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Cytosolic GAPDH as a redox-dependent regulator of energy metabolism
Markus Schneider1,
Johannes Knuesting1,
Oliver Birkholz2,
Jürgen J. Heinisch3 &
Renate Scheibe ORCID: orcid.org/0000-0002-6140-61811
Plant cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GapC) displays redox-dependent changes in its subcellular localizations and activity. Apart from its fundamental role in glycolysis, it also exhibits moonlighting properties. Since the exceptional redox-sensitivity of GapC has been suggested to play a crucial role in its various functions, we here studied its redox-dependent subcellular localization and the influence of the redox-state on GapC protein interactions.
In mesophyll protoplasts from Arabidopsis thaliana, colocalization of GapC with mitochondria was more pronounced under reducing conditions than upon oxidative stress. In accordance, reduced GapC showed an increased affinity to the mitochondrial voltage-dependent anion-selective channel (VDAC) compared to the oxidized one. On the other hand, nuclear localization of GapC was increased under oxidizing conditions. The essential role of the catalytic cysteine for nuclear translocation was shown by using the corresponding cysteine mutants. Furthermore, interaction of GapC with the thioredoxin Trx-h3 as a candidate to revert the redox-modifications, occurred in the nucleus of oxidized protoplasts. In a yeast complementation assay, we could demonstrate that the plant-specific non-phosphorylating glyceraldehyde 3-P dehydrogenase (GapN) can substitute for glucose 6-P dehydrogenase to generate NADPH for re-reduction of the Trx system and ROS defense.
The preferred association of reduced, glycolytically active GapC with VDAC suggests a substrate-channeling metabolon at the mitochondrial surface for efficient energy generation. Increased occurrence of oxidized GapC in the nucleus points to a function in signal transduction and gene expression. Furthermore, the interaction of GapC with Trx-h3 in the nucleus indicates reversal of the oxidative cysteine modification after re-establishment of cellular homeostasis. Both, energy metabolism and signal transfer for long-term adjustment and protection from redox-imbalances are mediated by the various functions of GapC. The molecular properties of GapC as a redox-switch are key to its multiple roles in orchestrating energy metabolism.
Pathways of plant metabolism that generate energy equivalents both from light and from substrate in the various cellular compartments must be permanently adjusted to changing environmental factors and metabolic requirements. The electron-transport chains in chloroplasts and mitochondria are major sources of redox-imbalances and radical formation under stress. Signals arising from such imbalances are sensed and transferred to the nucleus. Altered gene expression will then help to maintain homeostasis of metabolic fluxes and cellular redox-state [1].
NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) is a highly conserved glycolytic enzyme in all kingdoms of life. Several non-glycolytic functions of GAPDH have also been observed, classifying this enzyme as prototypical example for moonlighting (for reviews see [2,3,4,5,6]:). In A. thaliana, GapC1 (At3g04120) and GapC2 (At1g13440) are the GAPDH isoforms residing in the cytosol. We previously characterized redox-dependent cysteine modifications of GapC, namely S-glutathionylation and S-nitrosylation, which result in inactivation of the enzymes [7]. In a yeast two-hybrid screen, VDAC was identified as an interaction partner of GapC [8]. Moreover, a fraction of GapC was shown to be localized to the nucleus of Arabidopsis mesophyll protoplasts [7, 9,10,11], and associated with the outer mitochondrial membrane (OMM) and the cytoskeleton [9]. S-sulfhydrylation of GapC has been observed to also result in nuclear localization of the modified protein [12].
GAPDH is most sensitive towards oxidation at its catalytic cysteine residue [13,14,15], and thus represents an initial redox sensor and a hub to induce the various responses that are required to maintain redox-homeostasis [15, 16]. Glutathionylation and nitrosylation of GapC were found in large-scale proteomic approaches, both in plants and animals [17,18,19,20]. GAPDH was also suggested to act as an H2O2 sensor by triggering the protective oxidative stress response and re-establishment of cellular homeostasis [21]. Modification of the catalytic cysteine of GapC, only occurring in the absence of the substrate, leads to reversible or irreversible inactivation of the enzyme, depending on the oxidant, as demonstrated in vitro [7]. In addition to the regulation of enzyme activities, such modifications are assumed to affect signaling cascades and other moonlighting functions.
Trx and NADPH-thioredoxin reductases (NTR) are universal mediators of cellular redox-modifications [22, 23]. For continuous re-reduction, reducing equivalents can be provided by NADPH-dependent reductases, e.g., glucose-6-P dehydrogenase (G6PDH) or the plant-specific irreversible non-phosphorylating glyceraldehyde-3 phosphate reductase (GapN; EC 1.2.1.9) [24].
In this study, we focused on the redox-dependent interactions of GapC with VDAC as a central regulator of the metabolic flux across the OMM [25, 26], as well as the nuclear translocation of GapC, and its interaction with Trx-h3 as a candidate for catalyzing the reversal of the transient oxidative modification [27]. We used both, in vitro approaches such as reflectance interferometry (RIf) as well as in vivo experiments by expressing fluorescent fusion proteins in isolated mesophyll protoplasts and by heterologous enzyme complementation in yeast. Oxidants and reductants, as well as cysteine mutants of GapC, were used to analyze the redox-dependent localization of GapC1 and GapC2 in the different cellular subcompartments and their interactions with VDAC3 and Trx-h3. The cellular localizations and interactions found under reducing and under oxidizing conditions might help to unravel the various functions of GapC in more detail.
Isolation and transient transformation of Arabidopsis protoplasts
Subcellular localization and interaction studies with fluorescent fusion proteins of GapC1, GapC2 and Trx-h3 were performed in mesophyll protoplasts isolated from leaves of 4-week-old wild-type A. thaliana plants grown under short-day conditions. Isolation and transformation of protoplasts were performed according to [28], with modifications described in [29].
Imaging of fluorescent fusion proteins in transiently transformed protoplasts
For GapC-localization studies, SmaI/XbaI fragments encoding C-terminal mEGFP-fusions of GapC1 and GapC2 were cloned into the binary transformation vector pBAR-35S. To express the fusion proteins in vivo, mesophyll protoplasts were transiently transformed with the pBAR-35S-mEGFP constructs and incubated for 16 h at 22 °C. Plant mitochondria were visualized by staining with 500 nM MitoTracker® Red FM (Thermo Fisher Scientific, Waltham, USA). The protoplasts were imaged using a confocal laser scanning microscope (cLSM) 510META (Carl Zeiss, Göttingen, Germany). The mEGFP fluorescence and chlorophyll autofluorescence were excited at 488 nm. The mEGFP emission was detected at 500–530 nm, chlorophyll autofluorescence was monitored in the range of 650–700 nm.
To quantify the nuclear localization of GapC1 and GapC2 fluorescent fusions, the basic leucine zipper protein bZIP63 was used as a nuclear marker protein. The bZIP63 cDNA from Arabidopsis was cloned as a SmaI/XbaI fragment into the binary vector pBAR-35S to yield a C-terminal mCherry fusion. The resulting plasmid was co-transformed with constructs encoding GapC-mEGFP fusion proteins, as indicated. The mCherry fluorescence was detected by excitation at 585 nm and emission at 600–620 nm. The mEGFP signal was determined in the cytosol and the nucleus for at least 50 protoplasts in three independent experiments as described above. The fluorescence intensities of the cytosolic and nuclear mEGFP signals were calculated as the ratio of each individual signal to the sum of both.
For split-YFP experiments, the cDNA sequences of the Arabidopsis proteins GapC1, GapC2 and Trx-h3 were cloned as BamHI/KpnI fragments into the vectors pUC-SPYNE or pUC-SPYCE [30] to generate C-terminal fusions with the N- or C-terminal part of YFP, respectively. bZIP63-mCherry was used as a nuclear marker as described above. To express the fusion proteins in vivo, mesophyll protoplasts were transiently co-transformed with the pBAR-35S constructs and incubated for 16 h at 22 °C. For detection, complemented YFP was excited at 514 nm, and emission was monitored at 525–540 nm.
For each experiment, cLSM raw data were processed with the Fiji software system [31].
Treatments of isolated protoplasts with redox-reagents
To analyze localization and interactions of the various proteins under different redox conditions, transformed Arabidopsis mesophyll protoplasts were treated with 10 mM DTT, 50 μM GSNO or 0.2 mM H2O2 by incubation at 22 °C for 40 min prior to cLSM analysis. In preliminary experiments, intactness of the protoplasts after the treatments was checked.
Expression and purification of recombinant GapC and Trx-h3 from A. thaliana
Expression and purification of the recombinant GapC and Trx-h3 proteins from A. thaliana were performed as previously described [7]. In the case of Trx-h3, the protein was stored in 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. GapC was stored in 50 mM Tris-HCl (pH 7.8), 5 mM EDTA, 150 mM KCl, 2 mM DTT, 140 μM NAD, 50% glycerol, and desalted before use.
Expression, refolding, purification and reconstitution of recombinant VDAC3 from A. thaliana
Overexpression, refolding and purification of Arabidopsis VDAC3 was performed according to [32, 33] with modifications. VDAC3 was overexpressed using the cold-shock expression system described by [34]. The Arabidopsis VDAC3 cDNA was cloned into the pCold I DNA vector (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) using SacI and XbaI. The E. coli mutant strain BL21(DE3)-omp9 [F−, ompT hsdSB (rB− mB−) gal dcm (DE3) ΔlamB ompF::Tn5 ΔompA ΔompC ΔompN::Ω] [35, 36] was transformed with the pCold I DNA-AtVDAC3 vector. Liquid LB medium (containing 100 μg ml− 1 ampicillin) was inoculated with a starter culture. Cells were cultivated at 37 °C under shaking at 200 rpm. After reaching an OD600 of 0.8–1.0 the cultures were incubated on ice for 30 min. Protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (1 mM) and transfer of the cultures to 15 °C. The cells were incubated at 15 °C and 200 rpm for further 24 h and finally harvested by centrifugation (5000 xg, 20 min, 4 °C). The cell pellet was shock-frozen in liquid nitrogen and stored at − 80 °C. For protein purification, the cells were thawed, emerged in resuspension buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA) and disrupted by ultrasonication (Vibracell, Fisher Scientific, Illkirch, France). The VDAC3-containing inclusion bodies were collected by centrifugation (25000 xg, 30 min, 4 °C), and washed three times with detergent-containing washing buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 2.5% (v/v) Triton X-100), followed by three washing steps using detergent-free washing buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl). The inclusion bodies were treated with denaturation buffer (25 mM NaH2PO4/Na2HPO4, pH 7.0, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 6 M guanidinium chloride). A 10-fold dilution of the guanidinium chloride was achieved by drop-wise addition to refolding buffer I (25 mM NaH2PO4/Na2HPO4, pH 7.0, 100 mM NaCl, 1 mM EDTA, 2.22% (v/v) lauryl-dimethyl-amine oxide (LDAO)). This mixture was stirred for 18 h at 4 °C. A further 10-fold dilution of the guanidinium chloride by drop-wise addition to refolding buffer II (25 mM NaH2PO4/Na2HPO4, pH 7.0, 10 mM NaCl, 1 mM EDTA, 0.1% (v/v) LDAO) and stirring for 18 h at 4 °C followed this step.. Insoluble material was removed by passage through a 0.22-μm filter. For cation-exchange chromatography, the sample was loaded onto a pre-equilibrated Fractogel® EMD SE Hicap (M) (Merck, Darmstadt, Germany) cation-exchange column connected to an ÄKTAprime plus chromatography system (GE Healthcare, Munich, Germany). Elution of the refolded VDAC3 proteins was achieved using a linear gradient from 10 mM NaCl to 1 M NaCl in the elution buffer (25 mM NaH2PO4/Na2HPO4, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% (v/v) LDAO). The VDAC3-containing fractions were pooled and concentrated using a 10-kDa Amicon concentrator (Merck, Darmstadt, Germany). Size-exclusion chromatography was performed by loading the refolded VDAC3 onto pre-equilibrated (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.05% LDAO) Superose™ 12 10/300 GL column (GE Healthcare, Munich, Germany), and the elution profiles were recorded at 280 nm. The protein-containing fractions concentrated using a 10-kDa Amicon concentrator (Merck, Darmstadt, Germany).
Preparation of VDAC3-containing proteoliposomes
For RIf experiments, the recombinant VDAC3 was reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes in a molar ratio of 1:1000 (protein:lipid). The VDAC3- containing proteoliposomes were formed by detergent extraction in the presence of heptakis-(2,6-di-O-methyl)-β-cyclodextrin (β-CD) (Sigma) [37]. A mixture of 5 mM of DOPC and 20 mM Triton X-100 in HBS buffer (20 mM Hepes-KOH, pH 7.4, 300 mM NaCl) and LDAO-solubilized recombinant VDAC3 was prepared before a 2-fold excess of β-CD over the detergent was added. The mixture was incubated for 5 min at room temperature and diluted in HBS buffer.
Reflectance interferometry (RIf)
Poly-L-lysine (PLL − PEG-AC) modification of glass coverslips required for RIf was performed as described by [38, 39]. All measurements were conducted in a home-built setup as described in detail by [40]. The polymer-supported membrane was formed by injection of the VDAC3-containing proteoliposomes followed by rinsing with HBS buffer. After formation of the polymer-supported membrane, pre-treated recombinant GapC was injected. For each GapC injection, the surface was regenerated by two washing steps with 0.1% (v/v) Triton X-100. Extraction of pseudo-first order kinetic constants was performed by separately fitting dissociation and association phases using BIAevaluation 3.1 software (GE Healthcare, Little Chalfont, UK).
Silanization of RIf transducer chips with (3-glycidyl oxypropyl)-trimethoxysilane, and subsequent reaction with bis-amino-PEG with an average molecular mass of 2,000 Da was carried out as previously described [41]. Terminal amines were converted into carboxylates by incubation with 1 M succinic anhydride in 20 mM Hepes-KOH, pH 7.0, before the surface was NHS-activated by incubation with equimolar amounts of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (each 500 mM in dimethylformamide (DMF)) for 30 min. After washing with DMF, recombinant GapC1 at μmolar concentrations was added for immobilization. After 15 min at room temperature, excess protein was removed by rinsing with HBS buffer.
Yeast strains and constructs for heterologous expression of the GapN encoding gene
To study phenotypic rescue in yeast, a derivative of the strain HD56-5A [42], was employed which carries a deletion of the sole ZWF1 gene encoding glucose-6 phosphate dehydrogenase. To obtain this deletion, the one-step gene deletion method using a PCR product obtained from the template pUG6 [43] with the primer pair 16.235/16.236 (Additional file 1: Table S1) was applied to the homozygous diploid strain DHD5 [44]. Sporulation and tetrad analysis yielded the haploid segregant HOD269-1C (MATa ura3–52 his3–11, 15 leu2–3,112 zwf1::kanMX), which was used as a recipient strain for transformation with different plasmids as indicated in the results section. As a basis for heterologous gene expression, the vector pJJH2064 was used, which carries an optimized yeast PFK2 promoter on a 576 bp EcoRI/BamHI DNA fragment obtained from custom-made string synthesis (Geneart, Thermo Fisher Scientific, Waltham, USA) introduced into the CEN/ARS plasmid YCplac33 [45]. The promoter sequence was chosen according to previous analyses indicating that it allows for full and constitutive expression of downstream genes [46]. Point mutations were inserted as required to eliminate internal restriction sites from the native promoter sequence. String synthesis was also employed to obtain the sequence of the Arabidopsis gene encoding GapN adapted to the yeast codon usage and flanked by a BamHI restriction site immediately preceding the ATG translation start codon and a HindIII site following the respective translation stop codons. The gene was then cloned as BamHI/HindIII-fragment into pJJH2064, which was introduced into the yeast recipient strain by selecting for uracil prototrophy. As controls, the vector not carrying an insertion, YCplac33 carrying the native yeast ZWF1 gene including its flanking regions obtained by PCR with the primer pair 16.232/16.233 (Additional file 1: Table S1) from genomic yeast DNA cloned as a SacI/SalI fragment, or the yeast ZWF1 gene introduced into pJJH2064 under the control of the PFK2 promoter as a BamHI/HindIII fragment obtained with the oligonucleotide pair 16.234/16.233 (Additional file 1: Table S1) from genomic yeast DNA were used.
To assess the capacity of the different constructs to rescue the oxidative-stress sensitive phenotype of the yeast zwf1 deletion strain, transformants were grown overnight in 3 ml of synthetic complete medium lacking uracil (SCura; 2% glucose, 0.67% yeast nitrogen base with ammonium sulfate, supplemented with standard amino acids and adenine) according to [47], diluted to an OD600 of approximately 0.1 in fresh medium and grown for another 4–6 h. Cells were again diluted to an OD600 of 0.1 and used to prepare four 10-fold serial dilutions. Five μl of each dilution was spotted on SCura plates containing 1.5% agar with H2O2 added at the concentrations as indicated. Growth was recorded by scanning the plates after 3 days of incubation at 30 °C.
For determination of specific enzyme activities, crude extracts were prepared from 20 ml of yeast cells growing exponentially in SCura at 30 °C. Cells were harvested, washed with 50 mM Tris-HCl, pH 7.5, and broken with glass beads as described previously [48]. Protein concentrations in crude extracts were determined with the Microbiuret method [49] using bovine serum albumin as a standard. G6PDH activity was measured by following NADP reduction at 340 nm after addition of 2.5 mM glucose 6-phosphate as the substrate to the reaction mixture, which consisted of 50 mM Tris-HCl, pH 7.5, and 0.4 mM NADP and appropriate amounts of crude extract. Similarly, GapN activity was determined in the same buffer by recording NADP reduction. To the latter, 0.5 U/ml aldolase was added as an ancillary enzyme, and the reaction was started with 2.5 mM fructose 1,6-bisphosphate.
Association of GapC1 with mitochondria under different redox conditions
The GapC protein was previously found to associate with plant mitochondria [50, 51]. However, the role of this interaction remained unclear. Since metabolic fluxes and the cellular redox-state are closely related, we first analyzed the redox-dependence of the co-localization between GapC and mitochondria. For this purpose, isolated Arabidopsis mesophyll protoplasts were transformed with a GapC1-mEGFP fusion construct under the control of the S35-promoter. Simultaneously, mitochondria were stained with MitoTracker® Red FM to detect colocalizations. To assess the influence of the different redox conditions, protoplasts were pretreated with 0.2 mM H2O2 or 10 mM DTT. Fluorescent signals resulting from the fusion-proteins were detectable in the cytosol under reducing and oxidizing conditions (Fig. 1). Colocalization of GapC1-mEGFP with mitochondria was also found under both conditions, as indicated by white spots in the merged images. However, GapC1 was found to colocalize more frequently with mitochondria in a reducing environment, after DTT treatment of the protoplasts, than in the oxidized sample (H2O2 treatment) (Fig. 1). These findings suggest that the GapC1 protein is predominantly recruited to the OMM under reducing conditions, in contrast to its presumed oxidized state. For quantification of this effect, we chose an in vitro approach (see below).
Effect of the redox-state on colocalization of GapC1 with mitochondria in Arabidopsis protoplasts. Protoplasts were transiently transformed with p35S-GapC1-mEGFP. Mitochondrial staining was performed by incubating the protoplasts in 500 nM MitoTracker® Red FM for 40 min. The protoplasts were pre-treated with 10 mM DTT or 0.2 mM H2O2 for 30 min to establish defined oxidizing and reducing conditions. Fluorescence signals are displayed in false colors: GapC1-mEGFP (cyan), MitoTracker® Red FM (magenta); chlorophyll autofluorescence (yellow). To estimate a tendency for the occurrence of colocalization, protoplasts (n ≥ 50) wereinspected. A representative image is shown for each condition.and Colocalization events are visualized in the merged picture as white regions. The scale bar is set to 10 μm
Binding constants of GapC1 interacting with VDAC3 under reducing and oxidizing conditions
In earlier studies, an association of the GapC protein with the OMM via VDAC3 had been demonstrated in a split-YFP assay with mesophyll protoplasts kept under reducing conditions [9]. In the light of the redox-dependent GapC localization to the OMM (Fig. 1), we decided to determine the binding kinetics of GapC1 to the mitochondrial VDAC3 under reducing and oxidizing conditions by RIf. In this approach, recombinant VDAC had been refolded and incorporated into a lipid bilayer, thus representing the native state on a solid support membrane. Binding and dissociation of GapC1 to reconstituted VDAC3 was recorded in a flow chamber by measuring changes in white-light interference in a mass- and protein-sensitive manner (Fig. 2a, Additional file 2: Figure S1). Different amounts of reduced (pretreatment with 2 mM DTT) and oxidized (pretreatment with 0.5 mM H2O2 + 0.5 mM GSH) GapC1 protein were injected to determine the redox-dependent effects on the strength of the interaction with the VDAC3 resident in a lipid bilayer (Fig. 2a). Binding properties of GapC1 to a protein-free lipid membrane served as a negative control. Thus, the equilibrium constants (KD) for the interaction of reduced and oxidized GapC1 with VDAC3 could be determined. The KD value of oxidized GapC1 ranged at 556.3 ± 69.9 nM, while a lower KD value of 236.0 ± 19.8 nM was obtained for reduced GapC1 (Fig. 2b, Additional file 2: Figure S1). This indicates a twofold increase of the affinity for VDAC3 of reduced GapC1 compared to the oxidized form.
Redox-dependent binding of GapC1 to VDAC3. a Association and dissociation behavior of reduced and oxidized recombinant GapC1 added to bilayer-incorporated recombinant VDAC3 at the indicated concentrations is shown as original traces. For reduction, samples were pre-treated with 2 mM DTT, for oxidation with 0.5 mM H2O2 + 0.5 mM GSH. b Equilibrium dissociation constants (KD) of pre-treated GapC1 binding to VDAC3 were obtained as described in Material & Methods. Error bars represent SD, p-value by Students t-test p ≤ 0.005
Nuclear localization of GapC isoforms and their cysteine mutants under different redox conditions
Besides their interaction with VDACs at the mitochondrial surface, GapC isoforms can also translocate into the nucleus. Therefore, GapC-mEGFP fusion proteins were expressed in Arabidopsis mesophyll protoplasts to follow their subcellular distribution under different redox-conditions. For this purpose, the protoplasts were pretreated with 10 mM DTT or 0.2 mM H2O2, before analysis by cLSM. Both, GapC1- and GapC2-mEGFP fusion proteins showed fluorescent signals in the cytosol, but also in the nucleus. Nuclear localization was confirmed by the overlap with the fluorescent signals of the nuclear bZip63-mCherry marker (Fig. 3a, b). In protoplasts subjected to 10 mM DTT, the fluorescent signals appeared predominantly in the cytosol, while pretreatment of the protoplasts with 0.2 mM H2O2 resulted in an increased nuclear localization of GapC1-mEGFP and GapC2-mEGFP fusion proteins (Fig. 3a, b).
Subcellular localization of GapC under reducing and oxidizing conditions. Isolated mesophyll protoplasts of A. thaliana were transiently transformed with (a) GapC1-mEGFP or (b) GapC2-mEGFP fusion proteins (cyan). The nucleus was labeled with bZip63 fused to mCherry (magenta). Chlorophyll autofluorescence is shown in yellow. Transformed protoplasts were treated with 10 mM DTT or 0.2 mM H2O2 for 30 min. Nuclear colocalization events are shown in white. The scale bar is set to 10 μm
For quantification of the redox-dependent localization of GapC1/2, single and double Cys mutants of GapC1 and GapC2 were also included to determine the influence of presence and redox-state of the key cysteine residues on the nuclear localization of GapC. At least 50 protoplasts were analyzed for subcellular localization of each case. In accordance with the previous observations, wild-type GapC1 and GapC2 showed a significant increase of nuclear localization under oxidizing conditions compared to the reducing conditions (Fig. 4a, b). GSNO was included in this experiment, since ROS can initiate NO formation in vivo leading to the formation of GSNO by nitrosylation of a small portion of the GSH pool. Both thiol oxidants, GSNO and H2O2, led to similar distributions of the GapC isoforms between cytosol and nucleus. In contrast, protoplasts carrying GapC with either the C156S mutation or the C156S/C160S-double mutation did not respond with an increased translocation of the GapC isoforms to the nucleus upon oxidation. It should be mentioned that under reducing conditions all mutant enzymes tended to be more abundant in the nucleus than their wild-type counterparts. Taken together, these findings suggest that the catalytic cysteine residue C156 is required for sensing the cellular redox state. Its oxidative modification is required to initiate nuclear translocation of the cytosolic GapC isoforms.
Redox-dependent distribution of GapC between cytosol and nucleus. For quantification of nuclear localization, mesophyll protoplasts were transiently transformed with GapC-mEGFP fusions of the isoforms GapC1 (a), and GapC2 (b), and their corresponding single (C156S and C160S) and double (C156S/C160S) cysteine mutants. The subcellular localization was determined after the treatment with different redox reagents (10 mM DTT; 0.2 mM H2O2 or 0.05 mM GSNO). (n ≥ 50 analyzed protoplasts. The sum of cytosolic and nuclear mEGFP-signal corresponds to 100% for each experiment; mean ± SD)
Interaction of GapC with Trx-h3
GapC has been found as a target of Trx, which can convert the enzyme back into its reduced state [27, 52,53,54]. Trx, in turn, would be re-reduced by NADPH as mediated by the NTR system [23]. To fulfill its function in vivo, Trx would have to interact with GapC. To test this assumption, split-YFP assays were applied to isolated Arabidopsis mesophyll protoplasts subjected to various redox conditions. The protoplasts were transformed with constructs for fusion proteins of the N-terminal half of YFP (YFPNE) fused to either GapC1 or GapC2 and the C-terminal half (YFPCE) fused to Trx-h3. The bZip63-mCherry nuclear marker was used as a control. Treatment of the transformed mesophyll protoplasts with 20 mM DTT resulted in the interaction of Trx-h3-YFPCE with GapC1-YFPNE and GapC2-YFPNE, respectively, as indicated by the YFP fluorescence in the cytosol (Fig. 5a, b). Treatment with 0.2 mM H2O2 or 0.05 mM GSNO to establish an oxidative environment led to enhancement of the nuclear fluorescence signal, generated by the interaction of Trx-h3 with either GapC1 or GapC2 in the nucleus. Nevertheless, a significant portion of these interactions could still be detected in the cytosol under these conditions (Fig. 5a, b). This result indicates that Trx-h3 might be able to remove the oxidative modifications at the active-site cysteine C156 of GapC, both in the cytosol and in the nucleus.
Redox-dependent localization of GapC and Trx-h3. Vectors encoding (a) GapC1- or (b) GapC2-fusion constructs of YFPNE, and Trx-h3-YFPCE were co-transformed into mesophyll protoplasts. The fluorescent signals from the YFP complementation are shown in cyan. The nuclei are shown in magenta as labeled by bZip63 fused to mCherry. Colocalization events from the complemented YFP and the nuclear-located bZip63-mCherry proteins are shown in white. Transformed protoplasts were treated with 0.2 mM H2O2 or 10 mM DTT for 30 min. Scale bars were calibrated to 10 μm
The interaction of GapC with Trx-h3 was further analyzed in vitro by RIf. Association and dissociation of Trx-h3 to the immobilized GapC1 protein was recorded at different concentrations of Trx-h3 (Fig. 6). Due to the transient interactions, the KD had to be determined by the equilibrium binding signals using a Scatchard plot and yielded a value of 40 μM (Additional file 3: Figure S2). In contrast, immobilization of the unrelated yellow fluorescent protein mCitrine to the surface did not lead to any significant association of Trx-h3.
Binding of Trx-h3 to GapC1. Binding kinetics of Trx-h3 to immobilized GapC1 were monitored by RIf spectroscopy. The experiment was performed in 20 mM HBS-buffer (100 mM NaCl, 0.01% Triton X-100, pH 7.5) supplemented with 2 mM DTT. The GapC1 protein was immobilized via NHS. Association and dissociation reactions were recorded at different Trx-h3 concentrations
Complementation of a yeast zwf1 deletion strain lacking G6PDH by expression of plant cytosolic GapN
The reduced cofactor NADPH is required to recover cells from oxidative stress. It provides electrons for re-reduction of oxidized glutathione (GSSG) and GSNO, and the reversal of oxidative modifications at GapC by Trx and NTR [22]. Any enzyme or signaling activity of GapC affected by its oxidation, or that of its orthologs from other organisms, can thus be reverted when the cells recover from the stress situation, and the metabolic flux can be switched back to active glycolysis. The oxidative branch of the pentose pathway, with G6PDH as a key enzyme, generates NADPH in all organisms, in human and in yeast cells represented by single isoforms and in plants by six isoforms [55]. In addition, plants and bacteria possess a highly stable GapN [54, 56], which is a prime candidate for additional NADPH production in stressful conditions.
To study this putative function of GapN in a system devoid of background activity, we constructed a yeast zwf1 deletion mutant, which lacks its endogenous G6PDH [57, 58]. As a positive control, we employed a similar construct carrying the native ZWF1 gene from S. cerevisiae. All genes were expressed from a single-copy CEN/ARS plasmid under the control of the constitutive PKF2 promoter, in a derivative of a system previously employed for the heterologous expression of PFK genes from patients with Tarui's disease in yeast [59] introduced into the zwf1 deletion. Drop-dilution assays of the different transformants on plates containing H2O2 demonstrated that the zwf1 deletion carrying an empty vector was highly sensitive (Fig. 7). This sensitivity was not only rescued by the native Zwf1 but also to a considerable extent by the plant enzyme GapN. This result indicates that GapN from plants can generate sufficient NADPH to mediate resistance against oxidative stress conditions even in yeast cells. The weaker suppression of the oxidative stress sensitivity by GapN as compared to the complementation capacity of the native Zwf1 could be attributed to two lines of reasoning: i) the native ZWF1 gene expressed under the control of the yeast PFK2 promoter generates an enzyme with a specific G6PDH activity of 303 ± 10 mU/mg protein. On the other hand, specific GapN activity only amounted to 10 ± 2 mU/mg protein, which is near the limitof detection in the assay used and may thus generate much less NADPH than the Zwf1 reaction. ii) Since GapN activity is not present in normal yeast cells, diversion of the flux from the NADH-generating glycolytic reaction of the yeast's GAPDH isoforms may interfere with the yeast's metabolism and lead to a reduced capacity of antioxidant defense. Nevertheless, the increased resistance of the transformants with GapN activity as compared to those with the vector control clearly demonstrates its ability to generate sufficient NADPH in yeast to suppress some of the negative effects of H2O2 exposure.
Complementation of a yeast zwf1-deletion strain by plant GapN. Serial drop dilution assays of the yeast strain HOD269-1C carrying the vector without insertion (pJJH2064), the plasmid with the yeast endogenous ZWF1 gene under the control of the constitutive PFK2 promoter (pJJH2111), or the plasmid with the GapN-encoding sequence under the control of the PFK2 promoter (pJJH2118) were performed. Cells were adjusted to an OD600 of 0.1 as described in Materials & Methods. Serial dilutions were dropped onto SCura plates containing the indicated concentrations of hydrogen peroxide. Images were taken after incubation for 3 days at 30 °C
We here demonstrate evidence that the GapC isoforms of Arabidopsis thaliana localize to different cellular compartments depending on their oxidation state. This may be related to moonlighting functions apart from the catalysis of the glycolytic reaction. Thus, active glycolytic metabolons have been suggested to form in the vicinity of the mitochondrial surface. When the redox-state of the cell starts to be shifted into oxidizing conditions upon stress, a portion of the redox-sensitive GapC will be oxidized and can translocate into the nucleus, where it possibly initiates a changed gene expression.
Association of GapC with the outer membrane of plant mitochondria had been shown previously [9, 50, 51]. We observed an enhanced colocalization of GapC1 with mitochondria under reducing conditions when cells are in a metabolically active state compared to oxidizing conditions. This indicates the formation of metabolons by which glycolysis is directly taking place in proximity to mitochondria. Such metabolons are built of glycolytic enzymes, channeling the intermediates efficiently through the pathway and enabling import of the products into mitochondria for further oxidation in the respiratory chain. VDAC might serve as a docking platform for such metabolons. The present in vitro interaction study revealed that reduced GapC exhibits a higher affinity for VDAC compared to the oxidized GapC. In fact, this supports the hypothesis that reduced GapC forms an active glycolytic metabolon attached to the OMM via VDAC3.
In colocalization experiments presented here, GapC was found to accumulate in the nucleus upon oxidative stress. Furthermore, GapC had been detected in nuclei of cadmium-treated roots [10], and in calcium- and NO-stressed tobacco BY-2 cells [11]. There is now a growing number of publications describing the "moonlighting" functions of GAPDH in the nucleus [2, 3, 5, 16]. As a well-studied example in the mammalian system, an association of S-nitrosylated GAPDH and SIAH1, an E3-ubiquitin ligase, leads to nuclear translocation and induction of cell death [60]. In fact, a similar protein, namely SINAL7, was identified as a binding partner of GapC in plants [61]. In rice, GapC was found to be responsible for transcriptional activation of glycolytic genes under oxidative stress [62].
In various Trx-interaction studies, GapC had been found as a target protein [27, 52]. Due to the transient nature of the nuclear localization, the interaction with a redox-mediating compound such as Trx must be assumed for the reversal of the cysteine modifications. Trx type-h was found in the nucleus of oxidatively stressed Arabidopsis cells [63, 64]. It had been suggested that denitrosylation of modified proteins in animal cells is catalyzed by the thioredoxin system [65]. In Chlamydomonas, cytosolic Trx-h isoforms even seem to possess some specificity of their nuclear functions in response to different types of DNA damage [64]. If GapC fulfills a moonlighting function by regulation of nuclear gene expression, interaction with Trx-h3 could be interpreted in view of the reversal of the oxidative modification and concomitant export from the nucleus, thus aborting its access to the transcription machinery after a stress period.
As soon as any disturbance of the metabolic flux becomes evident, H2O2 or NO is formed leading to modifications of the most sensitive thiols of GAPDH [13, 66], triose-phosphate isomerase [67] or pyruvate kinase M2 [68]. These examples demonstrate that the central glycolytic pathway is flexibly adjusted by linking the cellular redox-state and the substrate pool sizes at multiple steps. Due to the concomitant inactivation upon oxidative stress, glucose oxidation is redirected towards the OPP for NADPH generation by G6PDH [69]. Alternatively, the oxidation of GAP via GapN is an additional source for NADPH in plants, especially under increasing oxidant levels [54] (Fig. 8).
Pathways of carbohydrate oxidation. In unstressed cells (left panel), glycolysis and respiration are reinforced by the formation of a metabolon at the mitochondria attached to VDAC. In a stressed cell (right panel), GapC serves as a redox-sensor and mediates signal transfer to the nucleus, where genes required for poising and ROS defense are induced. NADPH is provided by GapN, specific for plants. In both cases, NADPH can also be provided by the OPP
Due to its redox-sensitive catalytic cysteine, GapC provides a connection of redox-state and energy metabolism [13, 16]. Therefore, GapC might serve as a central hub for energy supply and signal transfer to execute the various cellular fates [70, 71]. Redox-dependent processes are now accepted as major players in a multitude of cellular processes, and as important signal transmitters to induce readjustment of homeostasis upon oxidative stress [72]. Various sources, duration, and levels of stress can be transmitted by a specific post-translational modification code that is highly dynamic. Stress stimuli could lead to a differential modification pattern on a single protein. The idea of a modification code combining modifications also of other residues with the various redox-modifications of cysteine is tempting. Obviously, such a code can only be investigated and deciphered in vivo. Yet, only a minor proportion of each protein in question is expected to be modified for triggering the specific response. To solve this problem, high-resolution analysis of all single GAPDH molecules would be required to identify different modification patterns in subfractions of the cellular GAPDH pool.
An equally challenging task is the exact determination of the stimuli that provoke each modification, resulting in a complex interplay of different factors in vivo, and different output in each case. These modifications are additionally dependent on metabolites, microenvironmental conditions and/or macromolecular crowding effects [73, 74]. To prove any effect in such a complex scenario in vivo and to mimick a realistic microenvironment in vitro, will be a difficult task for the future. The investigation of the switching ability between GAPDH activity in the central metabolism and its role as a redox-sensor by classical genetic methods carries the intrinsic problem that these functions are both dependent upon the active-site cysteine of the protein. GAPDH-knockout mutants show many pleiotropic effects which are not distinguishable in relation to their origin due to the dual role of the active-site cysteine. Any effects could be either caused by the lack of the moonlighting functions of GAPDH or an impeded central metabolism when glycolysis is not functional.
The glycolytic enzyme GapC is a versatile hub to sense and counteract metabolic and redox imbalances. Both pieces of information are integrated into signaling pathways for a proper response. Many different input signals evoke multiple cellular responses, ranging from improved growth to induction of programmed cell death (PCD). In particular in plants, integration of signals from environmental impact and developmental state in the various photoautotrophic and heterotrophic tissues requires effective regulatory systems that respond accordingly to maintain homeostasis [75]. The occurrence of S-glutathionylation, S-sulfhydration, and/or S-nitrosylation at the catalytic cysteine and/or the neighboring cysteine of GAPDH could serve as a redox code [76]. The degree and kind of stress might even be reflected in a specific pattern of the various possible modifications of sensitive cysteines, thus transferring the actual information in a finely graduated way to obtain the proper output. Based on such a system, a wide spectrum of environmental challenges and metabolic imbalances can be responded to at the various levels of regulation to regain and maintain homeostasis. In the worst case, execution of PCD to prevent further ROS formation in damaged cells, when metabolism gets out of control, might then be also an appropriate response.
A transient inactivation of GAPDH suggests that the glycolytic pathway will be down-regulated during oxidative or nitrosative stress. For the time of imbalance, this leads to a redirection of the metabolic flux away from glycolysis and ATP production into the OPP pathway for rapid generation of NADPH required for GSSG reduction poising, and repair [69]. Additionally, apart from the oxidation of G6P through the activity of G6PDH, the required NADPH can be provided by oxidation of triose-P through the cytosolic GapN. Ectopic expression of GapN enables the yeast strain lacking G6PDH to persist oxidative stress conditions by its capability to provide NADPH (Fig. 8).
On the other hand, oxidative modifications affect the binding properties of GapC and its subcellular localization. Due to the various post-translational modifications, transient formation of subcompartments or metabolons, changing cellular localizations and moonlighting functions can be initiated [77]. Upon regain of homeostasis, signals need to be converted back to normal, and regular energy metabolism should recover. This requires the reversal of GapC modifications and ending of its moonlighting functions. Here, the NADPH-generating systems for regeneration of reduced thioredoxins and reversal of cysteine modifications are also required.
In the present study, we could demonstrate the redox-dependence of the cellular localization of GapC, resulting in increased nuclear localization of both isoforms under oxidizing conditions, and the interaction with Trx-h3 in the nucleus. Binding of GapC isoforms to the mitochondrial VDAC, on the other hand, was more pronounced under reducing conditions as would be the case upon recovery. The efficiency of the glycolytic flux is then optimized by the formation of a metabolon in close vicinity of the mitochondria.
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We would like to thank Minhee Kang for performing initial experiments. Provision of the E. coli BL21 (DE3)-omp9 strain from Prof. Dr. Lars-Oliver Essen (Philipps-Universität, Marburg, Germany) is acknowledged. We thank the Department of Biophysics for access to their RIf facilities, and Andrea Murra for technical assistance.
Our research was funded by the German research foundation (DFG) within the Collaborative Research Centre (CRC) 944 "Physiology and dynamics of cellular micro-compartments".
Data and materials are available from the authors upon request.
Division of Plant Physiology, Department of Biology and Chemistry, Osnabrück University, Barbarastr. 11, 49076, Osnabrück, Germany
, Johannes Knuesting
& Renate Scheibe
Division of Biophysics, Department of Biology and Chemistry, Osnabrück University, Barbarastr. 11, 49076, Osnabrück, Germany
Oliver Birkholz
Division of Genetics, Department of Biology and Chemistry, Osnabrück University, Barbarastr. 11, 49076, Osnabrück, Germany
Jürgen J. Heinisch
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MS, JK and JJH performed the experiments. MS, JK, and JJH analyzed the data. OB helped with RIf data evaluation. MS, JK, JJH and RS designed the project and wrote the article.
Correspondence to Renate Scheibe.
Table S1. Primer used for the amplification of the ZWF1 gene encoding the glucose-6-phosphate dehydrogenase. Primer sequences used to generate the different yeast strains for the yeast complementation assay. (DOCX 14 kb)
Figure S1. Redox-dependent binding of GapC1 to VDAC3. Binding kinetics of the GapC1-VDAC3 interaction were monitored by RIf spectroscopy. (A) After the injection of VDAC3-containing proteoliposomes (gray), the formation of a polymer-supported membrane and the binding of GapC1 to VDAC3 (light blue) were recorded by label-free RIf detection. (B) Representative association and dissociation behavior of reduced and oxidized GapC1 and VDAC3 with the respective residuals. (PPTX 169 kb)
Figure S2. Determination of the KD for the interaction of GapC1 with Trx-h3. The values of the equilibrium binding signals (Γeq) for each experiment were plotted against the ratio of the binding signal to the concentration of Trx-h3 (Γeq/c). The KD value of the corresponding interaction of Trx-h3 with GapC1 was extracted from the slope of the corresponding. (PPTX 50 kb)
Schneider, M., Knuesting, J., Birkholz, O. et al. Cytosolic GAPDH as a redox-dependent regulator of energy metabolism. BMC Plant Biol 18, 184 (2018) doi:10.1186/s12870-018-1390-6
Metabolon
GapN


Joe Martins discovered music and fishing around the same time � when he was about 9.
Fishing was a by-product of growing up surrounded by water in Newport. He fished for mackerel from the Goat Island Causeway. It was a natural form of recreation being so close to the water.
�Here we get to do all the things you can�t do in Kansas,� he said.
Martins, a Rogers High School graduate, holds a bachelor�s degree in natural resources from the University of Rhode Island.
His passion for fishing led to his purchase of Point Jude Lures in 2004, after he learned to make the tins that resemble fish prey in order to draw fish to lines. Martins, also known as �Point Jude Joe,� makes the lures at home and sells them to retailers across the region.
Winter provides him extra time for music. He plays saxophone, bass, guitar and some clarinet in his band Triple Threat, which will perform Saturday at the Gas Lamp Grille. He also sits in during local blues jams and with local bands such as the Merge.
On Sunday, he headed to the Met Cafe in Pawtucket to see bands including John Cafferty and the Beaver Brown Band and Roomful of Blues inducted into the Rhode Island Music Hall of Fame. It�s a small state, and Martins knows a lot of the players. Earlier this month, he jammed with the James Montgomery Blues Band at Chan�s in Woonsocket.
Martins said he lost interest in playing for a while in the 1990s, but became rejuvenated when a friend suggested he stop by a blues jam at Rusty�s in Middletown. The next time he went, he packed his horn and sat in. At 56, he plays for fun and some extra cash.
�Playing in your 50s is a lot different from playing in your 20s,� he said. �It�s more enjoyable in a lot of ways. You don�t have the same expectations. I�m not expecting to end up on a big TV show, the way the Beatles did on the �Ed Sullivan Show,� and that�s fine with me. I enjoy playing music, mostly blues and classic rock, the stuff I grew up with.


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Hello! My name is Jessenya Guerra and I am the Editor-in-Chief of The Lutrinae, California State University, Monterey Bay's (CSUMB) student-run newspaper. Whether this is your first semester here, or your last, I am here to say welcome or welcome back and thank you for reading The Lutrinae! I thoroughly enjoyed my first semester as Editor-in-Chief and can't wait for what this second semester has in store for us.
I wanted to give a special thanks to our faculty advisor Sam Robinson who, because of her new position as humanities and communication department chair, will be leaving us. She has been a remarkable faculty advisor and is an inspiration to me and many other students. I would also like to welcome our new advisor, Sara Gobets. I know that Gobets is excited to be at CSUMB and to teach us all that she can.
I would also like to take this chance to invite any and all students that are interested in joining The Lutrinae team. We would love to have our team grow regardless of experience, major or writing level. We are simply looking for students that are dedicated to providing CSUMB, and the CSUMB community, with news and entertainment content.
I am looking forward to the issues that The Lutrinae staff and contributors put forth this semester and ask for your continued support. Our team of students works hard every week to put out a newspaper for the CSUMB community and I am so proud to be a part of that team. I wish all of you a successful, safe and sane spring 2019 semester.


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Wrangler® Is First To Adopt Dry Indigo® Denim
GREENSBORO, N.C. — September 18, 2018 — Wrangler® today confirmed an agreement for denim fabric to be produced later this year with a new foam-dyeing process that will eliminate 99 percent of the water typically used in indigo-dyeing. Wrangler is the first brand to embrace the innovative technology, which is expected to transform the denim industry.
"While we have been able to reduce 3 billion liters of water in product finishing during the past 10 years, we know that more needs to be done across the entire supply chain," said Wrangler President Tom Waldron. "Foam technology reduces water consumption and pollution further upstream, helping our fabric suppliers to dramatically minimize the impacts of making denim fabric blue."
Recognizing the potential of this breakthrough, Wrangler and the Walmart Foundation provided Texas Tech University with early-stage funding for development of the foam-dying process. The iconic denim brand helped introduce fabric mills to the latest technology and now will incorporate the first foam-dyed denim into a line of jeans launching in 2019.
"We invested in the development of this innovation, because we believe it can drastically change the denim industry for the better," Waldron said. "We're grateful to have an industry-leading partner in Royo, with whom we are taking this revolutionary step towards more sustainable denim."
Tejidos Royo, a Spanish fabric mill with a reputation for prioritizing environmental performance, will be the first to integrate the foam-dye process, which it calls Dry Indigo®. Royo is scheduled to receive the foam-dye equipment in October and expects to begin supplying Wrangler with denim before the end of the year.
"We're excited Wrangler is dedicating an entire line of jeans to this innovation," said Tejidos Royo Sales Director Jose Royo. "Our Dry Indigo process nearly erases the environmental impact of denim dyeing and represents the next generation of denim production."
The first line of foam-dyed jeans will be Wrangler's most recent action to minimize environmental impact and save precious resources. Among the brand's demonstrated sustainability activities are the ongoing work in U.S. sustainable cotton and a commitment to reduce water usage by five billion liters by 2020. According to Royo, applying indigo dye to raw denim with foam instead of water will eliminate the need for the tens of millions of gallons of water typically consumed by conventional wet-dye systems.
Source: Wrangler

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Home » Gaming Review » Apex Legends Heirloom Review Heirloom Set
Apex Legends Heirloom Review Heirloom Set
Apex Legends Heirloom Review will focus on the new apex legends heirloom set, and how the game can be both played and still have many of the same benefits of playing previous games. apex legends heirloom review | game | characters | play | also} APEX has always been a good game to play, in that there are many different characters to choose from. It also has an excellent balance between the energy system, as well as the health system. Because of this, players are not only constantly doing something to increase their health, but they are also doing everything possible to reduce their energy costs. This keeps the game very fun and active because the player never knows what he or she will come up against next.
This AWeber Heirloom Review will focus on the new apex legends heirloom set, and how the game can be both played and still have many of the same benefits of playing previous games. apex legends heirloom review | game | characters | play | also} APEX has always been a good game to play, in that there are many different characters to choose from. It also has an excellent balance between the energy system, as well as the health system. Because of this, players are not only constantly doing something to increase their health, but they are also doing everything possible to reduce their energy costs. This keeps the game very fun and active because the player never knows what he or she will come up against next.
However, all of this has changed with the new apex heirloom set. These characters now level up the same, but also have a special move for each level. Apex Legends Heirloom Items The moves are powerful, but they do not have an effect on the player's health or energy. Instead, they only affect the player's characters. For example, the Ice Wall that the characters can put up is only a defense mechanism and does nothing to actually help them fight off enemies or do anything else in the game.
Apex Legends Reviews & Near Me Apex Legends 2021 New
Apex Legends Heirloom leaves players with two main choices when choosing an apex heirloom set. They can either choose a character that is extremely powerful for the amount of energy it costs to use, or they can choose one with an extremely strong defense. It is also interesting to note that the characters in this game all cost approximately the same amount of energy.
Apex Legends Heirloom This type of heirloom set will be very valuable to players that want a bit of everything in one package. However, players who are just starting out or want something more challenging should stick to the lower-end characters in the beginning while they work on building up their skills. The power of the higher-level characters in the game is not worth losing a lot of energy if you are just a beginner in the game.
Finally, I want to share an heirloom review of the Apex leveling guide that I have created. This guide was created specifically to help new players learn the ins and outs of the game. I have been playing Aion: Tower of Eternity since early October of 2021. Since then, I have gained some valuable knowledge by using a leveling guide and heirloom items. If you would like to learn more about the game in my Aion Heirloom Guide, you can find it below.
Please Add a comment of Apex Legends Heirloom, Please let us know if you need help with this. We and other thread size will help.
For more related Apex Legends, please Related Posts check below.
Apex Legends Heirloom Do you have a positive or negative opinion about this product?
Yes, I bought the product from Amazon when it first came out. I am still using it and I am very satisfied ... Apex Legends Heirloom
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Surrounded by the famous buzz of London's West End, employees of these offices are never far from boutiques, retailers and eateries. The site is just seven minutes from Oxford Circus and Piccadilly tube stations while Tottenham Court Road station is only a minute further.
Smart and affordable office space is available at 80 Berwick Street, Soho, London W1F just minutes to Oxford Circus and Piccadilly Circus underground stations with easy access to Charing Cross rail station. This period property offers serviced office space to rent that comes with highspeed internet, IT support, meeting rooms and quality furnished offices to create a professional looking environment.
If you would like to view this Berwick Street building or find out how much it costs to rent offices in Soho, simply fill out the Quick Quotes form or call us on 020 3053 3893.
We have commercial property with currently available offices to rent in prestigious locations throughout West Central London. Fill out the Quick Quotes form or give us a call to find out more about the workspace on offer at this building on Berwick Street or click here for more Soho office space.


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Home Articles Car & Driving Technology Waymo to Test Autonomous Vehicles in Atlanta
Waymo to Test Autonomous Vehicles in Atlanta
By: Staff Writer February 2, 2018
Officials at Waymo announced in late January that they would be testing their self-driving minivans on Atlanta's congested, dangerous roadways.
When it comes to autonomous cars in the United States, Waymo, the self-driving branch of Google, is leading the way in both research and development. The company's name stands for the "way forward in mobility."
On Jan. 22, Waymo officials announced plans to find that way forward in Atlanta. The city is ranked fourth for worst traffic in the country with commuters spending an average of 70.8 hours per year in on-road congestion, which is only exacerbated by a climbing number or roadway deaths across the Peach State. The snarls and bumper-to-bumper quagmires will put the company's autonomous Chrysler Pacifica hybrid minivans to the ultimate test.
"We are thrilled to welcome Waymo to our state because fully self-driving vehicle technology holds tremendous potential to improve road safety, and we are proud Georgia is paving the way for the future of transportation," wrote Georgia Governor Nathan Deal in a statement e-mailed to Forbes.
The Atlanta-bound Pacificas will have Level 4 autonomy, which means they can drive themselves in almost all situations but still have a steering wheel and pedals. In mid-January, one was sent ahead to map the city, driving through Atlanta's vehicle-choked streets while its laser-based scanning systems cataloged 3-D images and maps of the planned test areas.
Additionally, Waymo's engineers have 4 million miles of total experience to draw from when developing and managing their efforts in Atlanta.
The self-driving start-up has tested its technology on roads in regions across the country, including Austin, Texas; the Northern California tech enclave of Mountain View; and Phoenix, Arizona. Still, none can match Atlanta for congestion and difficulty level.
For now, Waymo will deploy the autonomous minivans in Atlanta strictly for testing purposes; the vehicles will not pick up passengers just yet. In the Phoenix suburb of Chandler, however, the company's Pacificas have moved beyond testing and into a 100-vehicle on-demand ride service, though always with a human driver in the vehicle to monitor the situation. (Waymo officials intend to launch a totally driverless service there soon.)
Going from having a human being behind the wheel "just in case" to commuting without a driver is a big step in the world of autonomous vehicles. However, the cars' potential positive impact on safety—and traffic congestion—could improve the country's roadways by leaps and bounds.


A Gateway in to Pluscarden Abbey.
Ben Buie, Isle of Mull.
Living in Elgin in the Laich of Moray, North East Scotland, I am fortunate to have a diversity of Scottish scenery and Nature to explore and photograph, from mountains to sea shore.
I shoot most of my images on a Sony A99ii. I have also started to shoot monochrome images using a Mamiya RZ67.
Although I shoot mainly Heritage, Landscape, and Nature I hope that with a scan through my galleries you will find something to focus on that pleases or inspires you.
I maintain copyright of all images on this site as per the copyright, designs and patents act 1988.


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This new search interface has 2 new labels namely "Settings" and "Tools". In the current interface (as displayed in the below screenshot), there is a gear icon that has a drop down containing the search settings option. The new interface is more user friendly as many users remain unaware about the search settings label being hidden inside the gear icon.


Jquery image zoom plugins create awesome zooming effects. Visitors to your website are able to see the detailed look of your images. The effects created while hovering the image is great to look at. These jquery scripts are a necessity for designer, photography and art websites. Below is the list of best jquery image zoom plugins.
This image zoom plugin allows you to have zoom effect in 3 ways. Which are inner, standard and follow. The plugin is responsive.It has lot of flexible options. You can customize as per your needs.
Glasscase is a responsive jQuery image zoom plugin. It is easy to set up and customize. Features include are one click to fullscreen, define zoom movement & transition, easy to position thumbnails etc. The plugin is available at codecanyon for $12.
Etalage is a premium jquery image zoom plugin. You can customize the plugin with various options. It's features include are optional image zoom movements, add captions, automatic generation of small thumbnails etc.
Zoome is a image zoom plugin with hover effects.The hover effects are blur, transparent and grayscale. You have the option of zooming in and zooming out with mouse wheel. The plugin is responsive with flexible options.
Pinch Zoomer is a jquery plugin that zooms the image by pinching and mouse scroll. The image is zoomed in the same image window. The plugin is compatible for desktop, mobiles and tablets.
Optic zoom is a magnifier type zooming plugin. It is responsive. There are various magnifying options. The visitors of your website will surely find the zooming an attractive feature.
Nuvuzoom allows you to zoom the image with in and out animation effect. The image zooms on hover. Simple yet impressive.
Zoomwizard is a lightweight flexible plugin.There are 3 ways to zoom image using this plugin. Easy to customize.


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Tim September 28, 2017 Blogging tips
Why is Twitter changing its character limit?
Twitter announced on Tuesday that they are testing an extended character limit of 280 characters per tweet, as opposed to the current 140. Why? And is it a bad thing?
Firstly, this isn't a feature that is available to everyone yet. It is being tested with a small set of users. So if you're one of them, consider yourself lucky. Based on previous roll-outs of new features, the rest of us will have to wait a few months before we can get our hands (thumbs?) on those extra characters.
Nonetheless within minutes of the announcement Twitter users were up in arms. #Outraged. WTF. Angry emoji.
This is nothing new. Social media users are outraged whenever their favourite platform introduces a change. Facebook News Feed. The Instagram algorithm. Pretty much anything Twitter does. It's funny how us techies can be so resistant to change, isn't it?
A few calmer voices wondered aloud – or, at least, wrote in 140 or fewer characters – why Twitter is prioritising a feature that hardly anyone has asked for over finding a better way to deal with trolls and hate speech. Or the universally desired 'edit tweet' function.
It's not about users, it's about advertisers
Yes, the 140-character limit is Twitter's defining feature and people have an emotional attachment to it. But opinion is divided. Many users see the brevity this enforces as a strength. It keeps timelines free-flowing and conversations snappy. Others – in particular business users – are frustrated by the constraints imposed by the limit, as it can make it nearly impossible to convey complex messages in a single tweet.
Guess who pays the bills?
Let's take a step back for a minute. Twitter has lost its lustre in recent years. There is no clear strategy, other than to reinforce its niche as a key source of real-time news and conversation. Growth has plateaued – currently 328 million active users and going nowhere fast – while its rivals continue to grow. Facebook passed two billion users recently, Instagram has just topped 800 million. Twitter is no longer the popular new kid, as investors fawn over Snapchat and others.
Without user growth, without a clear strategy, question marks hang over Twitter's future profitability, which is reliant on advertising revenue streams that are dwarfed by Facebook and outpaced by Instagram. Without confidence in Twitter's business model, investment dries up. Without financial backing, well, you get the idea.
So priority number one is to make Twitter attractive to business advertisers again. Yes, Twitter is saying it's about improving user experience – but I rather suspect that is only a secondary motivation.
The storm of social media outrage will pass soon enough. It always does. Will users abandon Twitter in droves? Unlikely. Life will go on.
Why do businesses need longer tweets?
So, back to business (literally). What does Twitter have to do to make itself more attractive to businesses?
Companies use Twitter for a variety of reasons. It is a global communications channel. It is an advertising platform. It is increasingly a customer service channel.
In each of these cases, only having 140 characters to play with is a problem.
For instance, from a comms and advertising standpoint, the character limit restricts how much copy you can include in a tweet. Include a URL link and a hashtag and your 140 characters is reduced to about 100 for your ad text. It's not much.
There are 47 characters in this sentence alone.
What's happening is a natural extension to the changes we have seen over the past year. Replying to someone using their @handle or including an image used to eat into the 140-character limit. They don't any more. These changes alone freed up valuable real estate for actual words.
Now imagine how liberating an additional 140 characters will be for advertisers. Give marketers more space and they will be able to communicate more sophisticated messages and achieve better results, which means they will spend more on Twitter ads. More revenue, better growth prospects, more investment. Bingo!
It's also great for languages that tend to have longer words. A 140-character limit can be problematic at times in English in a way it simply isn't in a 'compact' language such as Japanese. But a 140-character message in English may be well above the limit in Spanish or, worse still, German, a language known for its long words. There is a reason why Twitter isn't particularly widely used in German-speaking countries.
What does this mean for bloggers?
So what implications can we expect from a blogger's perspective? I think we will see two key changes.
The first of these is a renaissance of the hashtag, particularly for sponsored activity. When brands ask to include their campaign hashtags it restricts what we can put into tweets, especially if they ask for more than one hashtag or to mention their company's @handle. With 280 characters, there will be plenty of space for extra hashtags and for bloggers to compose compelling tweets.
The second thing we will see is a shift towards longer tweets – obviously – but then, I suspect, the pendulum will swing back towards shorter ones. Maybe not as short as we have now, but certainly less than 280 characters.
Smart advertisers and bloggers will already know that with any form of social media, there is a noticeable drop-off in engagement as posts get longer. Recent research suggests that tweets under 100 characters in length drive on average 17% more engagement.
It makes sense if you think about it. If you can convey in 150 characters what takes someone else 280, readers are more likely to absorb and respond to the one that takes less time to read. So while it will unquestionably be beneficial to have an extra 140 characters available, just because you can use them all doesn't mean you should do.
And here's the rub. Even when the new character limit is introduced, brevity will still be king on Twitter.
I don't think this change will be as earth-shatteringly horrible as many seem to think. In fact, I think that within a few weeks we will barely notice the difference. As the saying goes: the more things change, the more they stay the same.
Posted in Blogging tips and tagged Blogging, family, fatherhood, parenthood, Parenting, social media, Twitter. Bookmark the permalink.
Transit Address	 on September 28, 2017 at 6:56 am
If they're doing so,it's a great move indeed
Tim	 on September 29, 2017 at 10:16 pm
The jury's out from a user point of view. But I think there will be quite a few benefits, despite all the people who are convinced it's the end of the world as we know it.
RachelSwirl	 on September 28, 2017 at 1:20 pm
To be honest I'm really chuffed that Twitter is extending their character allowance. Change can only be a good thing right?!
That's it. I think we should be open to change – instead so many social media users seem to think that any change is the end of the world.
Exactly !
dad|THEORY	 on September 28, 2017 at 2:59 pm
I've long though that an increase to 141 characters would do it for me…that crucial extra character might turn my inane nonsense into profound profundity. I'd get lost in 280!!
I think we'll find that most people will rarely use the full 280. But it will be nice to have a few extra characters to play with!
RawChildhood	 on September 29, 2017 at 8:23 am
Great post, personally I don't like the new limit. I can definitely see how it would be beneficial when doing sponsored tweets but that's probably about it. We're going to start seeing those silly chain mail stuff that's usually on Facebook by the fools of Twitter. I guess we will have to wait and see!
I'm willing to wait and see how it goes. Ultimately whether it works or not will be down to how users adopt it. Hopefully we won't get to see too much of the silly stuff.
RelentlesslyPurple	 on September 29, 2017 at 9:16 am
I'm not fussed either way in all honesty! #ThatFridayLinky
Phew. 95% of people see to be outraged, just because. I think it will be fine.
We all moan when something changes yet we all make the most of changes that benefit us. Everyone will adjust.
David @ DadvWorld	 on September 29, 2017 at 1:32 pm
I'm a huge advocate for change! I think trials, new concepts and changes show willingness to evolve and learn. I often feel people pay too much attention to particular platforms and rule sets, it's all part of the game to evolve your own message to put online. I'll roll with it, when I'm finished smashing in Instagram Stories 🙂
Great post Tim 🙂
Cheers David. i think it's odd sometimes how anti-change we can be, especially when it is tech adopters moaning about changes to tech. Change is good, and Twitter more than most needs to change more if it is going to survive and thrive.
Its Adam Again - Dad Days Out	 on September 29, 2017 at 9:17 pm
If it is released, I will miss the challenge of condensing a message for my own or business marketing. My concern is that it is more a structural change towards microblogging, designed to increase stats such as the amount of time people spend on the app as they trawl through lengthier messages or the additional hashtags that will be used!
My concern is that all social channels are gradually merging into each other and losing their unique appeal. The more recent changes have been sensible and I can see how for B2B marketing and campaigns longer messages would be beneficial and draw new users, it may just detract from the very thing that has brought Twitter to where it is now. That for me, would be a shame.
Having said all that – what do I know? I still have a Google+ account! lol
#thatfridaylinky
I don't know. Even with this new change, Twitter will still have a very different look and feel to Facebook or Insta. Some of their recent changes, such as suggesting content at the top of your timeline, has already significantly increased user dwell time. I'm not sure this change will do much to increase user time on platform but it will be interesting to see if users still respond better to shorter tweets, as they do now. I think we will still see the majority of posts under 140 – at some point people will realise that just because you have an extra 140 characters doesn't mean you have to (or should) use them.
Its Adam Again - Dad Days Out	 on October 1, 2017 at 9:18 pm
I agree with the above, but if the change is to draw a new audience who don't like Twitter in its current form because of the character restriction, I would expect that the longer posts to increase in my feed. If it was purely for business advertising they could apply this to sponsored posts or business accounts as a trial – so I expect they believe there is an audience who is keen to join the platform.
Tim	 on October 6, 2017 at 10:02 pm
Oh, definitely. Especially in countries like Germany, where the extra characters will attract users who have previously been put off.
Tracy	 on October 2, 2017 at 4:23 pm
I'd be happy with a few more characters and an edit button for those early morning errors ha ha
I certainly wouldn't say no to an edit button!
Nigel	 on October 2, 2017 at 5:17 pm
If I'm honest I'm not bother Thank you for linking to #ThatFridayLinky Please come back next week
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