deformation. With the increase of deformation temperature, the mobility of dislocation increase and more slip systems can be activated. Thus, the c+a glide mechanism becomes the main way and twinning ceases this role at high-temperature compression. The dislocation source can produce a large number of dislocations at high strain rate, which cannot bypass the hard SiC nanoparticles distributed evenly in the matrix alloy and form a dislocation tangle. When the number of dislocations pinned by SiC nanoparticles reaches a certain level, tangled dislocations generate a mass of dislocation cells. As the surrounding stress field increases, dislocation cells develop into unstable sub-structures. These sub-structures can further form low-angle grain boundaries and, finally, recrystallized grains. Correspondingly, at high deformation temperature the matrix alloy is softened and a pinning effect of the SiC nanoparticles on the dislocations is weakened. In this situation, the SiC nanoparticles play a role in pinning grain boundaries and inhibiting grain growth.The comparison of OM images of AZ91 and nano-SiCp/AZ91 composite is given in 17Al12 phases tend to precipitate preferentially in the form of fine particles. In this current work, precipitated spherical particles are mainly distributed in the deformed microstructure of AZ91 alloy. With respect to nano-SiCp/AZ91 composite, Mg17Al12 phases mainly exist in three forms: dispersed block, fine spherical particles and streamline shape. It should be noted that a large number of spherical second phases precipitate along the recrystallized grain boundaries both in the AZ91 alloy and nano-SiCp/AZ91 composite in 17Al12 phases. The strain increases continuously and the stress field increases gradually during the precipitation of Mg17Al12 phases [17Al12 phases, while the precipitation of Mg17Al12 phases can promote DRX, and the two aspects are mutually promotive. EDS analysis of the region “A” with red rectangular frame in 17Al12 phases precipitate in this area. However, due to the low content of SiC nanoparticles (1 vol. %) and slow strain rate (0.01 s−1) the number of dislocation tangles is low, and size as well as amount of precipitated phases in the form of streamline shape is low. In −1, it can be found that the Mg17Al12 phases in block form are distributed within the recrystallized area surrounding by fine Mg17Al12 phases. At high-temperature deformation, magnesium alloy can coordinate deformation by grain boundary sliding [17Al12 phases are broken and flow with the grain boundary sliding at slow strain rate and high deformation temperature.2 phases . When th sliding . After h−1 as shown in −1. The difference of flow stresses between AZ91 and its composite is decreased when strain rate is below 0.1 s−1. −1 at 673 K, the matrix has completed a greater extent of recrystallization compared with that of the AZ91 with a large amount of primary coarse grains  [θ/dσ)/σ curve based on the θ-σ curve, and the stress at the lowest point on the curve is the critical stress. Then the critical strain is determined on the stress-strain curve according to the critical stress. Therefore, by applying work hardening rate, critical stresses of AZ91 alloy and nano-SiCp/AZ91 composite under different compression conditions can be judged according to the critical stress pattern of magnesium alloy [θ and σ for AZ91 alloy and nano-SiCp/AZ91 composite under different compression conditions. In order to investigate the effect of SiC nanoparticles on critical condition of recrystallization, critical strain of AZ91 alloy and its composite during hot compression is compared in this work as shown in Based on the above phenomenon, deformation temperature and strain rate have an important effect on the deformation behavior for both AZ91 alloy and nano-SiCp/AZ91 composite. During hot deformation the appearance of peak stress in the micro-SiCp reinforced magnesium matrix composites indicates the occurrence of DRX . When the stages : slidingess (σp) . Poliak ess (σp)  gave theum alloy . Figure The relationship between strain rate and flow stress during thermal deformation can be described by constitutive equations . There aA1, A2, A3, α and β are material constant, δ is flow stress, n1 and n are stress exponents, Q is the activation energy during hot deformation, R is gas constant (8.31 J mol−1 K−1) and T is absolute temperature. The calculation of activation energy and stress exponent can be used to determine the deformation mechanism at high temperature. Three typical constitutive equations are shown in Equations (1)–(3) :(1)ε˙=AT can be combined by the Zener–Hollomon parameter The effects of the strain rate Taking natural logarithm for both sides of Equation (1), it can be expressed by:n1 can be obtained by the plot of The value of stress exponent Taking natural logarithm for both sides of Equation (2), it can be expressed by:β can be obtained by fitting the curve of n1 and β are 8.0 and 0.10532, respectively. Thus, the value of α can be determined according to formula α = n1/β. The value of material constant Taking natural logarithm for both sides of Equation (3), it can be expressed by:n can be obtained by fitting the curve of By substituting the value of α into Equation (7), the value of stress exponent Equation (8) is obtained based on Equation (7), which can be expressed:n for AZ91 alloy can be obtained by linear regression analysis of Linear regression analysis was performed on the curve of Q can be described by Equation (9):According to Equation (3), the activation energy n is related to different deformation mechanism [n = 2 for grain boundary glide, n = 3 for dislocation glide, n = 5 for dislocation climb and n = 8 for constant substructure model. When the deformation temperature ranges from 523 K to 623 K, the value of activation energy Q is close to the activation energy of lattice diffusion (QL = 135 kJ/mol) [n under these compression conditions is close to 5 and the deformation mechanism can be determined as the dislocation climbing mechanism controlled by lattice diffusion. When the deformation temperature is 673 K, the value of activation energy Q is higher than 200 kJ/mol. Due to the high deformation temperature recrystallization extent of the AZ91 alloy is high even at high strain rate, and the value of stress exponent n is 7.4 at 673 K  and (9), and Q for the composite ranges from 86 to 104.74 kJ/mol, which is close to the activation energy of grain boundary diffusion (Qgb = 82–105 kJ/mol) for pure magnesium [n at 523 K is 3.1, so deformation mechanism for the composite at this situation can be determined as dislocation glide. When the deformation temperature varies from 573 K to 673 K, the value of activation energy Q is larger than the activation energy of lattice diffusion (QL = 135 kJ/mol) [n for the composite is 4–6. Thus, the deformation mechanism of nano-SiCp/AZ91 composite is controlled by dislocation climb resulting from lattice diffusion. With respect to nano-SiCp/AZ91 composite, as shown in agnesium . The val kJ/mol) . This inQ at different deformation condition (ln(sinh(ασ)) according to Equations (3) and (4). Actual measurement and calculated value of flow stress are illustrated and fitted, as shown in n it is found that the dominant deformation mechanism is the same for both AZ91 alloy and composite, and the hot activation energy of the composite is significantly lower than that of the AZ91 alloy. This phenomenon can be attributed to the following reasons. Firstly, with the addition of SiC nanoparticle original microstructure of nano-SiCp/AZ91 composite is finer than that of AZ91 alloy. Secondly, SiC nanoparticles can hinder grain boundary migration during hot compression, leading to refined grains of the composite and reduced deformation resistance. Based on the above analysis, since the addition of SiC nanoparticles can reduce the critical stress and critical strain of the composite, recrystallization will occur more easily. In addition, it is reported that after adding SiC nanoparticles the nonuniformity of deformation in the matrix alloy increases with the increase of strain during the deformation process [By substituting activation energy ondition  into Equ process . The nonσ and strain rate In a hot-working process, the relationship between flow stress ion (10) .(10)σ=Km value is guaranteed by the coefficient. The power dissipation factor at the same temperature is represented by Equation (11) [The curve of ion (11) .(11)m=lm is variable. The microstructure during the material processing can be controlled according to the m value. The power dissipation map in a two-dimensional plane can be obtained using deformation temperature and strain rate, which is generally described as an iso-efficiency counter map. Equation (13) shows the criterion used to define the onset of flow instability [During the DMM the property for power dissipation due to microstructures evolution can be expressed by the dissipative efficiency of power , which ctability :(13)ζ, power dissipation efficiency increases with the increase of the compression temperature. This is because volume fraction of DRX in the composite increases with the decrease of strain rate and the increase of compression temperature. When the compression temperature interval is 623 K–673 K and the strain rate range is 0.001–0.01 s−1, the degree of DRX is large and the distribution of grain size is even, dislocation density decreases and the degree of the stress concentration is significantly weakened in the composite. Thus, this area can be regarded as a workability region for the composite since power dissipation efficiency is high within the area. By comparing −1, while peak dissipation efficiency of AZ91 alloy is slightly higher than that of the composite. Besides, the instability region of matrix alloy in the composite is reduced compared with that of AZ91 alloy at high temperature, indicating that the addition of SiC nanoparticles can improve the deformation ability at high temperature. At deformation condition of 523 K–573 K/0.1–1 s−1, it is difficult to activate the slip system in both the AZ91 alloy and composite, so twinning is the main deformation way in this situation. The occurrence of twins can improve deformation resistance and internal stress field, leading to the instability region. Meanwhile, compressive stress-strain curves of the AZ91 alloy and its composites indicate that flow stress at 523 K/0.1–1 s−1 is significantly higher than that at 523 K/0.001–0.01 s−1, which is consistent with the results of microstructure analysis. It can be also found that the instability region of the composite is significantly smaller than that of the AZ91 alloy under high temperature and high strain rate by comparing the processing maps of AZ91 alloy and composite. When the compression temperature is 623 K, the microstructure for the composite is mainly composed of DRX with some coarse original grains at high strain rate (0.1–1 s−1). With respect to AZ91 alloy, fine DRX grains are mainly distributed in the grain boundaries of the original coarse grains, forming a regional rheological structure. The stress concentration that exists in the rheological structure can produce regional rheology, resulting in flow instability.−1. It can be seen from 17Al12 phases surrounded by a large number of nanoparticles exist in the place where the crack initiates and propagates as shown in 17Al12 with large numbers of distributed at grain boundaries at low temperature and high strain rate can still impede dislocation mobility and grain boundary movement, resulting in the generation of intergranular crack.(1)The comparison of microstructures for AZ91 alloy and nano-SiCp/AZ91 composite shows that during low-temperature compression basal slip accompanied by twinning (giving c+a strain) is the main way for incoordinate deformation, while the c+a glide mechanisms becomes the main way and twinning ceases this role at high-temperature compression.(2)By comparing compressive stress-strain curves of AZ91 alloy and nano-SiCp/AZ91 composites, the addition of nanoparticles can improve the deformation ability of the matrix alloy under high-temperature conditions. Compared with AZ91 alloy the composite has a lower critical strain and reaches its critical strain earlier.(3)Hyperbolic sine can be applied to describe AZ91 alloy and composite under the present work. The dominant deformation mechanism is the same for both AZ91 alloy and composite, while the hot deformation activation energy of the composite is significantly lower than that of the AZ91 alloy.(4)−1, while peak dissipation efficiency of AZ91 alloy is slightly higher than that of the composite. Besides, the instability region of the composite is reduced compared with that of AZ91 alloy at high temperature.The AZ91 alloy and its composite have the same workability region of 600 K–673 K and 0.001–1 sIn the present work, hot compression of AZ91 alloy and nano-SiCp/AZ91 composite is carried out to investigate flow behavior and processing maps. The following conclusions are drawn:


So far these peculiar peroxidases are only found in the kingdom of Fungi. Here we present a phylogenetic reconstruction of the whole superfamily with focus on Hybrid B peroxidases. We analyse the domain assembly and putative structure and function of the newly discovered oligosaccharide binding domains. Two distinct carbohydrate binding modules (CBM21 and CBM34) are shown to occur in phytopathogenic ascomycetous orthologs of Hybrid B heme peroxidases only. Based on multiple sequence alignment and homology modeling the structure-function relationships are discussed with respect to physiological function. A concerted action of peroxide cleavage with specific cell-wall carbohydrate binding can support phytopathogens survival within the plant host.Heme peroxidases, essential peroxide converting oxidoreductases are divided into four independently evolved superfamilies. Within the largest one – the peroxidase-catalase superfamily - two hybrid lineages were described recently. Whereas Hybrid A heme peroxidases represent intermediate enzymes between ascorbate peroxidases and cytochrome  They differ in overall fold, active site architecture and enzymatic activities, catalysing the hydrogen peroxide-mediated one- and two-electron oxidation of a myriad of cationic or anionic inorganic and organic molecules or even proteins (Reactions 2O2 can be performed by some representatives (Reaction Peroxidases (EC 1.11.1.1–1.11.1.19) are essential peroxide converting oxidoreductases present in all domains of life. Four heme peroxidase superfamilies  arose independently during evolutionReaction .1\docume2O2-mediated formation of antimicrobial and halogenating oxidants  and many more putative sequences in general databases  thus providing the same systematic nomenclature as used in other superfamilies1. Family I is comprised of  catalase-peroxidases, ascorbate peroxidases, cytochrome c peroxidases and all their evolutionary intermediates. In Family II fungal secretory peroxidases including all manganese and lignin peroxidases and their evolutionary intermediates like versatile peroxidases are found, but also numerous other peroxidases described yet as “generic” (expected to be nonlignolityc)9. Finally, Family III is represented by plant secretory peroxidases with hundreds of closely related genes in almost all sequenced genomes of the plant kingdom.The peroxidase-catalase superfamily is the most abundant heme peroxidase superfamily currently counting over 8,800 unique annotated members in PeroxiBasec peroxidases16 and by (ii) Hybrid B heme peroxidases that were previously classified as Family I members13. However, recent analyses clearly demonstrated significant differences between Hybrid B peroxidases and Hybrid A or other Family I members15. In the present study we demonstrate that Hybrid B heme peroxidases are found solely in the kingdom of Fungi and are comprised of two domains, i.e. a conserved N-terminal catalytic peroxidase domain and a C-terminal carbohydrate-binding domain with a high variability. We present the phylogeny of these fungal enzymes, discuss their domain assembly and carbohydrate sequence motifs (CBMs) as well as their putative tertiary structures derived from homology modelling. Additionally, the physiological role of these oxidoreductases is discussed.From an evolutionary point of view the phylogeny of the intermediates positioned between the three families is highly interesting. Important turning points of gene evolution are represented by (i) hybrid A or ascorbate-cytochrome 17 and the second largest (super)family of hydrogen peroxide reducing heme enzymes . Because there were recent attempts to group quite different peroxidase and catalase sequences together in a cladogram (e.g. a neighbour-joining reconstruction18) it is important to note that the heme peroxidase superfamilies summarized in Table 2 as explained also in PeroxiBase documentation at http://peroxibase.toulouse.inra.fr/infos/documentation.php.The peroxidase-catalase superfamily annotated in databases as IPR002016 or PF00141 is currently represented by more than 23,300 protein sequences. As Table c peroxidases, manganese peroxidases, lignin peroxidases, versatile peroxidases, “generic” peroxidases, plant secretory peroxidases and hybrid peroxidases of type A & B) contain sequences from all domains of life. Definitively, there are now numerous novel members stemming from taxonomic lineages beyond bacteria, fungi and plants thus it is more appropriate to denominate the whole superfamily as peroxidase-catalase superfamily reflecting the dominance of peroxidase , which segregated in two different main clades, and various clades of ascorbate peroxidases. A recent study focused mainly on these divergent clades of ascorbate peroxidases15. Besides “classical” Family I ascorbate peroxidases and ascorbate-peroxidase related (APx-R) genes, which segregated in two well-supported clades  electron donor. With respect to the peroxidase domain APx-Ls might also represent pseudogenes15. Our present phylogenetic analysis shows that Metazoan (clearly non-plant) putative ascorbate peroxidases descended from the basal clades directly after the branches of “classical” intracellular ascorbate peroxidases from red & green alga and plants . In contrast, there is no known sequence of a Family II representative found in a phylogenetically basal lineage of Fungi yet. Family II enzymes  occur in Dikarya (Ascomycetes & Basidiomycetes) only. Thus, Hybrid B peroxidases appear to have older roots than all Family II members but clearly more sequences from all basal fungal lineages are necessary to strengthen this hypothesis.Recently, we have described the occurrence of Hybrid B peroxidases and started to analyse their phylogenyMagnaporthe oryzae causing rice and wheat blast (i.e. MagHyBpox1) and two other sequences from related hemibiotrophs (i.e. CfioHyBpox1 and CgloHyBpox3 – abbreviations explained in Supplementary Table 14. The WSC domain with the InterPro accession IPR002889 (or PF01822) was formerly described also as a putative carbohydrate binding domain. Mostly, it contains up to eight conserved cysteine residues that may be involved in several disulfide bridges. However, there is currently no evidence on its ability to specifically bind carbohydrates similar to above mentioned CBMs. A detailed functional analysis revealed that WSC proteins are typically highly O-glycosylated22 and that they can serve as cell wall integrity stress sensors23.The monophyletic Hybrid B peroxidase subfamily with currently 114 full-ORF-length representatives can be subdivided in 9 main clades. Two of them are chytridiomycetous, three are basidiomycetous, and remaining four ascomycetous. In the well-resolved solely ascomycetous clade #7 formed by sequences from phytopathogenic fungi  or even Lys69 – Tyr72 – His73 (SsclHyBpox numbering). The latter unique variant opens the question about the role of lysine in heterolytic peroxide bond cleavage. In any case, the distal histidine is apparently invariantly conserved in all known sequences of the whole superfamily. Besides this conserved triad an invariant Asn142 (BpKatG1 numbering) occurs at the distal side, which is involved in H-bonding and modulation of the pKa of the above mentioned catalytic His24.From the multiple sequence alignment Fig.  it is obAt the proximal side the heme ligand His279 Fig.  and its 25. They have a N-terminal catalytic heme domain and a shorter gen-duplicated homologous (heme-free) domain that supports the maintenance of the overall and heme cavity architecture26. The phylogenetic origin of the C-terminal domain is still under discussion15. Anyway, in course of further evolution this C-terminal domain was lost. There are rare exceptions detected only among Hybrid A peroxidases where the second domain still remained preserved27. However, the vast majority of members of the various subfamilies (except Hybrid B peroxidases) consist of only a single heme peroxidase domain (overview in PeroxiBase10http://peroxibase.toulouse.inra.fr/).Almost all members of this superfamily are one domain proteins consisting of the peroxidase domain only. Exceptions are catalase-peroxidases and Hybrid B peroxidases (and few Hybrid A members). At the basis of evolution of the whole superfamily two-domain bifunctional catalase-peroxidases are found14 it is now obvious that not all Hybrid B peroxidases contain a WSC domain and the variability in this region of the fused peroxidases is much higher than expected before.All Hybrid heme B peroxidases are fused proteins consisting of the highly conserved heme peroxidase domain and at least one non-homologous and non-catalytic C-terminal domain Figs  and 6. T28. It has to be emphasized that both CBM21 and CBM34 belong to the so-called raw starch-binding domains (SBD) found typically as modules of various microbial amylolytic enzymes29. Among 81 currently known CBM families there are at least 13 verified raw starch-binding domains as classified in the CAZy database (http://www.cazy.org/ 30) and, indeed, some of them have already been identified in non-amylolytic enzymes29. For example, CBM20 was found in the mammalian genethonin-131 and laforin32 as well as in fungal lytic polysaccharide monooxygenases33, whereas CBM48 was detected in the plant SEX4 protein32 and the β-subunit of AMP-activated protein kinase34.Our structural analysis Figs  and 8 clRhizopus oryzae glucoamylase (PDB code: 2DJM35) for MagHyBpox1 (residues Asp345-Asp426) and CBM34 from Thermoactinomyces vulgaris α-amylase TVA-I (PDB code: 1JI136) for both CfioHyBpox1 (residues Ile326-Gln426) and CgloHyBpox3 (residues Ile326-Glu426). In each case the models were selected in an effort to take into account the most appropriate combination of the three parameters confidence, sequence identity and alignment coverage.With regard to Hybrid B heme peroxidases the eventual presence of any CBM with assumed raw starch-binding capability is highly interesting. Despite the fact that MagHyBpox1 may contain CBM21, while both CfioHyBpox1 and CgloHyBpox3 possess CBM34, structural superimposition of their models with experimentally verified CBM21 and CBM34 templates clearly demonstrate that the overall respective folds, i.e. a typical immunoglobulin-like fold (β-sandwich) consisting of several antiparallel β-strands, have been preserved Figs  and 8C. 37. This is also the case of CBM34 , the relevant aromatic residues are present at both binding sites40. Comparison of saccharide binding residues from known CBM21s and CBM34s with putative CBMs from Hybrid B peroxidases  may represent a relict from a primordial CBM ancestor before the current CBMs specialized during evolution.However, there are several other aromatic residues, although temporarily with no assigned functional role, positioned “inside” the CBM, which are found in real amylolytic starch-binding domains and the Hybrid B peroxidase models Figs  and 8E. JZ969979.1, JZ970399.1 or DR621480.1). The physiologically observed oxidative burst accomplished by a prompt accumulation of reactive oxygen species mainly from the action of plant host NADPH oxidases represents the main streamline of the apoplastic immunity42. A concerted action of peroxide bond cleavage with a specific binding on integral cell-wall carbohydrates can counteract the plant defence pathways and allow the fungal pathogen to survive within the host tissue. Concerning the taxonomy spectrum of organisms found currently in the families CBM21 or CBM34, the former can be considered a eukaryotic family with a majority of various amylases of yeast and fungal origin, whereas the latter is yet a solely prokaryotic family with an unambiguous dominance of bacterial amylolytic enzymes30. To identify a homologue of CBM21, which is a typical fungal domain, among fungal Hybrid B peroxidases may thus not be so surprising, but to reveal a homologue of a typically bacterial CBM34 in a fungal hybrid B peroxidase should be of interest. Moreover, both CBM21 and CBM34 are best known as non-catalytic modules of amylolytic enzymes, which help their catalytic domains to bind and degrade raw starch or, in a wider sense, the α-glucans related to and/or derived from starch41. Since, however, the residues responsible for binding the α-glucans in both CBM21 and CBM34 have not been found to be conserved in their counterparts from Hybrid B peroxidases, it is possible to expect also some changes in target bound carbohydrates, even in terms of their stereochemistry, i.e. a change to β-glucans. To determine the exact role these CBMs may play in the function of Hybrid B peroxidases represents therefore a relevant challenge for experiments on purified proteins that are already being undertaken.A functional connection of a heme peroxidase with carbohydrate binding motifs thus observed among Hybrid B peroxidases from various important hemibiotrophic Ascomycetes might have significant impact for their phytopathogenicity. Transcripts of corresponding genes are currently detected in fungal families Magnaporthaceae & Glomerellaceae within mRNA libraries either non-induced or induced with some kind of oxidative stress (e.g. GenBank-EST database accession numbers c peroxidases) and monophyletic Hybrid B enzymes. The latter are unique fusion proteins containing a N-terminal highly conserved peroxidase domain and C-terminal domain comprised of variable carbohydrate binding motifs of two different types. The here observed unique domain fusion between a heme peroxidase and a CBM domain can open new horizons of future research exploring the physiological impact of the oligosaccharide binding domain(s) on the peroxidase function which might include hydrogen peroxide degradation during oxidative burst and/or site specific plant polymer degradation reactions in biotrophic and hemibiotrophic fungal pathogens.The phylogenetic reconstruction of the peroxidase-catalase superfamily reveals three well resolved families and two distantly related polyphyletic Hybrid A (ascorbate-cytochrome 10 at http://peroxibase.toulouse.inra.fr. Only in the case that a particular peroxidase sequence was not (yet) available in PeroxiBase corresponding Uniprot accession was used. All analysed peroxidase sequences are representative for the whole peroxidase-catalase superfamily divided in three main families and twelve subfamilies currently counting almost 8,800 manually annotated & curated sequences in PeroxiBase . Multiple sequence alignment of 500 selected full length protein sequences was performed with Muscle program43 implemented in MEGA 7 package. Optimized alignment parameters were: gap open −0.8 gap extend −0.05, hydrophobicity multiplier 0.9. Maximum of performed alignment iterations was set to 1,000. The used clustering method was UPGMB, for other interactions NJ and minimal diagonal length was set to 28. Alignment was inspected mainly for the presence of seven conserved catalytic residues on both distal and proximal sides involved in catalysis and binding of the heme prosthetic group44 and further refined in GeneDoc45. Ambiguously aligned regions were excluded from further analysis. After inspection and refinements the final alignment used for molecular phylogeny contained 500 full length sequences from all subfamilies of the peroxidase-catalase superfamily. For bifunctional catalase-peroxidases analysed thoroughly in previous studies14 only the sequences of N-terminal domain known to bind the prosthetic heme group were used and not their gene-duplicated C-terminal (heme-free) counterpart.All sequence data used for this analysis were collected from public databases. Protein sequences of herein analysed peroxidases were mainly from PeroxiBase46. Muscle-aligned protein sequences including all sequences with currently known 3D structures were subjected to Maximum-Likelihood (ML) method of this package. Following optimised parameters were applied: 100 bootstraps, WAG model20 of amino acid substitutions with four discrete gamma categories. The branch swap filter was set to very strong and the number of threads was set to 1. The branching patterns for particular subfamilies were presented with the Tree Explorer program of the MEGA 7 package in the rectangular form. The same protein alignment of 500 peroxidase sequences was then subjected to phylogenetic reconstruction using MrBayes 3.2.6 suite47. For calculating substitution rates the WAG model20 applying invariant gamma option was used with 4 discrete gamma categories. For diagnostics a relative burn-in of 25.0% was applied. Majority consensus tree was obtained from all credible topologies sampled by MrBayes over 3,000,000 generations with finally achieved standard deviation of split frequencies below 0.09 (recommended limit 0.10). Resulting trees were displayed and annotated with Interactive tree of life (iTOL v.348) in a circular form with transformed branches.Molecular phylogeny was first reconstructed using the MEGA package, version 7http://www.cbs.dtu.dk/services/NetAspGene/49). GT-AG consensus sequence for the borders between exons and introns was present in most but not all hybrid peroxidase genes. Detailed output for each particular gene is presented in PeroxiBase10.Search for donor & acceptor splice sites in (mostly) putative fungal hybrid peroxidase genes was performed. For this purpose the program suite NetAspGene 1.0 of the CBS server was used . The appropriate prediction database was chosen according to determined phylogenetic relationship of the analysed sequence. Those sequences that were found as intracellular with this approach were further subjected to subcellular localization analysis using TargetP 1.1 from the same online suite50.Putative signal sequences for protein secretion were revealed using the predictive algorithm of the program SignalP 4.1  and/or GenBank52; http://www.ncbi.nlm.nih.gov/genbank/). The partial alignment, covering only the predicted CBM domain, was performed using the program Clustal-Omega available at the European Bioinformatics Institute’s web-site (http://www.ebi.ac.uk/). In order to maximize similarities, the alignment was manually tuned taking into account previous bioinformatics studies55.All used CBM sequences were retrieved from the UniProt knowledge database56; http://www.rcsb.org/pdb/) for representatives of the individual CBM families, i.e.: CBM21 (PDB code: 2V8L40) and CBM34 (PDB code: 1UH438). Three-dimensional models for domains without experimental 3D structure were created with the Phyre-2 server57 (http://www.sbg.bio.ic.ac.uk/phyre2/) employing the “Normal” modelling mode. Obtained structures were superimposed using the program MultiProt58 (http://bioinfo3d.cs.tau.ac.il/MultiProt/) and displayed with the WebLab Viewer Lite programme (Accelrys Inc.).Three-dimensional structures were retrieved from the Protein Data Bank (PDB10 (http://peroxibase.toulouse.inra.fr/) and are listed in Supplementary Table 1.Of all peroxidases used in this work can be retrieved in PeroxiBaseTable with complete sequence listSupplementary Material