The sea ice-associated biomarker HBI III revealed an open-water signature, with highest concentrations near the mid-July ice edge. This proxy evaluation is an important step towards reliable palaeoenvironmental reconstructions that will, ultimately, contribute to better predictions for this High Arctic ecosystem in a warming climate.In order to establish a baseline for proxy-based reconstructions for the Young Sound–Tyrolerfjord system (Northeast Greenland), we analysed the spatial distribution of primary production and sea ice proxies in surface sediments from the fjord, against monitoring data from the Greenland Ecosystem Monitoring Programme. Clear spatial gradients in organic carbon and biogenic silica contents reflected marine influence, nutrient availability and river-induced turbidity, in good agreement with in situ measurements. The sea ice proxy IP The thinning and retreat of Arctic sea ice is one of the most striking consequences of recent climate change, and has a very significant impact on Arctic ecosystem functioning  lipid, which is synthesized in the sea ice matrix by some species of sea ice diatoms , which is likely produced by diatoms blooming in the open-water environment of the marginal ice zone  data. Examples of primary production tracers are the organic carbon content of sediments (TOC), which can be used as a basic proxy for organic matter production and mineralization, as well as biogenic silica, an indicator of siliceous—mainly diatom—production. A relatively large number of proxies may be used to trace the presence and variability of sea ice . One such proxy is based on the abundance of a biomolecule termed IPThe amount and quality of information available for the Young Sound fjord system provides an exceptional opportunity to evaluate the spatial distribution of these proxies against present-day conditions—a necessary first step for reliable palaeo-reconstructions. The main goal of this study is, thus, to provide a first evaluation of sedimentary signatures of primary production and seasonal sea ice along the fjord system (sediment core-top samples) in comparison with the present-day seasonal and interannual variability of these parameters (GEM data). This study defines a baseline for future palaeo-reconstruction studies using sediment core records from this region.As part of the MarineBasis program, a hydrographic transect covering approximately 120 km is sampled every year in early August to quantify physical and biochemical properties in the Young Sound–Tyrolerfjord system (since 2003). Vertical profiles of salinity, temperature, oxygen, turbidity, and fluorescence are measured at approximately 30 stations using a Seabird 19+ CTD (Conductivity–Temperature–Depth) instrument with the appropriate sensors. In 2014, the monitoring program was complemented by additional measurements conducted in July, September, and October.A total of 13 sediment core-top samples were selected from four sediment-coring campaigns in the Young Sound in the years 2013–2015  the sediment in a sodium pyrophosphate-solution and analysing the dispersed particles on a Malvern Mastersizer E/2000. The dispersion was wet-sieved at 1 mm prior to analysis in the Malvern Mastersizer and no large particles were retained on the sieve. The grain-size distribution of the samples is given here as % clay, silt and sand.210Pb, 226Ra and 137Cs via gamma spectrometry at the Gamma Dating Center, Department of Geosciences and Natural Resource Management, University of Copenhagen. The measurements were carried out on Canberra ultralow-background Ge-detectors. 210Pb was measured via its gamma-peak at 46.5 keV, 226Ra via the granddaughter 214Pb (peaks at 295 and 352 keV) and 137Cs via its peak at 661 keV.In order to confirm that the sediments integrate a present-day signal of processes occurring in the fjord, all samples were analysed for the activity of Two sample sets, one for total carbon (TC) analysis and one for total organic carbon (TOC) analysis, were established to determine the carbon contents of the sediments. Freeze-dried sample materials were placed in 10-ml test tubes and the samples were then rinsed twice with distilled water to remove the salt residue. The samples in the TOC set were further treated with 10% hydrogen chloride (HCl) for 24 h to remove inorganic carbon. After 24 h, the samples were checked for acidity with litmus paper and as all of them maintained acidic solution, no further acid treatments were carried out. The samples were rinsed with deionized water until the pH of the supernatant reached that of the deionized water. Both the TC and the TOC set of samples were then freeze-dried again. A known quantity (9–150 mg) of freeze-dried material was weighed and packed into a tin (Sn) capsule. Carbon contents were analysed using a MICRO CUBE Elemental Vario elemental analyzer (EA) at the Laboratory of Geochemistry in the University of Helsinki with lake sediment (LKSD-4) as a reference material. Uncertainty, based on duplicate and triplicate measurements of the actual samples, is within ±0.1%.2CO3 extraction method with mineral correction  according to the blue ammonium molybdate method (Mullin and Riley The amorphous or biogenic Si (BSi) concentration in the sediment was analysed from freeze-dried sediment samples using the 1% NaDeMaster . Samples2Cl2) and methanol (MeOH) . Extracts were pooled together and the solvent was removed by evaporation under a slow stream of nitrogen. The total extract was subsequently re-suspended in hexane and purified through an open column chromatography (SiO2). Hydrocarbons (including IP25 and HBI III) were eluted using hexane. Procedural blanks and standard sediments were analysed in between every 15 samples. Hydrocarbon fractions were analysed using an Agilent 7890 gas chromatograph (GC) fitted with a 30-m fused silica Agilent HP5-MS column (0.25 mm i.d. and 0.25 µm phase thickness) and coupled to an Agilent 5975C Series mass selective detector (MSD). Oven temperatures were programmed as follows: 40–300 °C at 10°C min−1, followed by an isothermal interval at 300 °C for 10 min. The data were collected using Chemstation and analysed using the MassHunter quantification software. IP25 and HBI III were identified on the basis of retention time and comparison of mass spectra with authenticated standards. Abundances were obtained by comparison between individual GC–MS responses relative to those of the internal standard. Data are reported here in ng g−1.Highly branched isoprenoid (HBI) analyses were conducted according to the procedure described in Belt et al. . Prior tSatellite images from 2014 show the typical seasonal sea ice development in Young Sound  did not show measurable content of unsupported 210Pb but this may be caused by the sandy nature of the sample .The presence of unsupported ple Fig. . AdditioTotal organic carbon (TOC) results helped to define three zones within the fjord Fig. . Zone A,The generally higher TOC values for YST1 to YST6 , corresponding to 0.3 and 1.9 wt% SiO2 (median 1.2 wt% SiO2). Biogenic silica (BSi) refers to the amorphous Si content in sediments. It is mainly used as a proxy for siliceous microfossil abundance, and it has been shown to be a useful proxy for diatom abundance and productivity in aquatic systems , biogenic silica reflects the interplay between the available dissolved silica from glacial meltwater and light availability. Three major glacial rivers discharge into the fjord and the total combined runoff from land has been estimated to 0.9–1.4 kmDuring the summer, phytoplankton assemblages are largely dominated by diatoms in the outer part of the fjord, while dinoflagellates and ciliates increase in abundance towards station YST7 and colonial chrysophytes become the most abundant group around station YST12  and Pleurosigma (P. stuxbergii var. rhomboides). Within the MarineBasis framework of the GEM Programme, phytoplankton identification counts have been performed since 2004, and the dominant diatom species at the water/sea ice interface have also been reported for the Young Sound  production, supporting an in situ source of this biomarker (our unpublished results).IPent Fig. ; Table 2n et al.  as pan-A25 concentrations in the sediments are generally low along the fjord; highest concentrations are found in the outer fjord region, where conditions are closer to those of a marine setting  diatoms living in the sea ice, HBI III is likely derived from marine open-water planktic diatoms [only known producers at the moment belong to Pleurosigma and Rhizosolenia ]. The similarities between the spatial distributions of biogenic silica and HBI III support an open-water diatom production signature for this proxy. Indeed, the distribution of HBI III reflects the phytoplankton bloom occurring in end-July/August in the fjord, as seen by the fluorescence values  values ranged from 0 to 0.7 ng gent Fig. ; Table 2es Figs. , 4 and ces Figs. . Intereses Figs. .It has been suggested that HBI III-producing diatoms bloom in higher abundance in open-water areas near the sea ice edge  were analysed from 13 core-top samples along a transect from the outer Young Sound to the inner Tyrolfjorden. These proxies showed an overall good reflection of present-day conditions in the fjord, and a clear integration of the multiple factors (land–ocean–ice interactions) affecting this High Arctic ecosystem.Primary production proxies (organic carbon and biogenic silica), and sea ice-related biomarkers  with the highest concentration in the outer part of the fjord. This indicates that the fjord environment is somewhat unfavourable for the growth of sea ice diatoms producing IP25. This is possibly a consequence of poor light availability due to snow cover and low salinity levels resulting from the high runoff and meltwater input. We thus recommend caution when attempting to reconstruct past sea ice variability in fjord systems using IP25 records, as our results clearly indicate that these cannot simply be extrapolated to open ocean conditions. It is expected that future warming and enhanced melt in this region will lead to changes in sea ice cover, exchange with the ocean, and an increased runoff from land—with an unpredictable impact on primary productivity in the fjord, also for sea ice-related biota. This set of proxies, applied to sediment records covering past warm climate periods  have the potential to constrain future scenarios for the Young Sound region in a warming climate.Organic carbon contents were <1.5% and followed a clear gradient, with the highest values closer to the fjord entrance . Biogenic silica—a proxy for siliceous, mainly diatom production—was generally higher at the outer fjord stations and sites in the middle part of the ford, where there is a combination of high input from the glacial rivers (source of dissolved silica), lower turbidity than in the innermost fjord region, and some degree of exchange with more saline and nutrient-rich waters from the outer fjord/Greenland Sea. The biomarker HBI III followed a pattern roughly similar to that of biogenic silica and appears to reflect diatom production during the ice-free period, notably the annual bloom that starts outside the fjord mouth and penetrates into the middle part of the fjord. Nonetheless, higher concentrations of this biomarker were found near the mid-July ice edge. The sea ice proxy IP


OsTrx23, T55GOsTrx20, K48DOsTrx20 and T55G-K48D OsTrx20 as well as wild types OsTrx23 and OsTrx20 were tested in the reaction containing either NADPH/NTR or glutathione (GSH). The results revealed that reduction rate of T55GOsTrx20 was remarkably decreased by GSH as compared to WtOsTrx20 highlighting the critical role of Thr-55 in interaction of OsTrx20 with GSH. On the other hand a significant decrease in the reduction rate of G41TOsTrx23 was observed in reaction containing NADPH-dependent thioredoxin reductase as compared with readuction rate of WtOsTrx23. These results suggest that first residue after N-terminal active site Cys is one of the critical residue in determination of system that Trxs can be reduced in. Thioredoxins (Trxs) are small ubiquitous oxidoreductase proteins with two redox-active Cys residues in a conserved active site (WCG/PPC) that regulate numerous target proteins via thiol/disulﬁde exchanges in the cells of prokaryotes and eukaryotes. The isoforms OsTrx23 with a typical active site (WCGPC) and OsTrx20 with an atypical active site (WCTPC) are two Trx h- type isoforms in rice that were previously found to be reduced by NADPH-dependent thioredoxin reductase and GSH/Grx system, respectively. In the present work the reduction of mutants G41T Medicago truncatula that have been previously reported to be reduced by a GSH-dependent pathway .A multiple alignment among the amino acid sequences of rice Trx h isoforms demonstrated that the active site sequences of OsTrx1, OsTrx10, OsTrx15, OsTrx18, OsTrx23, OsTrx24, and OsTrx26 corresponds to classical Trx-active site motif (Trp-Cys-Gly -Pro-Cys). In the active site sequence of OsTrx20, however, Gly has been replaced with Thr. Moreover, in other plant Trx h isoforms a conserved Asn/Asp is observed before active site. This amino acid has been replaced by a Lys in OsTrx20 .OsTrx20, WtOsTrx23, OsNTRB and mutants K48DOsTrx20,T55GOsTrx20, T55G-K48DOsTrx20 and G41TOsTrx23 with a His.tag at the N-terminus were found in the soluble fraction of the E. coli transformant culture after induction with IPTG. The theoretical molecular mass of WtOsTrx20, WtOsTrx23, OsNTRB, K48DOsTrx20, T55GOsTrx20, T55G-K48DOsTrx20 and G41TOsTrx23 were 17.5, 15.4, 39.9, 17.5, 17.5, 17.5 and 15.4 KDa respectively. SDS-PAGE of cell extracts revealed a prominent polypeptide band of the expected molecular mass (The proteins Wt650) in insulin-disulfide reduction assays containing GSH in the presence of WtOsTrx20, T55GOsTrx20, K48DOsTrx20 or T55G-K48DOsTrx23  are not reduced by NADPH/NTR system but they can be reactivated by GSH/Grx system , 7. In cOsTrx23 by OsNTRB decreases in comparison to WtOsTrx23 suggesting the presence of Thr in this position impairs the interaction of OsTrx23 with OsNTRB. In addition we showed that the isoform WtOsTrx20 was actively reduced by GSH and substitution of Thr55 with Gly in OsTrx20 resulted in remarkable decrease of rate of reduction by GSH. These results supports the critical role of residues situated between two active site Cys in redox activity of Trxs [In this work, we showed that the rate of reduction of G41T of Trxs . and higTaken together the presence of atypical active site for some h-type Trx isoforms, with Thr or Ser substitution for the largely conserved Gly in the first position after N-terminal active site Cys might be an evolution change that enables these isofroms to be reduced by GSH and adapt them in the cell organelles lacking cytoplasmic/ mitochondrial NTR isoforms.The authors declared no conflict of interest.


Botrytis cinerea is a filamentous plant pathogen, which infects hundreds of plant species; within its lifestyle, the production of reactive oxygen species (ROS) and a balanced redox homeostasis are essential parameters. The pathogen is capable of coping with the plant’s oxidative burst and even produces its own ROS to enhance the plant’s oxidative burst. Highly conserved NADPH oxidase (Nox) complexes produce the reactive molecules. The membrane-associated complexes regulate a large variety of vegetative and pathogenic processes. Besides their commonly accepted function at the plasma membrane, recent studies reveal that Nox complexes are also active at the membrane of the endoplasmic reticulum. In this study, we identified the essential ER protein BcPdi1 as new interaction partner of the NoxA complex in B. cinerea. Mutants that lack this ER chaperone display overlapping phenotypes to mutants of the NoxA signaling pathway. The protein appears to be involved in all major developmental processes, such as the formation of sclerotia, conidial anastomosis tubes and infection cushions (IC’s) and is needed for full virulence. Moreover, expression analyses and reporter gene studies indicate that BcPdi1 affects the redox homeostasis and unfolded protein response (UPR)-related genes. Besides the close association between BcPdi1 and BcNoxA, interaction studies provide evidence that the ER protein might likewise be involved in Ca2+ regulated processes. Finally, we were able to show that the potential key functions of the protein BcPdi1 might be affected by its phosphorylation state. Botrytis cinerea, causative agent of the gray mold disease. The fungus has a necrotrophic lifestyle and benefits from high humidity and moderate temperatures. During vegetative growth, as well as during infection of plant material, various signaling molecules and cascades contribute to the developmental processes. Besides abiotic factors such as light and temperature  by transporting electrons across the lipid bilayer on to molecular oxygen , the regulatory subunit (p67phox – BcNoxR), adaptor proteins (p22phox – BcNoxD) as well as for putative transient members of Nox complexes such as the scaffold protein IQGAP (BcIqg1)  . Additio(BcIqg1) . While t(BcIqg1) , Alzheim(BcIqg1)  and athe(BcIqg1) , the Nox2+) signaling processes. Since analyses in plants reveal a tight and recurrent connection between ROS and Ca2+ signaling pathways  that this oxidative burst appears to rely on elevated Ca2+ levels. Moreover, we elucidated that increasing Ca2+ concentrations promote a slight oxidative burst inside the endoplasmic reticulum (ER), mediated by the catalytic subunit BcNoxA  proteins by the oxidative protein folding machinery. Primarily responsible for the reduction, isomerization or oxidation of proteins within this process is the protein disulfide isomerase (PDI). In its oxidized form, PDI is able to function as a posttranslational modifying chaperone, which facilitates correct protein folding and catalyzes disulfide exchange reactions. During this process PDI is reduced and has to be regenerated by the oxidoreductin 1 (Ero1) . PDIs artrations , apoptottrations  and stretrations . In mammSaccharomyces cerevisiae  and infection cushions (IC’s), as well as for full virulence. Finally, we were able to show that the potential key function of the protein BcPdi1 may be affected by its phosphorylation state, which likewise is necessary for the maintenance of the redox homeostasis and the transmission of signals.Here, we report the occurrence of physical interactions between members of different signaling pathways. The close association of the PDI BcPdi1 to CaBotrytis cinerea Pers.:Fr. [Botryotinia fuckeliana (de Bary)Whetzel] B05.10 was collected from Vitis vinifera (Supplementary Table S1.vinifera  and was Phaseolus vulgaris). Cultures were grown for 6–8 days at 20°C under light conditions  to obtain conidia. Sclerotia were induced by incubating the strains for 3 weeks at 20°C in darkness. For DNA preparation, the strains were grown for 3–4 days at 18°C on CM agar overlayed with Cellophane. For stress experiments, the CM was supplemented with agents inducing osmotic, oxidative, cell wall, membrane or ionic stress. Minimal medium was prepared after Czapek Dox .For cultivation synthetic complete medium (CM)  was usedbcpdi1, the 5′/3′ flank were amplified with the primer 1/2 and 3/4 (Supplementary Table S2) as well as the resistance cassette with the primer pair 16/17. Recombinational cloning was done with S. cerevisiae FY834 and the vector backbone of pRS426 (EcoRI/XhoI). For complementation, bcpdi1 was expressed under its native promotor (primer 10/15) as well as under the constitutive oliC promotor of Aspergillus nidulans (primer 9/10). The constructs for introducing mutated phosphorylation sites were amplified using the primer 9/12 and 10/11 (Δbcpdi1-C-M1), the primer 9/14 and 10/13 (Δbcpdi1-C-M2), 9/12, 11/14 and 10/13 (Δbcpdi1-C-M1/2), 10/29 and 9/28 (Δbcpdi1-C-DA). For the generation of PDI in loco complementation constructs that were fused to the genetically encoded biosensor roGFP2, six different fragments were generated by the primer pairs 1/11, 12/32, 33/34, 35/36, 37/38, 39/4 (PDI_phospho_null) or 1/28, 29/30, 31/32, 33/34, 35/36, 37/4 (PDI_phospho_mimic).For the generation of deletion and complementation constructs, the yeast homologous recombination system was used . For genS. cerevisiae FY834, positive transformants were selected on SD medium lacking uracil since the strain is auxotrophic for this amino acid. Total DNA was isolated and transformed in Escherichia coli. Re-isolation was done by the Nucleo spin® plasmid easypure kit  and correct assembly was tested by sequencing.After transforming Botrytis cinerea was transformed with 40–60 μg of plasmid DNA as described previously (eviously . Selectieviously . CorrectPhaseolus vulgaris L.) were infected according to For characterization of the different mutant strains various growth, differentiation and pathogenicity assays were performed. For testing defects in pathogenicity, French bean plants . Samples of 20 μg were loaded to an agarose gel and transferred to Hybond-N+ membranes. Northern blot hybridizations were done according to the method of TM Blue-White DPX  in McIlvaine standard buffer . Visualization of ICs was done with the 20× objective lens, while germinated conidia were analyzed with 40× or 63× magnification. ER staining was accomplished using the ER-Trackerd buffer . The sam1) as it was shown before (roGFP2 measurements were done using an inverted microscope (Leica DMIRE2) equipped with a Leica TCS SP2 scan head . Conidia were prepared as described previously . Resultsn before .S. cerevisiae for anchoring the proteins at the membrane. Interaction vectors (prey/bait) were generated according to the manufacturer’s protocol. Both vectors  were restricted with SfiI and ligated with likewise restricted PCR products (for details see Supplementary Table S2). The ligated products were transformed in E. coli and selected with the appropriate selection marker.Protein–Protein interactions were investigated by a split-ubiquitin based yeast-two-hybrid system . The proteins of interest were fused to the C- or N-terminal half of ubiquitin. Upon interaction, ubiquitin congregates and the transcription factor controlling reporter genes  can be released. Proteins without transmembrane regions are additionally fused to a transmembrane domain of the oligosaccharyltransferase subunit 4 (Ost4) of S. cerevisiae NMY51 according to the manufacturer’s protocol (DUALSystems Biotech) with a modified version of the lithium-acetate method . For drop tests, established colonies were grown overnight in SD medium lacking leucine and tryptophan. OD600 was adjusted to 1 and cells were pelleted. The medium was removed and starvation was induced by the addition of sorbitol (1 M): incubation then took place for at least 5 h at 30°C and 200 rpm on a shaker. Subsequently samples were diluted up to 1:1000 and 10 μl of each dilution were dropped on selective medium  and control plates (SD-leu-trp). Plates were incubated for 3–4 days at 30°C.For interaction assays, prey and bait were transformed in e method . Yeast coliC promotor (A. nidulans) and reporter genes were fused to the N- or C terminus of the respective protein. Fusion proteins were generated by the yeast recombination system. Whereas bcnoxA was cloned into the vector pNAH_OCT (restricted with NcoI) containing mcherry as reporter gene, bcpdi1 was cloned into the vector pNDN_OGG (restricted with NcoI) with gfp as fluorescent marker.For co-IP experiments, proteins of interest were transformed into the wild type strain. The expression is controlled by the constitutive After transformation, the strain expressing both constructs was purified by single spore isolation and grown on CM medium with Cellophane overlay for 3–4 days. The mycelium was harvested, lyophilized and proteins were isolated by grinding and applying disruption buffer . For the NoxA_mcherry control, the extract was stored until Western blotting. The protein extract of the PDI_GFP control as well as of the strain expressing both constructs was loaded to μMACS GFP beads/columns and purified according to the instruction manual of the μMACS GFP tagged protein isolation kit . After Western blotting, detection was accomplished using the anti-gfp antibody . For mcherry detection, the anti-mcherry antibody  was used as primary antibody and for the secondary antibody the donkey-anti-rabbit antibody  was used. Visualization was conducted using the enhanced chemi-luminescence (ECL) detection .B. cinerea strain B05.10 were obtained by the database Ensembl2. For the analysis of the sequences, different programs were used: Signal peptides were predicted by SOSUIsignal  probably contains two transmembrane domains with a small cytosolic loop , with both ends located inside the ER. Its two catalytic domains (CXXC-motif) are predicted to be located in the cytosolic loop  and inside the ER, respectively . Moreover, two cytosolic phosphorylation sites are predicted . Interestingly, the first putative phosphorylation site is highly conserved among different fungal species  while the second one is poorly conserved.Since our studies had revealed that the NoxA complex has a distinct function inside the ER, putative targets/partners were investigated. An essential ER protein, which in mammalian systems is associated with subunits of the Nox complex  is the Pbcpdi1 were generated . Transformants were checked via diagnostic PCR  and Southern blot analysis . Since all mutants displayed an identical phenotype, the results of the mutant Δbcpdi1_T2 are presented. In order to elucidate the role of the predicted cytosolic phosphorylation sites of the protein, mutants with mutated phosphorylation sites were generated. The key amino acids were replaced by an alanine to mimic the constitutively dephosphorylated state (phospho null) . Moreover, the putative first phosphorylation site was transferred into its constitutively phosphorylated form (phospho mimic) by replacing threonine by glutamic acid (T65E).For functional characterization, deletion mutants of To check whether BcPdi1 is involved in stress response pathways, the radial growth was determined on CM containing various stress agents.bcpdi1 mutant on CM was reduced by 20% in comparison to the wild type. Whereas the complemented strain, Δbcpdi1-C-M2 and Δbcpdi1-C-DA grew as well as the wild type, the colony diameters of Δbcpdi1-C-M1 (87.5%) and Δbcpdi1-C-M1/2 (83.5%) were slightly reduced.The radial growth of the Δbcpdi1 led to a higher sensitivity of the mutants to oxidative and osmotic stress. The addition of 10 mM H2O2 and on 1 M NaCl was sufficient to reduce the rate of radial growth of Δbcpdi1 significantly, but the addition of menadione had no effect. While the complemented strain displayed wild type-like growth on all selective media, the mutation of the first predicted phosphorylation site correlated with the deletion mutant’s phenotype. The mutation of the second putative phosphorylation site had no effect . The ‘phospho mimic’ mutants mostly resembled the growth behavior of the wild type. However, on NaCl-supplemented medium the mutant grew much better than all other strains. In contrast to oxidative and osmotic stress, the addition of Calcofluor White (2 mg/ml) as a cell wall stressor, or SDS (0.02%) as a membrane stressor did not differentially influence the radial growth of the mutants compared to the wild type.The deletion of Botrytis cinerea is able to infect its host from spore- or mycelium-mediated penetration processes. Whereas appressoria are formed after the short germination of a fungal spore, so-called ICs are mycelium-derived penetration structures that facilitate the breakthrough of the plant cell wall . For this study, spores were inoculated on onion epidermal layers and fungal material was stained with lactophenol blue after 16 h, allowing discrimination between the stained surface hyphae and those left unstained after penetration of the host. The mutants were able to penetrate the plant’s surface but the subsequent colonization was severely inhibited . A similar phenotype was recorded for the strains Δbcpdi1-C-M1 as well as Δbcpdi1-C-M1/2. In marked contrast, the complemented strain (Δbcpdi1-C) and Δbcpdi1-C-M2 appeared to infect in the manner of the wild type.The deletion of Figure 2B). However, since all plants were infected with the same concentration of conidiospores, there is no causal correlation between reduced production of conidia and impaired virulence.All strains that displayed a retarded colonization of plant tissue produced significantly fewer spores than the wild type and the strains that restored the wild type phenotype . Primary lesions were not visible until the 3rd day of infection. Similar results were obtained for Δbcpdi1-C-M1 and Δbcpdi1-C-M1/2.When studying the mycelium-mediated infection process of bcpdi1, Δbcpdi1-C-M1, and Δbcpdi1-C-M1/2 were unable to produce wild type-like ICs, but instead produced twisted hyphae which were not targeted toward one infection site as is normal for the wild type .The defects in infecting plant tissue may be due to the impaired production of ICs. All strains that were unaffected in the mycelium-mediated infection process were still able to produce ICs. In contrast, the mutant strains Δbcpdi1-C, were still capable of forming CATs, the deletion mutant and the strains Δbcpdi1-C-M1, Δbcpdi1-C-M1/2, and Δbcpdi1-C-DA were impaired in the formation of hyphal fusions. The mutant Δbcpdi1-C-M2 was still able to form the hyphal structures, but produced markedly less CATs than the wild type .Since it has been shown previously that members of the NoxA complex as well as putative interaction partners are impaired in the formation of CATs , the difbcpdi1-C . All other strains were defective in the production of sclerotia. Surprisingly, the mutant strain Δbcpdi1-C-M2 also failed to complement the defect in sclerotial development, although this mutant behave as the wild type in all other respects.Apart from hyphal fusions, BcPdi1 also affects the production of perennial survival structures called sclerotia. These structures, which are produced under constant darkness, were formed by the wild type and the complemented strain ΔTo summarize, BcPdi1 affects important vegetative and pathogenic processes, such as sclerotial formation, hyphal fusions and penetration structures. Essential for these developmental processes is the cytosolic region of BcPdi1 containing the two putative phosphorylation sites as well as one catalytically active site. Whereas the second predicted phosphorylation site seems to have an exclusive effect on the production of sclerotia, the first site seems to be essential for all essential protein functions.oliC promoter. Both were transformed into the deletion mutant’s background. The wild type phenotype was fully restored by both complementation constructs (Data not shown). Additionally, in both cases the target protein localized to identical structures. The strain BcPdi1-C was used since the fluorescence of the protein under the constitutive oliC promoter was much brighter for localization studies and later experiments. Apart from re-integrating bcpdi1 into its wild type version, GFP-fusion constructs with the mutated alleles  were generated.To study the localization pattern of BcPdi1, a GFP fusion construct was generated under the control of the native promoter (1 kb upstream of the ORF) and the constitutive Figure 4) belonging to the ER (verified by co-staining with ER tracker) and was additionally found in the nuclear envelope (verified by co-staining with the DNA dye Hoechst 33342 – Data not shown). Although the mutation of the second putative phosphorylation site (M2) into its phospho null form had no influence on the localization, the pattern changed upon mutation of M1. BcPdi1_M1 was detected in a faint network of filamentous structures belonging to the ER, but was hardly visible in the nuclear envelope. Moreover, the proteins seemed to adhere as aggregates that were detected throughout the hyphae. Similar results were obtained for the BcPdi1_GFP fusion construct containing both mutated phosphorylation sites.BcPdi1 localized to filamentous structures .The localization pattern of the phospho mimic mutants differed from all other observed PDI-constructs. The fusion constructs localized to filamentous structures, which show high similarity to mitochondria. Co-localization with the mitoIDred mito tracker as well as with the ER tracker revealed that those structures are overlapping with ER and mitochondrial structures  appeared like the wild type and the deletion mutant (data not shown) whereas the phospho mimic version (Δbcpdi1-C-DA_roGFP2) exhibited an elevated 395/488ratio .To elucidate the role of BcPdi1 both inside and outside the ER, studies with the genetically encoded biosensor roGFP2 were performed. The modified version of the reporter gene was suitable for the visualization of the glutathione pool, which mirrors the current redox state of the monitored compartment . These s2 is sufficient to induce an oxidative burst inside the cytosol, the mutants  were also tested under those conditions (50 mM CaCl2). Surprisingly, both patterns of the mutants did not resemble the wild type control. In the wild type, the addition of CaCl2 induced a massive oxidative burst, changing the 395/488ratio from 0.3 to 1.4. In comparison, the 395/488ratio of Δbcpdi1-C-M1_roGFP2 remained unaffected as the deletion mutant Δbcpdi1 , and the 395/488ratio of the phospho mimic version (Δbcpdi1-C-DA_roGFP2) was slightly shifted to a more oxidized state than the wild type . The induction via CaCl2 led only to minor changes of the 395/488ratio.Since previous studies with mutants of both Nox complexes revealed that CaCl395/488ratio of Δbcpdi1-C-DA_roGFP2 increased in comparison to the wild type, we did expression studies with known marker genes of the UPR. Since previous studies in plant cells revealed that changes in the redox state are due to the activity of the UPR machinery (bcpdi1 mutants expressing roGFP2 with respect to UPR activity. We used the putative B. cinerea homologs of the genes BiP/Kar2 (Bcin13g00960) and Hac1 (Bcin08g06730). Whereas BiP/Kar2 is a chaperone responsible for the correct folding of proteins . In summary, the results suggest that BcPdi1 is involved in the maintenance of the cytosolic redox state and may play a role in the UPR.To elucidate, why the basic achinery  we analyproteins , the traproteins , 1998. BFigure 6A and Supplementary Figure S2). The positive interaction of BcNoxA and BcPdi1 was confirmed in co-IP experiments . Strains expressing BcNoxA.mcherry (lane 1) or BcPdi1_GFP (lane 2) were used as controls  . Additional interactions were shown for BcPdi1 with thioredoxin 2 (BcTrx2), an oxidoreductin (BcEro1) and the catalytic subunit of the calcineurin complex (BcCnA).To examine whether the overlapping functions between the Nox complex and PDI are based on physical/direct interaction of these proteins we used a yeast-two hybrid approach; since membrane associated proteins are involved, we chose the split-ubiquitin system which is optimized for monitoring interactions between transmembrane proteins. The protein BcPdi1 was shown to interact with the catalytic subunit of the NoxA complex, and to a minor extent with BcNoxB . Again, the first putative phosphorylation site was transferred into its putatively dominant active form (phospho mimic) (T65E).Figure 6 and Supplementary Figure S2).Interaction tests were done comparatively with the mutated versions of BcPdi1. While the mutation of the second predicted phosphorylation site (M2) had no effect on any of the interactions, mutation of M1 (T65A) and the phospho mimic led to the loss of the interaction with BcCnA. There was no effect on the binding to NoxA/B, Trx2, and Ero1.  seems to bind to the cytosolic region of the protein and in particular to a segment exhibiting a single putative phosphorylation site.To summarize these results it is clear that BcPdi1 interacts with members of various signaling cascades such as Nox-, Cabcpdi1 is similar to those of ΔbcnoxA and ΔbcnoxD mutants suggesting a link between Nox signaling and BcPdi1 in these differentiation processes. A cytosolic phosphorylation site, which is highly conserved among different species, clearly is essential for the function of BcPdi1. In its phospho null form, the respective mutants behave like Δbcpdi1. In contrast, the phospho mimic mostly restores the wild type phenotype.In conclusion, we present evidence that BcPdi1 is involved in all major developmental processes, such as the formation of conidia, sclerotia and CATs. Moreover, the ER protein affects the virulence of the necrotrophic plant pathogen and mediates stress resistance to oxidative and osmotic stress. The phenotype of ΔB. cinerea .In this study, we identified the ER protein BcPdi1 as new interaction partner of BcNoxA. In yeast-two hybrid experiments, as well as by co-immunoprecipitation, the ER protein was found to directly bind to the catalytic subunit of the NoxA complex. In other fungi, PDI is essential for viability  and is aa reesei . Moreovea reesei . A more a reesei . In B. cn system . In contbcpdi1 may be attributed to the predicted two catalytic active domains located inside and outside of the ER. Therefore, the protein may influence signaling components which are located inside the ER, such as BcTrx2  . In contrast, the first putative P-site appeared to be essential for binding to the phosphatase and for every observed phenotype that had previously been described for the deletion mutant. It is noteworthy that both approaches, mimicking the phosphorylated and dephosphorylated version of the predicted P-site, are unable to restore growth typical of the wild type. Whereas the phospho null version resembles the phenotype of the deletion mutant, the phospho mimic version restored the wild type phenotype only partially. Thus, the results indicate that phosphorylation of BcPdi1 at the first predicted P-site is essential for its function, but it is remarkable that mimicking the phosphorylation does not have the opposite effect. The mutants displayed an intermediate phenotype between the deletion mutant  and the wild type . This intermediate effect may be because the phosphorylation is only mimicked by the replacement of threonine by glutamic acid. Two indications that there may be even more complexity behind the phenotype was the localization of the protein and the cytosolic redox state of the PDI mutants. The protein localization of the BcPdi1_GFP fusion construct was monitored in the nuclear envelope as well as in filamentous structures of the ER . Whereas the mutation of the second putative P-site had no influence on the localization pattern, in the mutants mimicking the phosphorylation of the first predicted P-site the protein was localized in small filamentous structures and hardly detectable in the nuclear envelope. Co-staining with ER- and mito-tracker agents revealed that both compartments seem to overlap. This phenomenon has previously been observed in mammalian cells in which the formation of tight mitochondria-ER complexes was dependent on stress conditions and involved in the regulation of the redox homeostasis and cell death  and Δbcpdi1-C-M1 appeared to be as in the wild type, the mutants mimicking the phosphorylation of the first predicted P-site exhibited a higher 395/488ratio inside the cytosol. This unbalanced redox equilibrium became even more obvious under Ca2+ inducing conditions. Here in the wild type an oxidative burst is detectable upon Ca2+ stimulation. In previous studies, we were able to show that both Nox complexes contribute to the changes in redox status upon Ca2+ exposure. Surprisingly, the deletion mutant and Δbcpdi1-C-M1 also seem to be insensitive to the addition of CaCl2, suggesting that BcPdi1 is part of the linear signal messaging of Ca2+ stress in close association to BcNoxA and BcNoxB. All mutants mimicking the phosphorylation of the first putative P-site displayed a very different pattern. A much higher 395/488ratio and the absence of a wild type-like oxidative burst were monitored. To prove which cellular processes might contribute to this elevated 395/488ratio, the expression of key genes of the UPR were analyzed . Therefore, the mutation of the first putative P-site may hinder the function of the PDI so that the redox homeostasis is unbalanced and rescue programs, such as the UPR, might be induced.Nevertheless, the obtained data support the hypothesis that both BcNoxA and BcPdi1 are acting in the same signaling pathway(s). Consequently, all major developmental processes are affected in a similar way. Besides the impact on ICs, conidiospore production and formation of CATs, both proteins impact on virulence and formation of sclerotia. The more severe impact on virulence of Δs BcTrx2 , at the s BcTrx2 , as welligure 6) . Since dype like , the obsvirulent . To unrall death . To verianalyzed . NortherB. cinerea. The phenotype of the deletion mutant resembles the ones of ΔbcnoxA and ΔbcnoxD and display – together with the physical interaction of BcNoxA and BcPdi1 – a strong link between ROS signaling and ER proteins and processes. The evidence at present is inconclusive as to which direction certain signals were forwarded in the pathways. Starting points for further studies might be the different tie-points between ROS, the ER and Ca2+. Therefore, not only does the physical interaction of BcPdi1 and BcCnA hint to a rather close association, but even more to the collaboration of BcNoxA, BcNoxB and BcPdi1 upon exposure to Ca2+. Future studies should investigate how Ca2+ affects the regulation of Nox complexes, ER proteins and the redox homeostasis.In summary, we show in this work that BcPdi1 is an ER protein, which is involved in several growth and differentiation processes such as formation of CATs, ICs, conidia, sclerotia as well as in virulence of RM: conception, methodology, writing of the manuscript and realization of all experiments. PT: conception, review and editing. Final approval: RM and PT.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.


While the etiology of developmental coordination disorder (DCD) is yet to be established, brain-behavior modeling provides a cogent argument that neuropathology may subserve the motor difficulties typical of DCD. We argue that a number of the core behavioral features of the DCD profile  are consistent with difficulties regulating inhibition within the primary motor cortex (M1). This study aimed to be the first account of the integrity of cortical inhibition in motor cortices in DCD.The sample consisted of eight adults with DCD aged (18–30 years) and 10 aged matched neurotypical controls. Participants received a common battery of single and paired-pulse transcranial magnetic stimulation from which a series of neurophysiological measures classically used to measure intra-  and inter hemispheric  cortical inhibition of the M1 at rest were recorded.ratios.While no group differences were observed for any measure of intrahemispheric cortical inhibition, individuals with DCD demonstrated significantly reduced interhemispheric cortical inhibition relative to controls, shown by consistently lower ISPOur findings are consistent with the view that regulation of cortical inhibition of M1 activity may be atypical in individuals with DCD, indicating differential GABAergic operation. This effect, however, appears to be select to cortical inhibition. Importantly, our data support the notion that reduced interhemispheric M1 cortical inhibition may at least partly explain commonly reported difficulties with bimanual motor control in DCD. The neurochemical implications and limitations of this evidence will be discussed. Diagnosin vivo neurological measurement of individuals with DCD has increased substantially over the last decade (via fMRI or EEG) when individuals with DCD perform perceptuo-motor and cognitive tasks, or the structural integrity of motor circuitry (via MRI). While promising, these data remain sparse and inconsistent. Accordingly, the “neural signature” of DCD is yet to be established  and 10 typically developing (TD) controls . All participants self-reported being right handed, gave written informed consent, and were screened to ensure they were free of TMS contraindications. No participants reported taking medication that would contraindicate TMS, and none reported experiencing any negative side-effects during or following TMS. The project received ethical clearance from Deakin University Human Research Ethics Committee.The sample comprised 8 adults with DCD , targeting both individuals with motor difficulties and typical motor skill. All participants were screened using methods that have been successful by ours and other research groups in identifying adults with DCD –94. Pros95%: 21.26Mean ± 3.27) and child (CI95%: 4.26 Mean ± 0.86) ADC scores using 47 healthy young Australian adults (n) 8. While no participant had a formal diagnosis of DCD, we can be confident that participants in our DCD group met criteria as outlined earlier. Using the same screening procedure, all controls were confirmed to have age-appropriate motor abilities, as indicated by a percentile ranking above the 20th percentile for “Total Motor Composite” scores of the BOT-2 For LICI, each participant’s median peak-to-peak MEP in response to the second TMS pulse (the conditioned pulse) was divided by their median peak-to-peak MEP in response to the first TMS pulse (the non-conditioned pulse). The resulting value was then multiplied by 100 and subtracted from 100 to represent the percentage of inhibition of the second suprathreshold test pulse [LICI (%)]duration) was defined as the mean of the temporal difference (in ms) between CSPonset and CSPoffset (CSPduration = CSPoffset − CSPonset). Furthermore, since the duration of silent periods depend on stimulus intensities  to reduce intersubject variability. The mean CSPratio was then calculated and used for subsequent analyses. Although not presented here, we note that the reported profile of between group comparisons for CSP did not differ appreciably without this normalizationFor CSP, the onset and offset of the silent period was determined using the objective graphical method described by Garvey et al. . The CSPensities , the CSPduration) was defined as the mean temporal difference between ISPonset and ISPoffset (ISPduration = ISPoffset − ISPonset). Given that the duration of ISPs also seems to be affected by stimulus intensities . The mean ISPratio was then calculated and used for subsequent analyses. Although not presented here, we note that the reported profile of between group comparisons for ISP did not differ appreciably without this normalizationThe graphical method described earlier was also used to determine onset and offset for ISPs . The durensities , 114, tht-tests for outcome measures where assumptions of normality were met . Where assumptions were violated, Mann–Whitney U-tests were adopted . Where significant group differences were observed, Pearson’s correlation analyses were conducted between those cortical inhibition measures and scale scores  of relevant subtests from the BOT-2 requiring the use of the FDI. Since group differences were only observed on measures of interhemispheric cortical inhibition (see below), the purpose of the subsequent analysis was to determine whether this effect in DCD was associated with motor performance deficits. Only those subtests  that placed greater demands on interhemispheric cortical inhibition  were subjected to correlations with our TMS measure of interhemispheric cortical inhibition.Group comparisons of intra- and interhemispheric cortical inhibition were conducted using Independent Samples All participants completed all measures. No group differences were observed for RMT or AMT. Baseline MEPs were also comparable between groups. As RMTs, AMTs and baseline MEPs were comparable between groups, and stimulator output percentages for SICI, LICI, CSP, and ISP are all based on RMT and AMT percentages, comparisons on stimulus intensities for SICI, LICI, CSP, and ISP were deemed redundant and were not conducted. See Table ratio] found no significant differences between groups , LICI (%), and CSPratios were significantly smaller in the DCD group CI95%: 3.81Mean ± 0.61 relative to TD controls CI95%: 6.08Mean ± 1.32 .In regards to interhemispheric cortical inhibition, mean ISPratios and “Manual Dexterity” scale scores of the BOT-2, r = 0.48, n = 18, and p = 0.044. A scatterplot illustrating this correlation is presented below in Figure Pearson’s correlation revealed a significant, medium to strong, positive correlation between mean ISPratios. As predicted, however, we observed significant differences in mean ISPratios between groups, a commonly adopted measure of interhemispheric cortical inhibition. Indeed, adults with DCD showed significantly smaller mean ISPratios in comparison to healthy controls. Interestingly, the reduced interhemispheric cortical inhibition in the DCD group was also associated with poorer performance on subtests of the BOT-2 requiring bimanual coordination, shown by a significant correlation between mean ISPratios and performance on the BOT-2 manual dexterity subtest across groups.The aim of this study was to investigate the integrity of intra- and interhemispheric cortical inhibition of the M1 in adults with DCD using a battery of single-pulse TMS and ppTMS protocols that are commonly implemented to measure these respective facets of cortical inhibition. Contrary to expectations, our results suggested that intrahemispheric cortical inhibition at rest is preserved in DCD, with no group differences observed on mean SICI, LICI, or CSPratio] failed to reveal statistically meaningful differences. This was particularly surprising given the behavioral phenotypes described earlier had been previously reported to rely on intrahemispheric inhibitory mechanisms within the M1  and LICI (%)] were taken with the participant at rest  while the other  was taken while participants performed a very simple motor task . Differences in resting-state cortical inhibition such as those adopted here have previously been detected between healthy controls and individuals with neurodevelopmental disorders where motor function is often impaired, such as ADHD . Furtherhibition , 116]. Thibition ], althouhibition ]. Since hibition , it is pratios between DCD and TD groups found that the DCD group had significantly smaller ISPratios when compared with their TD counterparts. As discussed, ISPs are commonly used as a metric for indexing cortical inhibitory mechanisms involved in the suppression of unwanted transcallosal communication of motor commands across M1 hemispheres during lateralized movement  fell below the 95% CI of the control group on mean ISPratios. Thus, not only do those with DCD demonstrate decreased interhemispheric inhibition of M1 activity at a group level, but the effect appears to hold for a substantial proportion of individuals in this group. Furthermore, we observed that smaller ISPratios were associated with decreased performance on a subtest of the BOT-2 requiring bimanual coordination, and where the FDI was also used as the main effector , with a compelling 30% of the variability in performance being explained by mean ISPratios  during lateralized unimanual motor tasks , 77 and While the results of the present are promising, we acknowledge that we can only generalize these findings to the dominant M1 (left in this case), since cortical inhibition of the non-dominant hemisphere was not evaluated. That is, while our study shows that interhemispheric inhibition of right-hemispheric activity from the dominant left hemisphere is reduced in DCD, it cannot be assumed that this is also the case for the opposite hemisphere. Indeed, asymmetry in interhemispheric cortical inhibition has been noted in both patient groups with reduced ISPs  and contratios and the “Manual Dexterity” subtest of the BOT-2 found that TMS evoked ISPs were significantly related to performance on subtests requiring fine bimanual coordination. Taken together, our findings suggest that not only do individuals with DCD demonstrate reduced interhemispheric cortical inhibition of M1 activity, but that this may predict reduced motor performance in this group. Directions for future work and clinical implications were discussed. Our data are partly consistent with the view that cortical inhibition of M1 activity may be compromised in individuals with DCD and are consistent with behavioral accounts of reduced motor inhibition in this group.This study was the first of its kind to examine the integrity of intra- and interhemispheric cortical inhibition in DCD using single-pulse TMS and ppTMS paradigms. While intrahemispheric cortical inhibition was comparable between DCD and TD groups, interhemispheric cortical inhibition was reduced in DCD. An additional correlation analysis conducted between ISPThis study was carried out in accordance with the recommendations of Deakin University Human Research Ethics Committee (DUHREC) with informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by DUHREC.JH, CH, and IF contributed to all stages of this study, including project conception and design, data collection, analysis, and final write-up of the manuscript. PE and W-pT contributed substantially to project conceptualization and manuscript preparation. PB contributed to data collection and manuscript preparation.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.


Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential.GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the  It is t-α-4.99.14.99.16) ; α-malto4.99.16) . The pol4.99.16) . Each po4.99.16) . B chainMycobacterium tuberculosis GlgE has attracted particular attention because of the demonstration that it is a genetically validated drug target  is the nucleophile that attacks the donor to liberate inorganic phosphate by trapping a β-maltosyl-enzyme intermediate. A neighboring Glu-423 acid/base catalytic side chain is well placed to assist by protonating the phosphate leaving group and then deprotonating an incoming acceptor substrate. Loss of activity resulting from mutation of Glu-423 supports this role in catalysis , suggesting that the linear cleft is not particularly important in the accommodation of the larger acceptor either.There are two conspicuous clefts that are adjacent to the donor binding site . The oneS. coelicolor GlgE competitively with regard to acceptor substrates, the acceptor binding site might be expected to overlap with this site. Significant interactions between cyclodextrins and GlgE involve the side chains of Trp-448 and Arg-449, so we explored the effect of mutations at these positions. When Trp-448 was substituted by either Ala or Glu, the enzyme lost almost all of its detectable activity . Be. Be19). activity , consistkcatapp was reduced 43-fold . Deb. DebS. cS. coelicolor possesses two copies of the glgE gene, making the genetic manipulation of the GlgE pathway in this organism less tractable than in the closely related S. venezuelae, which has only one copy (S. coelicolor (isoform 1) and S. venezuelae share 79% amino acid sequence identity together with very high conservation around the secondary binding site including the key Tyr residue . Without complementation with an intact glgE gene, the mutant strain is known not to produce α-glucan and to exhibit a delayed growth phenotype due to the accumulation of α-maltose 1-phosphate. However, the strain complemented with the GlgE variant grew normally and produced α-glucan in a manner similar to the wild-type strain . Surprisingly, neither of the two secondary sites is spatially conserved, being over 10 Å apart in the superposed structures. This implies evolutionary pressure for this function to exist even if there is no structural conservation. Although the oligosaccharide appears to be bound to the M. tuberculosis enzyme in the reverse orientation, the resolution of this structure is only 3.9 Å. Therefore, it seems feasible that it is in fact in the same orientation as that seen in the S. coelicolor enzyme, a possibility acknowledged by the authors of the other study , whereas those of subsites +5 and +6 in domain N of the neighboring subunit are quite different . It is likely that these differences lie behind the lack of inhibition of the M. tuberculosis enzyme by cyclodextrins  kinetic mechanism  polyethylene glycol 3350, 0.2 m sodium citrate, and 15% (v/v) ethylene glycol as described previously  by plunging into liquid nitrogen and stored in Unipuck cassettes (MiTeGen) prior to transport to the synchrotron. The crystals were subsequently transferred robotically to the goniostat on station I04-1 or I24 at the Diamond Light Source  and maintained at 100 K with a Cryojet cryocooler (Oxford Instruments). Diffraction data were recorded using a either a Pilatus 2 M or a 6 M detector (Dectris) and processed using the xia2 expert system (4CN6) (3ZST)  . Therefom (4CN6)  and manum (4CN6) . By contm (4CN6)  using on) (3ZST)  from a sCN6 . TheglgE-null mutant of S. venezuelae ATCC10712 was generated using the Redirect PCR targeting method . Strains were grown for 2 days on maltose/yeast extract/malt extract/John Innes Centre borehole water 2 solid medium (The g method  where theviously . The mutid pMS82  as descrid pMS82  except td medium  at 28 °Cd medium . Colonied medium . α-GlucaS. B. coordinated the study and wrote the paper. K. S., C. E. M. S., and D. M. L. edited the manuscript. K. S., M. T., A. G., and A. M. R. carried out the kinetic experiments summarized in


Several studies report aberrant expression of sine oculis homeobox (SIX) homolog family members during cancer development and progression. SIX4 participates in organ development, such as myogenesis and neurogenesis. However, the expression and clinical implication of SIX4 in colorectal cancer (CRC) remains unclear.The SIX4 expression levels in colorectal patients were assessed in nine different human cancer arrays and compared using patient survival data. SIX4 expression was silenced in two cell culture lines for invasion and wound healing assessment. Finally, bioinformatics assessments ascertained the pathways impacted by SIX4.SIX4 was upregulated in The Cancer Genome Atlas CRC cohort and other gene expression omnibus (GEO) cohorts. In addition, SIX4 expression significantly correlated with lymph node metastasis and advanced Tumor Node Metastasis (TNM) stages. Moreover, SIX4 overexpression was related to unfavorable prognosis in CRC patients. Silencing SIX4 inhibited CRC cell metastasis by surpressing AKT phosphorylation.SIX4 is upregulated in CRC and can be used as a prognosis biomarker. Colorectal cancer (CRC) is one of the most prevalent malignant neoplasms with both incidence and mortality ranked third in the world . DespiteThe sine oculis homeobox (SIX) homolog family is comprised of six members, SIX1, SIX2, SIX3, SIX4, SIX5, and SIX6 . There aSeveral studies report abnormal SIX family member expression during cancer formation and progression. SIX1 participates in invasion and proliferation during tumor development in non-small cell lung cancer . Ono et Despite recent efforts to understand the role of SIX4 as a transcriptional factor, little is known about the roles and expression of SIX4 in CRC. Therefore, the mechanism of SIX4 in CRC deserves further studies. In this study, we clarify SIX4 expression in CRC and then analyze the relationship between SIX4 expression and clinicopathological features. Moreover, the function and mechanism of SIX4 in CRC cell metastasis was revealed by bioinformatics analysis and in vitro assays.GSE5206 (GSE9348 (GSE20916 (GSE17536 (GSE6988 (GSE14333 (GSE20842 (GSE39582 (Level 3 HiSeq RNASeq data were downloaded from The Cancer Genome Atlas (TCGA) website  for 380 GSE5206 , GSE9348 and theGSE39582 , we obtat-test. In order to determine the prognosis significance of SIX4 in CRC patients, survival differences were validated through the Kaplan–Meier method. In order to select the relationships between SIX4 and AKT-related genes, we applied Pearson’s correlation analysis to the mRNA expression data. R package survival was used to analyze the relationship between SIX4 expression and survival from TCGA and other CRC cohorts. The fold change between different tumor progression stages and SIX4 expression levels were calculated. Gene ontology (GO) provides a framework for biology computation models. GO provides ontologies to describe gene functions and relationships. It classifies genes according to molecular function, cellular components, and biological processes. The list of all genes correlated with SIX4 was submitted to the database for annotation, visualization, and integrated discovery (DAVID) online tools for GO and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis  software was acquired from the Broad Institute at MIT. TCGA dataset, including 380 CRC samples, was analyzed by GSEA. The “hallmark gene sets” were used for running GSEA. The phenotype labels were generated according to the expression level of SIX4. Fresh CRC tissues and paired adjacent nontumor colorectal tissues were obtained from CRC patients at Tongji Hospital  between August 2013 and July 2014 after providing written informed consent. None of the patients received chemotherapy or radiotherapy before surgery. The use of tissues for this study was approved by the ethics committee of Tongji Hospital. A total of 39 fresh CRC tissues were frozen in liquid nitrogen until further use. The clinical information included age, gender, stage, tumor size, depth of invasion, lymph node metastasis, and distant metastasis .2 atmosphere. Cells were collected and analyzed after 72 h of culture.The SW48 and LoVo cell lines, human CRC cell lines, were purchased from the China Center for Type Culture Collection . We cultured the cell line at 37 °C in DMEM , which contained 10% fetal calf serum (Gibco) under 5% COThe SIX4-siRNA and control siRNA were constructed by Ruibo Company , and myr-AKT plasmid was a gift from Kira Gritsman . The siR−ΔΔCT) method to calculate the results.The Trizol reagent  was applied to total RNA extract from cultured cells. PrimeScript™ RT Master Mix  was used to reverse transcribe the total RNA to cDNA. SYBR Green Real-time PCR Master Mix  was used to amplify cDNA in a 20 μL reaction system. The primers were designed and synthesized by TsingKe . The primer information is as follows: SIX4 upstream 5′-GCATTGAACCCACCAAAAATGT-3′ and downstream 5′-GGAAGTAGACCCCAGTATGTCA-3′, GAPDH upstream 5′-GGAGCGAGATCCCTCCAAAAT-3′ and downstream 5′-GGCTGTTGTCATACTTCTCATGG-3′. GAPDH expression was used for normalization. Finally, we used the  buffer was used to extract the proteins from colorectal cells. We used the bicinchoninic acid assay to measure protein concentration. The proteins were separated using sodium dodecyl sulfate-PAGE electrophoresis and then transferred to a polyvinylidene difluoride membrane . Tris-buffered saline with Tween-20 and 5% nonfat milk was used to block the membrane at room temperature. After blocking for 1 h, we incubated with the following primary antibodies at 4 °C overnight: anti-SIX4, anti-T-AKT, anti-P-AKT, and anti-GAPDH . GAPDH was used to normalize total protein levels.5 cells in the presence or absence of SIX4 were cultured in the upper chamber. Meanwhile, we placed DMEM containing 20% Fetal Bovine Serum (FBS) in the lower chamber. After 24 h of incubation, cells in the upper chamber were fixed in 4% formaldehyde and stained with 0.05% crystal violet. We counted the number of cells on the lower side of the filters at 200× magnification.We performed invasion and migration assays using Boyden chambers with or without Matrigel  in 24-well dishes. Initially, 1 × 10We seeded the CRC cells in six-well plates at approximately 100% confluence. A 200 μL pipette tip was used to draw a linear scratch-wound. Next, phosphate buffer solution (PBS) was used to wash the cells gently, and serum-free medium was used to block cell proliferation. After 24 h of culture, the closure area was measured using a microscope by comparing the images from the start time point (0 h) to the last time point (24 h).t-tests were applied to describe the differences between different groups. Kaplan–Meier analysis was used to calculate the survival differences between divided groups. The Pearson correlation test was used to compare the correlation between the SIX4 and AKT pathway genes. We considered P < 0.05 as significant in all cases.R software, SPSS 17.0 statistic software (SPSS), and GraphPad Prism 5.0 software were used to conduct statistical analyses. The R packaged used in the present study were: ggplot2 , survivaGSE5206, GSE20916, GSE6988, and GSE20842. These results were consistent with TCGA CRC cohort   . Next, D < −0.3) . The res < −0.3) . KEGG pa < −0.3) . In addire >0.05 . Finallyre >0.05 .To confirm whether the effects of SIX4 on CRC cells is dependent on PI3K-AKT signaling, we simultaneously decreased SIX4 expression levels and activated the AKT signaling pathway. IGF-1 and myr-AKT were used to carry out the rescue assay. Notably, the western blot assay confirmed that IGF-1 and myr-AKT increased p-AKT expression, a key marker of PI3K-AKT . In addiSIX4 is a member of the homeobox family required for eye development . SIX4 isGSE39582, GSE17536, and GSE14333, verified this finding. Moreover, our study demonstrated that SIX4 expression correlated with lymph node metastasis and TNM stage in TCGA database, which was validated in the GSE5206, GSE14333, and GSE39582 cohorts. Therefore, SIX4 may be related to CRC lymph metastasis and predict CRC patient prognosis. In addition, we investigated SIX4 function by silencing SIX4 in CRC cells. The results show that knockdown of SIX4 can inhibit cell migration and invasion in SW48 and LoVo cells.The SIX family contains evolutionarily conserved transcription factors that play important roles in cell proliferation, differentiation, apoptosis, adhesion, and migration . We founr > 0.3 or r < −0.3). The results of the GO analysis with co-expression genes showed that the genes significantly focused on several GO terms including cell adhesion, biological adhesion, extracellular structure organization, and blood vessel development. The cellular component terms associated with SIX4-related genes were involved in ECM, proteinaceous ECM, and extracellular structure. The molecular functions for co-expressed genes included calcium ion binding, polysaccharide binding, pattern binding, and glycosaminoglycan binding. KEGG pathway analysis provided significant pathway information for SIX4 co-expressed genes, including ECM-receptor interaction, focal adhesions, PI3K-AKT signaling pathway, and osteoclast differentiation. GSEA analysis also demonstrated that the PI3K-AKT pathway might have an effect on SIX4’s function in CRC. PI3K-AKT signaling positively correlated with CRC progression. Several studies demonstrate that the PI3K-AKT signaling pathway is involved in the metastasis process (However, the mechanisms that SIX4 may regulate in cancer progression remained unclear. In order to clarify the SIX4-associated pathways, bioinformatics analysis was applied using TCGA RNA-sequencing data. The bioinformatics analysis included GO, KEGG, and GSEA. First, we selected the SIX4 co-expression genes via Pearson correlation analysis yielding 2,283 genes considered SIX4 co-expression genes ( process . Here, t process . Several process . The migIn conclusion, the present study demonstrated that SIX4 overexpression in CRC tissues correlated with lymph node metastasis and predicted poor prognosis in CRC patients. In addition, bioinformatics analyses, including GO, KEGG, and GSEA analysis, showed that SIX4 induces the PI3K-AKT pathway, which was confirmed by western blot assay. Our research contributes to finding new prognosis biomarkers for CRC and provides a novel molecular mechanism for CRC lymph metastasis.10.7717/peerj.3394/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.3394/supp-2Supplemental Information 2Click here for additional data file.