Phosphoglycerate kinase 1 (PGK1) catalyzes the reversible transfer of a phosphoryl group from 1, 3-bisphosphoglycerate  to ADP, producing 3-phosphoglycerate (3-PG) and ATP. PGK1 plays a key role in coordinating glycolytic energy production with one-carbon metabolism, serine biosynthesis, and cellular redox regulation. Here, we report that PGK1 is acetylated at lysine 220 (K220), which inhibits PGK1 activity by disrupting the binding with its substrate, ADP. We have identified KAT9 and HDAC3 as the potential acetyltransferase and deacetylase, respectively, for PGK1. Insulin promotes K220 deacetylation to stimulate PGK1 activity. We show that the PI3K/AKT/mTOR pathway regulates HDAC3 S424 phosphorylation, which promotes HDAC3-PGK1 interaction and PGK1 K220 deacetylation. Our study uncovers a previously unknown mechanism for the insulin and mTOR pathway in regulation of glycolytic ATP production and cellular redox potential via HDAC3-mediated PGK1 deacetylation. Insulin and mTOR use acetylation state to regulate phosphoglycerate kinase, a key enzyme that coordinates the balance between energy production, biosynthesis, and redox potential. Phosphoglycerate kinase (PGK1) catalyzes the reversible phosphotransfer reaction from 1, 3-bisphosphoglycerate  to ADP to form 3-phosphoglycerate (3-PG) and ATP. By controlling ATP and 3-PG levels, PGK1 plays an important role in coordinating energy production with biosynthesis and redox balance. In contrast to the extensive investigation of the transcriptional regulation of PGK1, little is known about its post-translational regulation. Here, we report that PGK1 is acetylated at lysine 220 (K220) and this acetylation inhibits PGK1 activity by disrupting the binding with its substrate, ADP. We have identified KAT9 and HDAC3 as the acetyltransferase and deacetylase, respectively, for PGK1. Moreover, we show there is molecular crosstalk between mTOR-mediated HDAC3 S424 phosphorylation and PGK1 K220 acetylation. Our study uncovers a previously unknown mechanism for the insulin and mTOR pathway in regulating glycolytic ATP production and cellular redox potential via HDAC3-mediated PGK1 deacetylation. Phosphoglycerate kinase EC 2.7.2.3; PGK) catalyzes the reversible phosphotransfer reaction from 1, 3-bisphosphoglycerate  to ADP to form 3-phosphoglycerate (3-PG) and ATP. The PGK-catalyzed reaction is the first ATP-yielding step of glycolysis and is essential for energy generation by the glycolytic pathway of aerobes and the fermentation of anaerobes in most living cells ; PGK cat. By contPGK is one of the most highly expressed genes and accounts for approximately 5% of the total mRNA and protein   analysis demonstrated that the recombinant PGK1K220ac protein was defective in binding with ADP ] . The K221(K220)] . Consistwith ADP . These rKAT genes [KAT genes was determined by quantitative RT-PCR (ATF2 (Gene ID: 1386), KAT5 (Gene ID: 10524), KAT6A (Gene ID: 7994), KAT9 (Gene ID: 55140), KAT12 (Gene ID: 9329), or KAT13B (Gene ID: 8202) led to an increase in cellular PGK1 enzyme activity , but not the other five KATs, significantly decreased the K220 acetylation level of endogenous PGK1 without changing its protein expression in HEK293T cells (The acetylation state of a given protein is controlled by the action of lysine acetyltransferases (KATs) and deacetylases (KDACs), enzymes that catalyze the addition and removal, respectively, of an acetyl group from a lysine residue. To search for potential KAT(s) which are responsible for PGK1 K220 acetylation, we generated a siRNA library with three siRNAs targeting each of the 19 human AT genes . The knoe RT-PCR . We founactivity , indicat3T cells . In a co3T cells . CollectY298H [HDAC3 increased the K220 acetylation level of endogenous PGK1 by >2-fold and decreased PGK1 activity by >50% in HEK293T cells  decreased and bottomed at 60 min after insulin injection, followed by a period of recovery in parallel with the blood glucose levels  resulted in a transient drop in blood glucose levels . Interese levels . Similare levels . TogetheKAT9 gene expression in HEK293T cells  pathway . In addiS424A mutant exhibited weak binding to PGK1 even upon insulin treatment  in glycolytic ATP production compared to wild-type rescued cells (p < 0.05), in PGK1K220Q mutant put-back cells compared to wild-type rescued cells (p < 0.01) in PGK1K220Q mutant put-back cells , while glycogen storage was significantly increased  in the PGK1K220Q mutant put-back cells when compared to wild-type rescued cells  stimulated by insulin treatment (p < 0.01)  . In contmulation . These fThe current study uncovers a biochemical mechanism for how acetylation controls the activity/function of PGK1. We have identified KAT9 and HDAC3 as the potential acetyltransferase and major deacetylase of PGK1, respectively. To the best of our knowledge, we show for the first time that acetylation of PGK1 plays an important role in modulating glycolytic energy production and cellular oxidative response.K220ac protein is defective in ADP binding, implying that neutralization of the positive charge of K220 by acetylation may disrupt ADP binding and thus inhibit PGK1 catalysis. It has to be noted that both acetylation mimetic K220Q substitution and deacetylation mimetic K220R substitution can significantly reduce PGK1 activity. This raises another possibility, that is, the steric hindrance that occurs when K220 is mutated to Q or R, and this will generate different interaction force and/or steric hindrance, thereby abolishing ADP binding and inhibiting PGK1 catalysis.We have identified K220 as an important regulatory acetylation site within the PGK1 protein. Human PGK1 exists as a monomer containing two nearly equal-sized domains corresponding to the N- and C-termini of the protein . 1, 3-BPOur study has provided novel insights into the role of K220 acetylation in controlling PGK1 activity/function in response to insulin. To the best of our knowledge, this is the first evidence linking the regulation of PGK1 to mTOR downstream of PI3K/AKT and insulin. In addition, we show a molecular crosstalk between mTOR-mediated HDAC3 S424 phosphorylation and PGK1 K220 acetylation.We have identified HDAC3 as the major deacetylase of PGK1. HDAC3 can form multi-protein complexes with the co-repressors SMRT and N-CoR and deacetylates histones, thereby regulating the transcription of a plethora of genes ,51. In aKAT9 expression nor KAT9-PGK1 interaction is changed upon insulin treatment.In addition, we have also identified KAT9 as a potential acetyltransferase of PGK1. KAT9/ELP3, which encodes the catalytic subunit of the histone acetyltransferase elongator complex, has previously been identified as an α-tubulin acetyltransferase in mouse neurons . PreviouPGK1 knockdown and put-back of the acetylation mimetic K220Q mutant as compared to wild-type rescued cells  were purchased from the Fudan Animal Center. Animals were given unrestricted access to a standard diet and tap water. Blood was collected from tail vein at different time points post intraperitoneal injection of insulin (5 U/kg body weight), and blood glucose levels were determined by using a glucose detection kit (Roche). Before being humanely killed, mice were anesthetized with sodium pentobarbital . After that, mouse livers and kidneys were harvested and then homogenized using the Tissuelyser-24 (Shanghai JingXin) in 0.5% NP-40 buffer containing protease inhibitor cocktail, and lysed on ice for 30 min. To determine the acetylation level of endogenous Pgk1, tissue lysates were incubated with the Pgk1 antibody (Santa Cruz) for 1 hr, followed by incubating with Protein-A beads (Upstate) for another 2 hr at 4°C.The Rabbit anti-pan-acetyllysine antibody was generated as previously described . To gene2, 0.25 mM NADH, 2.4 mM ATP, 12 mM 3-phosphoglycerate and 50 μg/ml glyceraldehyde-3-phosphate dehydrogenase in a total volume of 0.3 ml, and assayed at 25°C. By monitoring the reduction of NADH fluorescence , the specific PGK1 activity was measured using HITACHI F-4,600 fluorescence spectrophotometer.Flag-tagged PGK1 protein was overexpressed in HEK293T cells, immumoprecipitated and eluted by 250 mg/ml Flag peptides  dissolved in PBS (pH 7.5). The PGK1 activity assay was carried out as previously described . The reaE. coli as previously described [E. coli strain BL21 (DE3) was transformed with three plasmids, pAcKRS-3, pCDF PylT-1, and pTEV-8-PGK1. Cells were grown overnight in LB containing spectinomycin (50 μg/ml), kanamycin (50 μg/ml), and ampicillin (150 μg/ml) at 37°C till OD600 reached 0.6–0.8. The culture was added with 20 mM NAM and 10 mM N-acetyllysine (Sigma-Aldrich). Protein expression was induced at 37°C by addition of 0.5 mM of isopropyl-1-thio-D-galactopyranoside (IPTG) for 3 hr. Afterward, the cells were harvested, the K220-acetylated PGK1 protein was purified and then stored at −80°C till further analysis.The K220 site-specific acetylated PGK1 was expressed in escribed ,33. In s2. All solutions were thoroughly degassed before being used by centrifugation at 13,000 rpm for 20 min. The experiment was conducted by consecutively injecting 50 μM ADP solution into the calorimetric cell containing 50 μM purified PGK1. The titration enthalpy data was corrected for the small heat changes in control titrations of ADP solution into the dialysis buffer.ITC assay was performed by using a MicroCal VP-ITC type microcalorimeter . Briefly, temperature equilibration was allowed for 1–2 hr till to 20°C prior to the experiment. PGK1 protein and ADP (Sigma) were dialyzed against 40 mM Tris-HCl (pH 7.4), 50 mM KCl, and 10 mM MgClPGK1 knockdown cell pools in HEK293T cells, shRNA targeting PGK1 was constructed, and retrovirus was produced using a two-plasmid packaging system as previously described [PGK1 is 5′-GCTTCTGGGAACAAGGTTAAA-3′. The pMKO.1-puro shRNA construct was co-transfected with vectors expressing the gag and vsvg genes into HEK293T cells. Retroviral supernatant was harvested 36 hr after transfection, and mixed with 8 μg/mL polybrene to increase the infection efficiency. HEK293T cells were infected with the retrovirus and selected in 1 μg/ml puromycin for 1 wk.To generate stable escribed . The shRPGK1 knockdown and put-back stable cell pools in HEK293T cells, two silent nucleotide substitutions were introduced into Flag-tagged human wild-type or K220 mutant PGK1 in the sequence corresponding to the shRNA targeted region. Both shRNA resistant PGK1 were cloned into the retroviral pQCXIH-hygro vector and co-transfected with vectors expressing the gag and vsvg genes in HEK293T cells to produce retrovirus. Retroviral supernatant was harvested 36 hr after transfection, and mixed with 8 μg/mL polybrene to increase the infection efficiency. HEK293T cells with PGK1 knockdown were infected with the retrovirus and selected in 1 μg/ml puromycin and 2 μg/ml hygromycin for 4 wk.To generate + was measured by enzymatic cycling methods as previously described [6. After 24 hr, cells were lysed in 400 μl of extraction buffer  and centrifuged at 1,200 g for 15 min. 150 μl of the supernatant was incubated in a heating block for 30 min at 60°C for NADPH extraction. And then, 20 μl of the cell extract with 160 μl of NADP-cycling buffer  containing 1.3 U of G6PD was added to a 96-well plate. After incubation for 1 min at 30°C in darkness, 20 μl of 10 mM G6P was added to well, and measured the change of absorbance at 570 nm every 30 s for 10 min at 30°C by using SpectraMax M5 Microplate Reader (Molecular Devices). Subtracting NADPH (heated sample) from the total of NADP+ and NADPH (unheated sample) was the level of NADP+.The intracellular NADPH/NADPescribed ,77. Briet test. Almost all data shown represent the results obtained from triplicated independent experiments with standard deviation of the mean (mean ± S.D.). The values of p < 0.05 were considered statistically significant. The numerical data and statistical analysis used in all figures are included in Statistical analyses were performed with a two-tailed unpaired Student's S1 Data(XLSX)Click here for additional data file.S1 FigA) and trichostatin  (B) as indicated. After purification by immunoprecipitation, the acetylation level of ectopically expressed Flag-PGK1 was analyzed by western blot using a pan-anti-acetyllysine antibody (α-Pan-Ac) and normalized against Flag.Flag-PGK1 was overexpressed in HEK293T cells followed by treatment with nicotinamide  ((TIF)Click here for additional data file.S2 FigThe sequence alignment of PGK1 from different species was compared, and the putative acetylated lysine residues of PGK1 identified by MS study were colored in red.(TIF)Click here for additional data file.S3 Figp < 0.01 for the indicated comparison; n.s. = not significant. The numerical data and statistical analysis used in the figures are included in Flag-tagged wild-type PGK1, K75R or K220R mutant were each overexpressed in HEK293T cells, following treatments with or without NAM (10 mM for 4 hr) and TSA (5 μM for 12 hr). Acetylation levels and activity of PGK1 were determined by western blot and enzymatic activity assay, respectively. Relative PGK1 acetylation levels were normalized against Flag. Note that the acetylation levels of wild-type, K75R, and K220R PGK1 in cells treated without or with NAM + TSA (i.e. the α-Pan-Ac bands) were determined on the same gel, while the α-Flag loading control samples were loaded to two separate gels. Shown are average values with standard deviation (S.D.) of triplicated experiments. ** denotes (TIF)Click here for additional data file.S4 Fig(A) Different amounts of acetyl-K220 peptide or unmodified peptide were spotted on nitrocellulose membrane, and the antibody specificity was determined by dot-blot assay. (B) HEK293T cell lysates were separately used for western blot with the α-PGK1 antibody (lane 1), α-acetyl-PGK1 (K220) antibody (lane 2), the α-acetyl-PGK1 (K220) antibody incubated with acetylated peptide (lane 3), or unmodified peptide (lane 4). The image is comprised from four separate western blots. (C) Flag-tagged vector, wild-type, or K220Q/R mutant PGK1 was transfected into HEK293T cells and the acetylation level of each purified protein was measured by western blot using the site-specific α-acetyl-PGK1 (K220) antibody or pre-immune serum.(TIF)Click here for additional data file.S5 FigKAT gene was determined by quantitative real-time PCR at 48 hr post transfection. Shown are average values with standard deviation (S.D.) of triplicated experiments. The numerical data and statistical analysis used in the figures are included in The siRNA oligonucleotide was transiently transfected into HEK293T cells, and the mRNA expression of each (TIF)Click here for additional data file.S6 Fign = 3 per group). At the indicated time points post injection, mouse kidney samples were harvested, and the K220 acetylation levels of endogenous Pgk1 were examined by western blot. The relative Pgk1 K220 acetylation levels were normalized against endogenous Pgk1 protein. The numerical data and statistical analysis used in the figures are included in Insulin (5 U/kg body weight) was intraperitoneally injected into BALB/c wild-type mice ((TIF)Click here for additional data file.S7 FigA) HEK293T cells were treated with insulin at the indicated concentrations for 2 hr, and KAT9 mRNA expression was determined by quantitative real-time PCR. The expression of KAT9 was normalized against β-actin. Shown are average values with standard deviation (S.D.) of triplicated experiments. n.s. = not significant. (B) Flag-tagged PGK1 and/or HA-tagged KAT9 were transiently overexpressed in HEK293T cells, and the cells were then treated without or with insulin . PGK1 protein was purified by IP with Flag beads, following western blot to detect its K220 acetylation level and interaction with KAT9. The numerical data and statistical analysis used in the figures are included in ((TIF)Click here for additional data file.S8 FigHDAC3 mRNA expression was determined by quantitative real-time PCR. The expression of HDAC3 was normalized against β-actin. Shown are average values with standard deviation (S.D.) of triplicated experiments. n.s. = not significant. The numerical data and statistical analysis used in the figures are included in HEK293T cells were treated with insulin at the indicated concentrations for 2 hr, and (TIF)Click here for additional data file.S9 FigHA-tagged proteins of wild-type and HDAC3 S424A mutant were transiently overexpressed in HEK293T cells. Cell extracts were immunoprecipitated and assayed for the histone deacetylase activity of HDAC3 as described in (TIF)Click here for additional data file.S10 FigA, B) Inhibition of PI3K increases the K220 acetylation level of ectopically expressed PGK1 and impairs the protein interaction between ectopic proteins of PGK1 and HDAC3. Flag-tagged PGK1 and HA-tagged HDAC3 were transiently co-overexpressed in HEK293T cells, and then these transfected cells were treated with two PI3K inhibitors, LY294002 (A) or Wortmannin (B), at the indicated concentrations for 4 hr and 2 hr, respectively. PGK1 proteins were purified by Flag beads, following western blot to the detect K220 level acetylation of Flag-PGK1 and its interacting HA-HDAC3.((TIF)Click here for additional data file.S11 FigFlag-tagged PGK1 was ectopically expressed in HEK293T cells treated with EGF (50 μg/L) for the indicated time periods. Endogenous proteins of ERK1/2, S6K, HDAC3 and their phosphorylation were detected by western blotting. PGK1 proteins were purified by Flag beads, and PGK1 K220 acetylation was determined by western blot.(TIF)Click here for additional data file.S12 FigPGK1 were transfected with retroviral plasmids expressing the indicated proteins. PGK1 knockdown efficiency and re-expression were determined by western blot.Stable HEK293T cells with knockdown of endogenous (TIF)Click here for additional data file.S13 FigStable HEK293T cells were treated with rotenone (100 nM) for 6 hr, and then the percentage of viable cells was determined by flow cytometric analysis using Annexin V and propidium iodode (PI) antibodies as described in (TIF)Click here for additional data file.S14 FigPGK1 knocking-down and putting-back using a Seahorse Bioscience XFe 96 analyzer system as described in A). The glycolytic function of rescued cells expressing wild-type PGK1 (blue circle), K220Q mutant PGK1 (red circle), and K220R mutant PGK1 (green circle) was compared (B). The parameters reflecting glycolysis, glycolytic capacity, and glycolytic reserve were determined by calculating Rate Measurement Equation (C). Shown are average values with standard error of the mean (S.E.M.) of triplicated experiments. The numerical data and statistical analysis used in the figures are included in Extracellular acidification rate (ECAR) was determined in HEK293T cells with (TIF)Click here for additional data file.S15 FigA) and glycogen storage levels (B) in the indicated stable HEK293T cells were measured as described in p < 0.01 for the indicated comparisons. The numerical data and statistical analysis used in the figures are included in Relative glucose consumption ((TIF)Click here for additional data file.S16 FigA, B) Stable HEK293T cells were treated with menadione for the indicated concentrations and periods. The protein expression of PARP and p38 MAPK and their phosphorylation were determined by western blot analysis.((TIF)Click here for additional data file.S17 FigStable HEK293T cells were treated with menadione of the indicated concentration for 3 hr. The changes in cell morphology were monitored under the microscope. Scale bars are 200 μm.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 TextFor more details about antibodies, plasmids, cell culture and treatment, immunoprecipitation and western blotting, RNA isolation and quantitative real-time PCR, animal experiments, RNA interference, protein concentration determination, protein expression and purification, pull-down assay, in vitro deacetylation assay, HDAC3 activity assay, measurement of intracellular ATP levels, flow cytometry, liquid chromatography mass spectrometry (LC-MS), extracellular acidification rate (ECAR), glucose depletion assay, glycogen content measurement, measurement of intracellular ROS levels, or cell survival assay, please refer to S1 Text.(DOCX)Click here for additional data file.


Characterizing myocardial infarctions (MIs) on the basis of LGE CMR requires gadolinium infusion, which poses limitations in certain patient populations and imaging workflow. We hypothesized that T1 differences between MI and remote territories at 3T would enable reliable characterization of chronic MI.Canines (n = 29) underwent CMR at 7 days (acute) and 4 months (chronic) following reperfused MIs at 3T (n = 19) and 1.5T (n = 10). Contrast-free T1 maps  and LGE images  were acquired. MI location, size and transmurality were determined using Mean+5SD criterion relative to remote myocardium. T2 maps  were acquired to compare acute and chronic MIs. Commonly used imaging parameters were slice thickness = 6 mm and spatial resolution = 1.3 × 1.3 mm 2. Histological validation was sought to confirm the presence of replacement fibrosis within the chronic infarct zones.Contrast-free T1 maps and LGE images of a representative mid-ventricular slice, along with AHA 17-segment bulls-eye plots depicting the MI size and transmurality acquired from a canine scanned imaged 4 months post-MI at 3T are shown in Figure Contrast-free T1 maps at 3T can determine the location, size and transmurality of chronic MIs with high diagnostic accuracy.This work was supported in parts by grants from National Heart, Lung and Blood Institute (HL091989) and American Heart Association (SDG 0735099N).


Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of  Staphylococcus aureus is an important human pathogen causing skin infections and a variety of life-threatening diseases such as pneumonia, sepsis, and toxic shock syndrome. Previous study showed that the growth of S. aureus in abscesses is suppressed by the host antimicrobial protein calprotectin, which sequesters Zn and Mn from bacterial usage. During bacterial infection, calprotectin also plays an important role in the production of proinflammatory cytokines. Although the antimicrobial activity of calprotectin has been well defined, it is not known how the proinflammatory property of calprotectin affects staphylococcal infection. In this study, we found that the Zn-binding property of calprotectin increases the pathogenic potential of S. aureus by enhancing the activity of the SaeRS two component system in S. aureus. We also found that, under certain infection conditions, the proinflammatory property of calprotectin is rather detrimental to host survival. Our study illustrates that the important antimicrobial protein can be exploited by S. aureus to render the bacterium a more effective pathogen, and provides an example of the intricate tug-of-war between host and a bacterial pathogen. S. aureus is an important Gram-positive human pathogen colonizing the skin, anterior nares and other mucosal surfaces in approximately 30% of the human populations, causing a wide variety of diseases -ATP (2 μCi), the samples were incubated at room temperature for 15 min and subjected to SDS-PAGE (10%) and autoradiography.Maltose-binding protein fused SaeS (MBP-SaeS) was expressed in 32P]-ATP with a Micro Bio-Spin Chromatography Column (Bio-Rad), SaeR (9 μM) and various concentrations (0–0.5 mM) of metal ions were added. The resulting samples were incubated at room temperature for 15 min and subjected to SDS-PAGE (13%) and autoradiography.First, MBP-SaeS was phosphorylated as described above in the reaction buffer. After elimination of free  × 100.5) from wild type or S100A9-/- mice were seeded on 13 mm glass cover slips treated with 0.001% polylysine and allowed to settle and stimulated with 200 nM PMA for 4 h. Cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton X-100, and blocked overnight in PBS containing 10% goat serum, 5% cold water fish gelatin, 1% BSA, and 0.05% Tween 20. CP was detected by treatment with S100A9 antibody  and Cy3-conjugated secondary antibody (Invitrogen), while DNA was detected by DRAQ5 . Specimens were analyzed with Fluoview confocal microscope (Olympus).Neutrophils (2 ×10-/-) mice were acquired from the Skaar laboratory under the permission from University of Muenster. The CP-deficient mice were maintained in house as previously described . The experiments were performed on RNA pooled from 3 mice per group and repeated 3 times.The bacterial cells were harvested from peritoneal fluid as described above at 12 h post infection. Total RNA was isolated from harvested bacterial cells with a FastRNA pro blue kit (MP bio), and contaminating genomic DNA was eliminated with RNase-free DNase set (Qiagen). cDNA was generated from the purified RNA with SuperScript II RT (Invitrogen) and random primers (Applied Biosytems). PCR was performed on ABI PRISM 7000 sequence detection system (Applied Biosystems) using SYBR Green Master Mix (Applied Biosystems) and the primers listed in 5) from wild type or S100A9-/- mice were infected with S. aureus for 2 h (MOI = 10) or left uninfected. Then cells were cultured in RPMI containing gentamicin (50 μg ml-1) for an additional 16 h, and the supernatants were collected. To measure cytokines produced in murine peritoneum, the peritoneal fluids were separated by centrifugation at 200 ×g for 10 min, and the supernatants were collected. Cytokines in the resulting supernatants were quantified with corresponding sandwich ELISA kits by following the manufacturer’s recommendations. The kits for TNF-α, IL-6, IL-1b, IL-17A/F and IFN-γ were purchased from eBioscience, while the MIP-2 (CXCL-2) ELISA kit was purchased from Sigma.To measure cytokines produced from murine neutrophils, neutrophils  test was used. Differences were considered significant when p is smaller than 0.05.Statistical analyses were performed by using the software Prism 5 (GraphPad). For the analyses of SaeQ expression, P1 promoter activity, and cytokine productions, two groups were compared by unpaired, two-tailed Student’s S1 Fig32P]-ATP; then the response regulator SaeR and various concentrations of FeSO4, ZnSO4, and CuSO4 were added. After 15 min incubation, the phosphorylated protein levels were monitored by SDS-PAGE and autoradiography. N, no SaeR.MBP-SaeS was autophosphorylated with [γ-(TIF)Click here for additional data file.S2 FigThree biological replicates for each treatment  were indicated with the same color. The close proximity of Zn-CP treated samples to the samples treated by Zn indicates a dominant Zn effect over CP.(TIF)Click here for additional data file.S3 Fig(A) Schematic diagrams of the reporter plasmids. The arrow indicates the transcription start site. P1, P1 promoter. (B) GFP expression from the negative control  and the P1 reporter plasmid . S. aureus USA300 strains containing the plasmids were grown in RPMI medium for 16 h; then the expression of GFP was analyzed by flow cytometry. (C) Confirmation of the SaeRS repression by Fe and Zn. Cells were grown for 16 h in RPMI containing 400 μM CaCl2, 130 μM FeSO4, 130 μM MnSO4, or 400 μM ZnSO4. Then GFP expression was measured by flow cytometry.(TIF)Click here for additional data file.S4 Fig(A) Effect of CP on the migration of murine neutrophils. Mice were infected with S. aureus USA300 (2×108 cfu) by intraperitoneal injection. At 2 h and 12 h post infection, peritoneal lavage was carried out, and the proportion of neutrophil (B220-Gr-1+CD11b+) was measured by flow cytometry (left panel). The absolute numbers of neutrophil, counted by hemocytometer, was also presented (right panel). Error bars indicate standard error of the mean. (B) The effect of CP on neutrophil extracellular traps (NET) formation. Neutrophils isolated from bone marrow of C57BL/6 (WT) or C57BL/6 S100A9-/- (A9-/-) mice were stimulated with PMA (200 nM) for 4 h and stained with S100A9 antibody (green) and the DNA staining dye DRAQ5 (red). (C) NET-formation was stimulated by either PMA or S. aureus strain USA300 (MOI = 10) for 4 h; then the released DNA was quantified by Picogreen-dsDNA assay. (D) The effect of CP on bactericidal activity of murine neutrophils. Neutrophils were mixed with S. aureus strain USA300 (MOI = 10). At the time points indicated, neutrophils were lysed and spread on a tryptic soy agar. The data are from three pooled mice per genotype and represent three independent experiments.(TIF)Click here for additional data file.S5 Figt-test.Statistical analysis was carried out by unpaired, two-tailed Student’s (TIF)Click here for additional data file.S6 Fig-/- (A9-/-) mice were infected with 1 × 107 cfu of S. aureus USA300 (USA) or the sae deletion mutant (Δsae) via retro-orbital injection. The infected mice were observed for 14 days. The significance of murine survival was assessed by Log-rank (Mantel-Cox) test.C57BL/6 (WT) or C57BL/6 S100A9(TIF)Click here for additional data file.S7 Figfur, a fur transposon mutant, carrying pCL-P1gfp reporter plasmid were grown in RPMI until 0.5 OD600. The cultures were divided into two, and 50 μM FeSO4 was added to one of the cultures. After 4.5 h incubation at 37°C, the resulting cultures (100 μl) were used to measure GFP expression with a microplate reader . The fluorescence was normalized by OD600. WT, USA300; fur, USA300fur. AU, arbitrary unit.USA300 and USA300(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.


MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development.Tauopathies, such as Alzheimer’s disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the  Alzheimer’s disease, progressive supranuclear palsy, corticobasal degeneration and several other neurodegenerative diseases are characterized by the presence of intracellular aggregates of microtubule-associated protein tau leading to their grouping under the name of tauopathies . In the MAPT gene in cases with familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T). So far more than 55 mutations have been identified in MAPT that cause the autosomal dominant disease -derived neurons recapitulate the developmental pattern of brain tau, expressing initially the shortest 3R tau and later, after a few months in culture, both 3R and 4R adult brain tau isoforms. Furthermore, we show that two different mutations in MAPT, N279K and P301L, cause both common and specific phenotypes. In common, they show earlier neuronal maturation and altered anterograde mitochondrial axonal transport. Specifically, the P301L MAPT mutation shows thicker processes with varicosities some of which contain 4R tau and alpha-synuclein and abnormal mitochondrial retrograde transport. The N279K mutation, instead, presents specifically abnormal developmental expression of 4R tau, an imbalance in the 3R:4R tau isoform ratio and AT100+ve hyperphosphorylated tau aggregates. The previously unreported developmental expression of 4R tau in N279K neurons, the varicosity-like structures in the P301L neurons, and the common earlier maturation phenotype may critically contribute to the pathogenesis of frontotemporal dementia with MAPT mutations.Studies of tauopathies have mainly relied on post-mortem human samples or animal models that often do not express all six adult human brain tau isoforms. Mice, for example, only express tau isoforms with 4R in adult brain. Therefore, the identification of a human neuronal system in which to investigate the effects of Approval for ‘Generation of patient-specific stem cells for research in neurodegenerative disorders of the CNS’ was granted by the Hertfordshire Research Ethics Committee (ref n° 09/H0311/88).Handling of human tissue was according to the UK Human Tissue Act 2006. The work on human tissue was covered by Cambridge LREC ethical approval (ref n° 09/40).MAPT gene P301L mutation (https://www.coriell.org/). The characteristics of the subjects providing fibroblasts for IPSC generation are reported in MAPT locus or Ch17 were not directly involved in the genomic alterations, these IPSC lines were removed from further studies (Skin biopsies (3 mm punch biopsies) were obtained from two patients with the mutation A. Fibrob studies .Figure Supplementary Fig. 1).Culture of all hIPSC lines was carried out on irradiated mouse embryonic fibroblasts (MEFs) according to standard protocols . Briefly 0.1 µg/ml colcemid solution (Sigma) was added to each culture for 2 h in growth medium. Pluripotent stem cell colonies were then dissociated into single cells with Accutase  and plated on 0.1% gelatine-coated dishes for 1 h at 37°C, in an incubator with 5% CO2. The IPSC suspension was then collected into tubes and following centrifugation at 1200 rpm for 7 min pellets were resuspended into 5 ml of a 55 mM KCl hypotonic solution, centrifuged again at 1200 rpm for 7 min and fixed with 3:1 methanol:acetic acid before delivery to the Cytogenetics Laboratory for processing.Karyotyping was performed by the Cytogenetics Laboratory at Addenbrooke’s Hospital  (TM substrate (Life Technologies) in mouse embryonic fibroblast-conditioned medium with 10 ng/ml FGF2 (Peprotech) and Rock inhibitor Y-27632 (Tocris). Once cells reached 90% confluence, medium was replaced with N2/B27 medium supplemented with 500 ng/ml mouse Noggin-CF chimera (R&D Systems) and 10 mM SB431542 (Tocris) to initiate neural induction (Day 0). On Day 12, neuroepithelial cells were dissociated with dispase (Sigma) and seeded on laminin-coated 6-well plates in N2/B7 medium. The resulting monolayer of neural precursor cells, forming tube-like rosette structures, was passaged up to three times for expansion. To enhance neuronal maturation, on Day 20, neural precursor cells were exposed to 10 µM Notch inhibitor  for 96 h. Neurons were then maintained up to 200 days with a fresh medium change every other day. βIII-tubulin staining showed that more than 90% of cells in the cultures were neurons in both control and MAPT mutant IPSC lines. Neuronal identity was further confirmed by staining with neuronal markers, electrophysiology and RNA-seq .Differentiation of human IPSCs into cortical neurons was carried out using a protocol adapted from Total RNA was extracted from IPSC lines at different time points during neuronal differentiation using the RNeasy® mini kit (Qiagen) according to the manufacturer’s protocol and used for both semiquantitative and quantitative PCR. Three repeat (3R) and 4R tau mRNA expression were detected by semi-quantitative RT-PCR using the One step RT-PCR kit (Qiagen) with the following primers: forward 5′-AAGTCGCCGTCTTCCGCCAAG-3′; reverse 5′-GTCCAGGGACCCAATCTTCGA-3′. RT-PCR products were detected on a 1.5% agarose gel: 4R and 3R tau RT-PCR products were 381 and 288 bp, respectively.MAPT was used. For 4R mRNA detection, the FAM ABI probe (ID Hs00902312_m1) that binds to the exon 9–exon 10 junction was used. MAPT mRNA values were normalized to those of human GADPH VIC TaqMan® probe (Applied Biosystems) simultaneously amplified in each sample. Triplicates were performed for each sample.For quantitative PCR, the RNA concentration was determined using a NanoDrop and 1 µg RNA was reverse transcribed to cDNA using SuperScript® First-Strand cDNA Synthesis for quantitative RT-PCR (Life Technologies). Quantitative PCR was performed with 2 × TaqMan® Fast Universal PCR Master Mix, no AmpErase UNG (Life Technologies). For 3R mRNA detection, the FAM ABI probe (ID Hs00902192_m1) that binds to the exon 9–exon 11 junction of The following primary antibodies were used: anti-3R tau , anti-4R tau , phosphorylation-dependent anti-tau antibodies AT8  and AT100 , anti-βIII-tubulin , anti-alpha-synuclein antibody syn1 , anti-β-amyloid precursor protein , anti-Oct-4 antibody , anti-Tra1-60 antibody , anti-SSEA4 antibody , anti-FUS antibody  and anti-TDP-43 antibody . Immunocytochemistry was performed as previously described  from frontal cortex were deparaffinized, rehydrated and endogenous peroxidase activity blocked with methanol/0.3% H2O2. Immunohistochemistry was performed as previously reported (2O2 and sections counterstained with cresyl violet before mounting in DPX (Sigma-Aldrich). For immunofluorescence, following incubation with biotinylated secondary antibody, sections were incubated with fluorescent streptavidin . Paraffin sections from cerebral cortex of two control subjects  as previously described (Escherichia coli (Sigma-Aldrich) in 50 mM Tris-HCl and 5 mM MgCl2 for 3–4 h at 65°C. Proteins were separated by 10% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). Membranes were then probed with polyclonal anti-human tau antiserum  followed by peroxidase-conjugated secondary antibody . Soluble tau was also extracted as described above from the cerebral cortex of one of the patients with the P301L mutation  supplemented with complete protease inhibitor cocktail (Roche). Extracts were separated by 4–12% SDS-PAGE and immunoblotted using anti-MAP2 antibody . As loading control, blots were reprobed with an anti-actin antibody .For MAP2 protein immunoblotting, extracts were produced from control and 2. Time-lapse images of mitochondria were acquired every 6 s for 5 min . For each IPSC line, the proportion of stationary, anterograde and retrograde moving mitochondria were calculated as described previously .Human IPSC-derived neurons from control, P301L and N279K lines at 65 days of differentiation (d65) were labelled with 10 nM MitoTracker Orange (Invitrogen) for 5 min at 37°C. Cells were then washed three times and fresh culture medium was added. Mitochondrial dynamics along the neuronal processes were recorded with a 63× oil immersion objective using with a Hamamatsu EM CCD C9100 camera (Leica AF7000). The environment was controlled with a heated stage set at 37°C and 5% CO+ glyconate as internal solution for the electrodes. Cultures were superfused with HEPES-buffered external solution bubbled with medical oxygen consisting of 144 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1 mM NaH2PO4, 2.5 mM CaCl2, 10 mM glucose, pH 7.35 (adjusted with NaOH), with fresh addition of 2 mM MgCl2 prior to the recordings. In some experiments, 1 μM tetrodotoxin (TTX) was applied to block voltage-gated sodium channels and neuronal activity. Recordings were corrected for −14 mV junction potential and data were analysed using Clampfit 10.2 and Sigma Plot 11. Following completion of the recordings, coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature and further immunostained for either βIII-tubulin  or Neurofilament . Two-way ANOVA and Holm-Sidak post hoc tests were used for statistical analysis (Graph Pad Prism).Whole-cell current- and voltage-clamp recordings  were performed as previously described  at Day 65. Paired-end barcoded Illumina RNA libraries were prepared using the standard Illumina protocol and sequenced on the Illumina HiSeq 2000. In addition, RNA-seq expression data were downloaded from human normal brain temporal lobe mRNA and normal total brain RNA , according to the manufacturer’s instructions.Genomic DNA was extracted from each IPSC line with QIAamp DNA mini kit (Qiagen) according to the manufacturer’s protocol. Genetic analysis of H1 and H2 haplotypes SNP genotyping for rs9468, tagging the Supplementary Fig. 3).In control and P301L cells, 3R tau mRNA was present from Day 1 of neuronal differentiation (d1) while 4R tau mRNA appeared at Day 30 d30, B. N279K Tau protein detected by immunocytochemistry was present in all neurons at Day 30, 3R tau being found in cell bodies and neuronal processes D. HoweveSupplementary Fig. 4), this would be in agreement with the immunocytochemistry data indicating 4R tau expression at Day 55. It is possible that in blots where it is not visible the abundant 3R tau band masks the weaker 4R tau band. In N279K neurons bands corresponding to 3R and 4R tau isoforms were clearly visible at Day 55 . At Day 55, in control and P301L neurons the embryonic 0N3R tau isoform was strongly expressed, in some experiments a band corresponding to 0N4R tau could be clearly seen   H. Howevent pulse M, with tnt pulse I and E–G neurons C, that wP < 0.007 and P < 0.001, respectively)  A and C. ctively) B.FigureMorphological differences were evident between lines, although both N279K and P301L neurons had some thicker processes than control neurons. P301L neuronal processes were more contorted with varicosity-like structures appearing after Day 55, stained by βIII-tubulin, some containing 3R and 4R tau D and F. Alpha-synuclein aggregates in Parkinson’s disease A where iSupplementary Fig. 5).Immunohistochemistry for the amyloid precursor protein  performeMAPT mutant neurons  compared to controls (35.49% ± 4.78)  B. HoweveMAPT mutations mature faster and are more excitable than neurons from control subjects. Similarly, neurons from mice overexpressing human P301L tau  or tau mRNA splicing (N279K). P301L neurons are specifically characterized by abnormal varicosity-like structures, sometimes containing alpha-synuclein, amyloid precursor protein accumulation and deficits in retrograde axonal transport. Indeed, the absence of a retrograde axonal transport deficit in the N279K neurons could partially account for the finding of alpha-synuclein and amyloid precursor protein accumulation specifically in neurons with the P301L mutation. Interestingly, the varicosity-like structures in the P301L mutant neurons were also found in the brain of one of the patients from whom the P301L neurons were derived  and otheMAPT splicing mutations are associated with earlier developmental expression of 4R tau.The N279K neurons instead are characterized by premature developmental expression of 4R tau, changes in 3R:4R isoform ratio and AT100 staining of hyperphosphorylated tau, suggestive of tau aggregation possibly related to early 4R tau expression. The novel finding of abnormal developmental expression of 4R tau, together with the imbalance of 3R:4R tau, could critically contribute to FTDP-17T, adding a developmental twist to this complex disorder. Future studies will need to investigate whether other MAPT mutations cause common and specific phenotypes will improve the understanding of disease mechanisms prompting the search for new therapies for tauopathies.Our finding that the six adult tau isoforms can be expressed in human IPSC-derived neurons paves the way towards investigating their specific functions and contribution to dysfunction. Moreover, it is now feasible to co-culture IPSC-derived neurons with glial cells to clarify whether and how astrocytes, microglia and oligodendrocytes contribute to development of tau pathology. Our demonstration that


Here, we constructed fifteen TEVp5M variants with one or more of six rare arginine codons in the coding sequence replaced with abundant E. coli arginine codons. These codon variants expressed in either E. coli BL21 (DE3) or Rossetta (DE3) showed different solubility and activity. Supply of rare tRNAs increased the tendency of certain codon variants to form insoluble aggregates at early induction stage, as determined by the fused S-tag. About 32% increase in soluble protein production of M5 variant with four synonymously mutated arginine codons was identified in Rossetta (DE3) cells using GFP fusion reporter, comparable to that of TEVp5M. After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp5M on cleaving the native or modified recognition sequence incorporated between GST and E. coli diaminopropionate ammonialyase by enzyme-coupled assay, whereas purified M5 variant showed activity similar to the TEVp5M. Supply of rare tRNAs caused the decrease of activity of TEVp5M and M5 by about 21%. Our results revealed that engineering of highly soluble TEVp variants can be achieved by the combined mutations of amino acid residues and optimization of specific rare codons, whereas simple augment of rare tRNAs abundance resulted in partial loss of activity.It has been identified that a TEV protease (TEVp) variant, TEVp Escherichia coli is fundamentally important in the manufacture of a wide range of biotechnological and biomedical products, owing to its easy culture, fast growth, simple fermentation and easy genetic manipulation E. coli is a combination of several factors such as efficiency of transcription, mRNA stability, mRNA folding, codon usage, protein solubility and folding As the host, E. coli, tRNA levels for rare codons AGG, AGA, AUA, CCC, CUA and GGA restrain high-level production of the recombinant proteins E. coliIn E. coli is limited due to its low solubility 5M) with five mutated amino acid residues that displayed improved protein solubility and thermo-stability Fusion tags are frequently applied for improving protein solubility and facilitating rapid purification by affinity chromatography. To avoid potential interference with biological activity or crystallization, the fused tags need to be removed, which is often achieved by a specific protease that recognizes and cleaves the engineered sequence between the tag and target protein E. coli BL21(DE3) 5M.There are six rare arginine codons  in the TEVp coding sequence. With the assistance of solubility enhancer maltose-binding protein (MBP), a TEVp variant with the synonymous mutations of rare codons for R49 and R50 is found to display an enhanced soluble production in E. coli strains DH5α, BL21(DE3), BL21(DE3)plysS and Rosetta(DE3), the plasmids pETDuet-1 and pET-22b, and the FRETWorks S-tag Assay Kit were purchased from Novagen . Reagents used in plasmid construction, protein expression, and site-directed mutagenesis were bought from Takara . The affinity matrix nickel-nitrilotriacetic acid (Ni–NTA) superflow is a product of Qiagen . The YM-10 membrane was supplied by Amicon (USA). Pyridoxal 5′-phosphate and DL-α,β-diaminopropionate (DL-DAP) were from Sigma (USA). DNA sequencing was performed by Invitrogen .5M, pET28-GFP and pGST-DAL were constructed previously 5M coding sequence were conducted by PCR amplification, using pET28-TEVp5M as the template. The primers were listed in supplementary Nco I and Xho I, and inserted into Nco I/Sal I sites of pET28-GFP plasmid. The mutated gene encoding M3 or M10 variant was also inserted into Nco I/Xho I sites of pETDuet-1 vector. The inserted gene in pGST-DAL plasmid encodes the fusion protein GST-tevS1-DAL containing His6-tagged GST, a natural TEVp cleavage site and E. coli diaminopropionate ammonia-lyase (DAL). Two amino acid residues glycine and serine in the TEVp recognition sequence ENLYFQ-GS were replaced by aspartic acid and glycine. The fusion protein containing the modified TEVp recognition sequence was named as GST-tevS2-DAL. The sequence ENLYFQ-DG is weakly recognized by TEVp The plasmids such as pET28-TEVpE. coli BL21(DE3) cells expressing the TEVp5M or codon variant were cultured overnight at 37°C in 5 ml of Luria–Bertani (LB) medium, diluted to 5-fold and grown at 37°C. When OD600 reached about 0.5, cells were induced by 0.5 mM of IPTG. After culturing at 28°C for 12 h, cells in 5 ml of LB medium were collected by centrifugation and disrupted in 0.8 ml of buffer A . Pellets were washed three times with buffer A, solubilized by 0.2 ml of 8 M urea, and centrifuged to remove the precipitants. About 40 µg and 10 µg of proteins in supernatants and pellets respectively were analyzed by SDS-PAGE or Western blotting using mouse anti-His6 antibody. Protein concentration was determined by Coomassie brilliant blue G250, using bovine serum albumin as standard. The absorption of 8 M urea solution was applied as control and subtracted.Except where noted, production and extraction of recombinant proteins in this study were conducted as follows. E. coli BL21(DE3) or Rosseta (DE3) cells. Three colonies were selected randomly and the fluorescence of cells, soluble fractions and pellets were measured by an F-4500 fluorescence spectrometer . Excitation and emission was conducted at 488 nm and 515 nm. Cells harboring pET28-TEVp5M were used as the control and cell fluorescence was subtracted.Recombinant codon variant or that fused with the bacterially codon-optimized emerald green fluorescent protein (EmGFP) were expressed in either 5M or selected codon variant were overexpressed in E. coli BL21(DE3) plysS cells and induced for 10, 20, 30, 40, and 50 min respectively at 37°C. Soluble protein production was determined by measuring the fused S-tag amount, according to Novagen's S•Tag System protocol. The reaction mixture contained 20 µl of FRET assay buffer, 2 µl of FRET ArUAA substrate, 5 µl of S-tag grade S-protein, and 153 µl of sterile deionized water. Reactions were initiated by adding 20 µl of soluble fraction, kept 5 min in the dark, and quenched immediately by adding 20 µl of stop solution. The solution was diluted to 2 ml and recorded fluorescence value at excitation and emission wavelengths of 495–530 nm with an F-4500 fluorescence spectrometer The S-tagged TEVpE. coli cells overexpressing the TEVp variant, or fusion protein as TEVp substrate were collected, re-suspended in buffer B , sonicated and centrifuged. Recombinant proteins in supernatants were purified by Ni-NTA resin according to QIAexpressionist protocol. After cleavage of purified two fusion proteins by TEVp5M, the DAL was released from Ni-NTA using buffer B. Purified proteins were concentrated and exchanged with buffer A.5M and the three codon variants for cleaving modified sequence was analyzed after reacting at 25°C for 6 h. Purified GST-tevS2-DAL and TEVp construct were mixed with the mass ratio of 10∶1 5M in soluble fraction or purified form at 100°C for 5 min was applied as the control and subtracted.Activity of purified DAL as the fusion partner or without the fusion tag was measured using DL-α,-β-diaminopropionate as a substrate to react for 5 min at 37°C. Amount of pyruvate was measured with 2,4-dinitrophenylhydrazine. Absorbance at 520 nm was recorded in a U-2001 spectrometer  5M is more soluble than the TEVpS219V variant 5M coding sequence converting them to abundant arginine codons found in E. coli. The codon variants constructed include M1–M4 with one or two rare arginine codons being replaced with the optimized arginine codons, and M5–M15, which combined to the mutations in M1–M4 with additional codons , one CUA (leucine), one CCC and three GGA (glycine), present in the TEVp coding sequence (https://www.addgene.org/8827/sequences/). E. coli strain Rosetta (DE3) harbors the plasmid pRARE that supplies tRNAs for all of the six rare codons. To detect effect of rare tRNAs on enhancing protein expression level, we analyzed protein solubility of six codon variants in Rosetta (DE3). These variants displayed different soluble expression levels in BL21(DE3). As a result, soluble expression levels of all six variants tested were higher in Rosetta (DE3) than in BL21(DE3) , it became less fluorescent than M5 in Rosseta (DE3), indicating the rare tRNAs may preferably promote M5 experssion. The variant M5 contains four synonymously mutated codons at R49, R50, R101 and R105. About 32% increase in soluble yield of M5 variant was determined in Rossetta (DE3), compared to 15% in the BL21(DE3). Similarly, several other codon variants expressed as insoluble aggregates also emitted stronger fluorescence by co-expressing rare tRNAs.In addition to rare AGA and AGG codons for arginine, there are other four rare codons in L21(DE3) . Compari5M, M3, and M10 were analyzed following initial induction. One arginine codon at R80 was synonymously mutated in M3, and another mutated arginine codon at R159 was combined in M10. The soluble expression levels of the two codon variants were affected by simple augmentation of rare tRNAs, based on cell fluorescence using GFP reporter (E. coli strain BL21(DE3)plysS to inhibit background expression. The maximum expression levels of soluble TEVp5M, M3 and M10 were detected after induction at 37°C at 20 min, 10 min, and 30 min respectively. With induction time prolonged more than 30 min, soluble amounts of all three TEVp constructs were decreased (E. coli Rosetta (DE3), their soluble expression levels were all decreased after 10 min induction  showed slightly higher activity than TEVpn TEVp5M . Co-exprn TEVp5M . The difSDS-PAGE . Among t5M were selected for further purification. The GST-tevS1-DAL coupled assay showed that activity of purified variants M1 and M2 from E. coli BL21(DE3) was slightly less than that of the TEVp5M, while M5 displayed similar activity as TEVp5M  and obtained about 80 mg pure protein from 1L LB culture E. coli is affected by several factors including rare codons, transcriptional and translational rates, mRNA structure and stability. In this study, we investigated the possible effect of codon frequency to the production of active TEVp5M, using a combination of six rare arginine codons to generate a collection of fifteen variants with identical protein sequences. When all six rare arginine codons were substituted with abundant codons, the variant M15 was expressed mainly as inclusion bodies, as detected by SDS-PAGE and Western blotting. This partly explained why the codon-optimized TEVpS219V was not produced at high yield in E. coli.Previously, we have overexpressed three TEVp variants including the TEVpS219V in E. coli. However, quantitative protein amount in pellets could not be assessed reliably by this method, owing to the effect of target protein on GFP folding. A more sensitive approach such as using split GFP reporter To detect protein folding in vivo, we applied EmGFP reporter since it emits stronger fluorescence than other variants Because GFP requires a long lag phase (95 min) to form the chromophore E. coli, improvement of enzymatic activity is more desirable. The current study demonstrated that the co-expressed rare tRNAs can enhance soluble protein production, but not efficiently improve protein quality. Recently, it was proposed that screening codon variants is an effective approach to augmenting soluble expression level E. coli strains displayed higher activity in the crude extracts but less activity in the purified form than TEVp5M. In contrast, M5 showed similar activity to TEVp5M in the purified form, but higher activity in the crude extract. Therefore, it is essential to evaluate protein production and quality of the codon variants by analyzing the activity in supernatants and purified form.Even though expression levels of certain codon variants were elevated in 5M. Supply of rare tRNAs increased soluble production, but concomitantly caused partial activity loss for certain codon variants. The correlation between in vivo protein expression, solubility and folding, and cleavage efficiency provided a consistent view of the effect of synonymously mutations on the soluble production and function of the sixteen TEVp constructs.In conclusion, we confirmed that the synonymous rare-to-abundant arginine codon substitutions and tRNA abundance affected the active production and folding of TEVpFigure S15M and fifteen codon variants.SDS-PAGE analysis of the fusion protein GST-tevS1-DAL cleaved by soluble TEVp Proteins were overexpressed in either BL21(DE3) (A) or Rosseta (DE3) (B). His6-tagged GST-tevS1-DAL and cleaved products were indicated by arrows. Released DAL with glycine as the first amino acid residue was labeled as G-DAL. The His6-tagged GST with partial TEVp recognition sequence was labeled as GST.(TIF)Click here for additional data file.Figure S25M and three codon variants detected by SDS-PAGE.Cleavage of two protein substrates by purified TEVp The protease mutants were purified from E. coli BL21(DE3) for cleaving GST-tevS1-DAL (A) or GST-tevS2-DAL (B). The cleavage of two proteins substrates by purified variants from Rosseta (DE3) were also displayed (C and D). Protein substrate, cleaved products and TEVp constructs were indicated by arrows. After cleavage of GST-tevS2-DAL, released DAL with aspartic acid as the first amino acid residue was labeled as D-DAL.(TIF)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)β1 to induce activation, in the presence or absence of active vitamin D 2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)DOur results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis. Heart failure (HF) represents a growing health concern worldwide, with overall incidence rates of 1-2%, and 12% of individuals over 80 years [Increased fibrosis and aberrant wound healing response resolution are well characterized pathophysiological hallmarks of HF. Strategies to limit the persistent pro-fibrotic response observed throughout compensatory cardiac remodeling in HF may provide novel therapeutic strategies to stem the burden of this condition. Cardiac fibroblasts are the most numerous cell type in the heart. They contribute to extracellular matrix (ECM) development and maintenance through collagen synthesis and remodeling, providing the structural, mechanical and electrical integrity essential for the efficient translation of cardiac myocyte contraction into cardiac output. Their role in preservation of cardiac structure in both health and disease is essential for maintenance of end organ perfusion throughout the body. The critical role of cardiac fibroblasts in preserving cardiac function in response to injury highlights their potential as an attractive therapeutic target in efforts to modulate fibrosis in the setting of HF. In settings of injury, fibroblasts are activated, and undergo transformation to myofibroblasts, the latter being cell populations characterized by their increased synthetic and contractile properties. This fibrotic response is essential in maintaining the structure of the heart and preserving cardiac function in response to injury, however unresolved fibrotic remodeling, can lead to increased residual interstitial fibrosis and result in myocardial stiffness, imperfect electrical propagation and myocyte disarray. Transforming growth factor (TGF) β is a potent activator of fibroblasts, known to induce myofibroblastic activation and induce increased collagen deposition and wound contraction . TGFβ’s in vitro, and that vitamin D status would correlate with extent of interstitial cardiac fibrosis in patients with end stage HF. A wealth of data exist on the pro-fibrotic milieu present in myocardial remodeling, and targeting these progressive remodeling processes will be essential to attenuation of HF burden.Vitamin D, an endogenously produced hormone, has garnered increasing attention for its potential role in cardiovascular (CV) health , 10. CurPatients evaluated for cardiac transplantation for end stage HF at St. Paul’s Hospital, Vancouver, British Columbia between January 2005 and August 2009 were approached, and those providing written consent were enrolled in the study. Pre-transplant blood serum samples were collected from patients on the transplant list and myocardial samples were obtained from explanted native hearts at the time of transplantation. Twenty-three patients had serum samples collected within one month of transplantation, and myocardial tissue available for analysis, and were selected for inclusion. Subject demographics are presented in 2D3 were assayed by the Clinical Laboratory at St. Paul's Hospital  using an API 5000 System mass spectrometer  connected to a 20AD Dual Binary UFLC . 100μL of patient serum was precipitated with 350μL of a 95:5 acetonitrile:methanol solution containing 36ng/mL 25-hydroxy vitamin—D3-26,26,26,27,27,27-d6 internal standard. 2 MRM's were monitored for VitD3 and VitD2, 1 MRM was monitored for the internal standard. Quantitation was performed using a 1 point calibration curve for each MRM, linear-through-zero. Precision evaluation for this assay was performed using a modification of the CLSI EP-5A2 protocol. Quintuplicate analysis of pooled human samples (n = 20–30 per level) with clinically relevant levels were analyzed over 5 days. Within-run coefficient of variation (CV), between-run CV and total CV for 25(OH)D3 at 64.8 nmol/L was 3.7%, 0.4% and 3.7% respectively; at 119.1 nmol/L was 2.1%, 2.8% and 3.5% respectively. The within-run, between-run and total CV for 25(OH)D2 at 35.8 nmol/L was 3.7%, 5.8% and 6.9%, respectively.Circulating levels of 25(OH)Formalin fixed paraffin embedded (FFPE) transmural lateral left ventricular free wall (LVFW) myocardial samples were taken from explanted HF hearts and sectioned at 4 microns. Sections were deparaffinized and stained with hematoxylin and eosin for evaluation of general morphology, and picrosirius red for collagen staining and quantification of fibrosis.Collagen area of picrosirius red stained sections was quantified by colour segmentation using ImagePRO Plus . Briefly, 5 random fields per case were taken at X20 magnification using a digital spot camera . Images were captured by a single observer blinded to the vitamin D status of each patient, collagen area was calculated as a percentage of tissue area per field, excluding non-tissue area and blood vessels. Sections were evaluated by an experienced cardiac pathologist to exclude infarct and peri-infarct areas, and to ensure all measures represented interstitial fibrosis.3 2D3)  was reconstituted in 95% ethanol. All experiments used Transforming Growth Factor (TGF)β1  at 10ng/ml and 1,25(OH)2D3 at 1μM. 95% ethanol was used as vehicle control. Protein was extracted at 24 and 48 hours following treatment.Commercially available primary adult ventricular human cardiac fibroblasts (HCF-av) were used for all experiments . Cells were maintained in a humidified incubator at 37°C with 5% CO2 and used for experiments between passages 3–7 at 70% confluence. Cells were cultured on poly-L-lysine coated culture plates in fibroblast medium (FM) (Sciencell) containing 10% FBS, growth supplements and penicillin/streptomycin, as per manufacturer’s instructions. 1,25 dihydroxyvitamin DCells cultured in chamber slides were fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% triton X-100 for 20 minutes, and blocked for 30 minutes with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). F-actin was visualized using a 1:500 dilution of phalloidin-Alexa fluor 594 , incubated for 30 minutes at room temperature. Cells were coverslipped with VectaShield mounting medium containing DAPI  and images were captured using a Leica AOBS SP2 confocal microscope as described previously . Images Total protein was extracted from treated HCF-av cells as described previously , separat4 cells per well in treatment media containing 0.5mg/mL type I collagen solution . The collagen cell suspension was mixed by pipetting, and 1mL total volume per well was plated. Collagen gel contraction was monitored over 96 hours, and gels were imaged at 24, 48, 72 and 96 hour time points. Gel areas were calculated from digital images using Image-Pro Plus , and contraction calculated as a percentage of surface area relative to untreated control gels.Collagen gel contraction assays were performed as described previously by our group . In brie4 cells per well in 96-well plates. After 24 hours, media was replaced with fresh treatment media containing TGFβ1 at 10ng/mL, 1,25(OH)2D3 at 1μM, or both. Cells were incubated for 48 hours and then labeled with BrdU for 24 hours. BrdU incorporation was evaluated using the Cell Proliferation Assay Kit  as per manufacturer’s directions. Absorbance was read at 450nm using a VersaMax microplate reader .Cellular proliferation was assayed using 5-bromo-2-deoxyuridine (BrdU) incorporation. HCF-av were seeded at a density of 1 × 102D3 and myocardial fibrosis, BrdU absorbance, and Western blot densitometry data were analyzed ANOVA with post hoc Bonferroni multiple comparison correction. Collagen gel contraction assays were assessed using two-way ANOVA with Bonferroni post tests. Categorical data for all experiments were compared by the chi-square test. For all experiments, significance was set at P<0.05.Statistical analysis was performed using GraphPad Prism version 5.01 for Windows . Serum 25(OH)in vitro experiments were fibroblasts by Western blot for discoidin domain receptor 2 (DDR2). Western blot for VDR was used to confirm primary cardiac fibroblast expression of the vitamin D receptor. To confirm the validity of our model system, we examined whether our HCF-av cells expressed a functional VDR. CYP24 is a well known VDR target gene, which is upregulated in the presence of the VDR ligand 1,25(OH)2D3. Treatment of HCF-av cells with 1,25(OH)2D3 resulted in upregulation of CYP24, consistent with previous biomedical literature on vitamin D signaling 2D3 to determine whether treatment with active vitamin D could abrogate TGFβ1-mediated myofibroblast activation. TGFβ1 treatment resulted in significant upregulation of αSMA expression (p<0.05) at 48 hours following treatment. Co-treatment of cells with 1,25(OH)2D3 reduced αSMA levels to baseline (p<0.001) 2D3 demonstrated a reduction in stress fiber staining intensity in the presence of active vitamin D3 (TGFβp<0.001) . Myofibrtamin D3 .in vitro, mediated by the contractile phenotype of activated myofibroblasts. To examine whether vitamin D can act to inhibit these functional effects we performed a collagen gel contraction assay. Fibroblast contraction, as measured by this model, was significantly increased in cells treated with TGFβ1 at all timepoints. Concurrent treatment with TGFβ1 + 1,25(OH)2D3 reduced collagen gel contraction to untreated control levels throughout the 96 hour time course (p<0.05) 2D3  . To dete5(OH)2D3  suggesti1 signals via SMAD dependent and independent pathways. We investigated whether vitamin D abrogated the SMAD dependent pathway by evaluating differences in SMAD2 phosphorylation in the presence of active vitamin D by Western blot. Treatment of cells with TGFβ1 significantly increased pSMAD2 levels in HCF-av cells both 24 and 48 hours after treatment (2D3 significantly reduced pSMAD2 levels at both timepoints (p<0.001 relative to TGFβ1). Our results indicate that active vitamin D can significantly inhibit TGFβ1-mediated SMAD2 phosphorylation and in this way diminish TGFβ1 downstream signaling events and gene transcription.TGFβreatment  (p<0.0002D3 (2D3 levels <50nmol/L) had significantly higher levels of cardiac fibrosis than patients with circulating levels above 50nmol/L (p<0.05). There was no difference in fibrotic area between patients with insufficient vitamin D levels (50-74nmol/L) and those with vitamin D sufficiency (≥75nmol/L), suggesting that there is a threshold level above which additional benefit is not observed.Vitamin D status and area of interstitial fibrosis in explanted hearts was evaluated in 23 end-stage HF patients summarized in 2D3 . The datin vitro data indicate that vitamin D may inhibit TGFβ1-mediated SMAD2 phosphorylation, and can reduce both biochemical and functional measures of myofibroblastic activation.In this study we present mechanistic evidence of an anti-fibrotic effect of vitamin D in human cardiac fibroblasts and have shown that circulating vitamin D levels are negatively associated with myocardial fibrosis in end-stage HF. Our 2D3. We induced myofibroblastic activation of primary cardiac fibroblasts with TGFβ1, and demonstrated the expected increases in protein expression of αSMA, matrix remodeling proteins, and associated augmentation of contractile function measured using a gel contraction assay. These characteristic myofibroblastic features were diminished in the presence of 1,25(OH)2D3. Epidemiological data suggests a protective role for vitamin D in the setting of HF, however the direct effects of circulating vitamin on cardiac structure or function remain unknown. In light of our in vitro data we evaluated explanted end stage HF myocardial tissue to determine whether we could observe any alterations in fibrotic remodeling in relation to vitamin D status. Histological evaluation of interstitial fibrosis demonstrated an inverse relationship between circulating levels of the vitamin D intermediate 25(OH)D3 and extent of fibrotic remodeling.The role of vitamin D in the human heart has remained poorly defined, although the presence of vitamin D signaling machinery within the human myocardium implies a direct role for this hormone in cardiac physiology. We identified expression of a functional VDR in primary human cardiac fibroblasts, as demonstrated by upregulation of its known target gene CYP24 in the presence of the VDR ligand 1,25(OH)Evidence linking vitamin D to cardiovascular health has accumulated in recent years: numerous epidemiological studies report deficiency as a significant cardiovascular disease (CVD) risk factor, and, as well, vitamin D levels are inversely correlated with known CVD risk factors and with incidence of overt CVD , 24, 25.1 in these cells [Data from rodent models of myocardial injury suggest active vitamin D can modulate critical remodeling processes, including cardiac hypertrophy  and extrse cells , 30. An se cells . A recense cells . A similse cells . Our worse cells . Other wse cells  thus actse cells , 37, 38.1 signaling other than αSMA. It is possible other regulatory mechanisms are involved in the expression of these proteins. Further, we did not elucidate the precise mechanism involved in the anti-fibrotic effects of vitamin D and more studies will be required in this regard. Our observational data regarding fibrosis in end-stage HF does not provide evidence of a causative role of vitamin D, particularly given the fluctuations in vitamin D that can occur seasonally and in response to lifestyle changes, and the extended time frame involved in myocardial remodeling. It is possible that chronic vitamin D deficiency may promote a pro-fibrotic remodeling program. In this light, while our data contribute to an existing understanding vitamin D's role in the CV system, continued work will be required to better define the complex role this hormone plays in health and disease.This study is limited in a number of aspects. Notably we did not observe significant downregulation of matrix remodeling proteins associated with TGFβMyocardial fibrosis is a critical contributor to progressive remodeling and the development of HF. Currently there are no available therapeutic agents which target this process . GrowingS1 File(ZIP)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus  CDC2 is an essential gene that encodes the only CDK essential for mitotic cell cycle progression. However, the wheat scab fungus F. graminearum contains two CDC2 orthologs. The cdc2A and cdc2B deletion mutants had no defects in vegetative growth but deletion of both is lethal. Whereas the cdc2B mutant was normal, the cdc2A mutant was almost non-pathogenic, indicating that only Cdc2A is essential in infectious hyphae. Cdc2A and Cdc2B differ in subcellular localization and only localization of Cdc2A to the nucleus was increased in cells active in mitosis. Furthermore, F. graminearum uniquely has two orthologs of Ipl1 Aurora kinase and mutants deleted of orthologs of five essential yeast mitotic kinase genes were viable. However, most of these mutants were significantly reduced in virulence. Overall, our data indicate that F. graminearum differs from the model fungi in CDK and other key mitotic kinase genes, and cell cycle regulation is different between vegetative and infectious hyphae. This is the first report on two Cdc2 kinases in fungi and they differ in subcellular localization and functions during sexual reproduction and plant infection.In the model yeasts and filamentous fungi,  Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are two model organisms used extensively for cell cycle studies [CDC2 or CDC28) that is required for all cell cycle transitions [cdc2 or cdc28 null mutant is non-viable [In eukaryotic cells, progression through mitosis and cell cycle is governed by a complex regulatory system. The central components of this system are the cyclin-dependent kinases (CDKs) , 2 that  studies . Both ofnsitions , 4, 6. Tn-viable , 8. Othen-viable . In addin-viable –12.S. pombe. In S. cerevisiae, at least four M-cyclins (Clb1-Clb4) are required to orderly activate Cdc28 for progression into mitosis [S. cerevisiae has a single essential CAK gene CAK1, the fission yeast employs two partially redundant CAK systems, the Mcs6-Mcs2 complex and Csk1, to activate Cdc2 [S. cerevisiae but Csk1 is not essential for Cdc2 activity in S. pombe. KIN28, the ortholog of S. pombe msc6, lacks CAK activity in the budding yeast.Studies in the budding and fission yeasts have provided insights into the common cell cycle control mechanisms for eukaryotes. Nevertheless, distinctive features and unusual mechanisms of mitosis and cell cycle regulation also are apparent in the two yeast species , 5. For clins Clb-Clb4 areclins Clb-Clb4 areate Cdc2 , 14. CakS. cerevisiae or S. pombe are present in filamentous ascomycetes [Aspergillus nidulans and Neurospora crassa have a single CDC2 ortholog that regulates cell cycle progression and is essential for growth [Fusarium graminearum, a causal agent of wheat and barley head blight disease, contains two putative CDC2 orthologs that were named CDC2A and CDC2B here. Interestingly, only the cdc2A mutant was defective in sexual reproduction and plant infection [CDC2 orthologs in F. graminearum, in this study we examined the expression profiles and subcellular localizations of Cdc2A and Cdc2B in different growth and infection stages. Cdc2A and Cdc2B differed in subcellular localization and only Cdc2A is required for cell cycle regulation in infectious hyphae and asci. Functional studies suggested that both the N- and C-terminal regions of Cdc2A are important for its function during ascosporogenesis and pathogenesis but only the N-terminal region is responsible for its subcellular localization. Furthermore, other pivotal mitotic kinases of F. graminearum, such as CAK and Aurora kinases, also have distinctive features in comparison with those of the budding and fission yeasts. Overall, our data indicate that F. graminearum differs from the model yeasts and filamentous fungi in CDK and other key mitotic kinase genes, and cell cycle regulation is different between vegetative and infectious hyphae. Cdc2A but not Cdc2B plays a stage-specific role in regulating cell cycle progression during ascospore formation and plant infection.Most of the mitotic protein kinase genes identified in omycetes , 16. Simr growth , 18. Intr growth , we founnfection . To furtF. graminearum has two putative CDC2 orthologs that were named CDC2A (FGSG_08468) and CDC2B (FGSG_03132) in this study. The Cdc2A and Cdc2B proteins share 78% identity in amino acid sequences and both have typical CDK structural components, including the PSTAIRE helix, a critical cyclin interface, and the activation T-loop, as well as the inhibitory and activating phosphorylation sites .Synteny analysis showed that the a reesei , Metarhicdc2A and cdc2B deletion mutants were generated by the split-marker approach in a previous study of the F. graminearum kinome [CDC2A and CDC2B, three independent deletion mutants , the cdc2A mutant only caused limited stalk rot at the inoculation sites  examination. On wheat lemma surfaces, the cdc2A mutant had no obvious defects in the formation of penetration branches  and CDC2B1-142-CDC2A143-325-GFP (CDC2B/2A-GFP) chimeric constructs by replacement of the N- or C-terminal regions of CDC2A with those of CDC2B  was comparable to that of Cdc2B in the cdc2A/CDC2A-GFP transformant (C2A-N4). However, the inhibitory phosphorylation level of Cdc2A was obviously higher than that of Cdc2B , the inhibitory phosphorylation of Cdc2A and Cdc2B was completely blocked  was identified in F. graminearum. In yeast two-hybrid assays, FgCak1 interacted with both Cdc2A and Cdc2B , the expression of all three Cdc2-cyclin genes was down-regulated by 3 days-post-fertilization (dpf) but became up-regulated after 4 dpf . Recently, it has been shown that horizontal transfer of genes is prevalent in Fusarium species [CDKs are the central regulators of eukaryotic cell cycle. Although the model yeasts and filamentous fungi used for cell cycle studies have only one mitotic CDK responsible for regulating cell cycle transitions, hylogeny . However2A clade , indicat species , 34.Fusarium species  and FGSG_02399 (named IPL1B)  . In mammike gene . In mammAurora-B . In F. g residue . It is pCDC2A and CDC2B indicate that F. graminearum may differ from S. cerevisiae and other model fungi in the regulatory mechanism of mitotic cell cycle. Consistent with this observation, F. graminearum has two putative Aurora kinase genes that regulate chromatid segregation. Furthermore, we found that a number of putative mitotic kinase genes also may differ in functions between F. graminearum and S. cerevisiae. Similar to CDC2 and CAK1, the DBF2/DBF20, CDC15, MEC1, and RAD53 genes are essential in S. cerevisiae. In F. graminearum, the Fgdbf2, Fgcdc15 and Fgmec1 mutants were viable but significantly reduced in virulence [KIN3 ortholog was essential in F. graminearum but the yeast kin3 deletion mutant is viable [F. graminearum differs from the model yeasts in the function of several mitotic kinase genes. Like in other plant pathogens [F. graminearum.Overall, our studies with irulence . The KINs viable . These oathogens –45, cellF. graminearum and its derived transformants used in this study are listed in 5 spores/ml and used to inoculate flowering wheat heads of cultivar Xiaoyan22 or Norm and stalks of 8-week-old corn plants of cv. Pioneer 2375 as described [The wild-type strain PH-1 of escribed . Wheat sescribed  with minCDC2A and CDC2B gene replacement constructs were generated with the split-marker approach using the neomycin resistance gene (NEOR) as the selectable marker. Protoplasts of the cdc2A and cdc2B mutants were prepared and transformed with the CDC2B and CDC2A gene replacement constructs as described previously [The eviously , 39. Foreviously .CDC2A, CDC2B, and FgCAK1 were confirmed by DNA sequencing and transformed into protoplasts of the corresponding mutants. The resulting transformants harboring the fusion construct were identified by PCR and further confirmed by the presence of GFP signals. GFP signals in conidia, germ tubes, ascospores and infectious hyphae were observed with a BX51 Olympus epifluorescence microscope or an Olympus FV1000 confocal microscope. Nuclei were stained with Hoechst 33258 .To generate the GFP fusion constructs, genomic fragments containing the promoter and coding region of the target genes were amplified and cloned into pFL2 vector by the yeast gap repair approach . The resCDC2A/B and CDC2B/A chimeric alleles under the control of the CDC2A promoter by replacement of the N- or C-terminal regions of CDC2A with those of CDC2B by the yeast GAP repair approach [CDC2A/B-GFP and CDC2B/A-GFP constructs were verified by sequencing analysis and transformed into the cdc2A mutant. The resulting cdc2A CDC2A/2B or cdc2A CDC2B/2A transformants were confirmed by PCR and observation of GFP signals.Based on the sequence alignment of Cdc2 orthologs , we diviapproach , 68, resF. graminearum conidia (105 spores/ml). After inoculation, the seedlings were grown at 25°C in a growth chamber with 95% humidity and examined at 48 or 72 hpi with a BX51 Olympus epifluorescence microscope. The experiments were repeated at least 3 times. In each experiment, we examined at least ten samples for each strain.For penetration branches observation, glumes and lemmas were collected from inoculated wheat spikelets 24 h post-inoculation (hpi), and fixed with 4% glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) overnight at 4°C . After dCDC2A and CDC2B were assayed by qRT-PCR with the FgTUB2 beta-tubulin gene of F. graminearum [RNA samples were isolated from germlings, vegetative hyphae, mating cultures, and infected wheat heads with the TRIzol reagent  as described . First-sminearum  as the iCDC2A and CDC2B were amplified from first-strand cDNA of PH-1 synthesized as described [FgCAK1 was amplified and cloned into pGADT7 as the prey construct. The resulting bait and prey vectors were co-transformed in pairs into yeast strain AH109 (Clontech). To check for auto-activation, the bait construct of CDC2A and CDC2B were co-transformed with empty pGADT7 vector. The resulting transformants were then assayed for growth on synthetic dropout (SD) medium lacking tryptophan, leucine, and histidine (SD-Trp-Leu-His) and β-galactosidase activities as described [The interactions of FgCak1 with Cdc2A and Cdc2B were assayed with the Matchmaker yeast two-hybrid system . The ORFs of escribed  and clonescribed .CDC2A- and CDC2B-GFP fusion constructs were generated by cloning genomic fragments containing the open reading frame (ORF) of these two genes into pFL2 by the yeast gap repair approach [FgCAK1 and CDC2B genes using the PFL7 vector. The resulting fusion constructs were confirmed by sequencing analysis and transformed in pairs into PH-1. The expression of both transforming vectors was analyzed by PCR and western blot analysis. Total proteins were isolated from strains expressing both transforming constructs and incubated with anti-Flag M2 beads  as described [The approach , 68. Simescribed . Westernescribed .Total proteins were isolated in the presence of phosphatase inhibitor cocktail (Sigma-Aldrich) from 18 h germlings grown in liquid complete medium (CM) cultures, separated on a 12.5% SDS-PAGE, and transferred to nitrocellulose membranes as described . For weswww.ncbi.nlm.nih.gov/tools/cobalt) and manually modified. The analysis of type I and type II functional divergence was performed with the Diverge 3.0 software [Multiple alignments of protein sequences were constructed with COBALT .Cyclin-dependent protein kinase Cdc2A , Cyclin-dependent protein kinase Cdc2B , Cyclin-dependent kinase-activating kinase FgCak1 , Aurora kinase Ipl1A , Aurora kinase Ipl1B , DNA damage response protein kinase FgRad53 , Mitosis entry checkpoint protein kinase FgMec1 , Transcription and stress response protein kinase FgDbf2 , Mitotic exit network protein kinase FgCdc15 , M phase inhibitor protein kinase FgWee1 , three putative Cdc2-cyclins , S1 Fighttp://fastml.tau.ac.il/) was shown above as the reference. Residues different from the reference are highlighted in color. The PSTAIRE helix and the T-loop are key landmark regions of Cdc2. ‘#’ and ‘*’ symbols mark the inhibitory (Y15) and activating (T181) phosphorylation sites, respectively. ‘$’ and ‘%’ symbols indicate amino acid sites with type I (highly conserved in one co-ortholog but variable in the other) and type II (highly conserved within both co-orthologs but diverged between them) functional divergence [The ancestral sequence reconstructed by the FASTML Server (vergence  at p > 0(PDF)Click here for additional data file.S2 FigF. oxysporum has many lineage-specific genomic regions [The phylogenetic tree was constructed with the protein sequences of catalytic domains. Each branch was marked with the p-values of approximate likelihood ratios (SH-aLRT). Branches with p-values less than 0.5 have been collapsed. Scale bar corresponds to 0.1 amino acid substitutions per site.  regions  and is t(PDF)Click here for additional data file.S3 FigCDC2A and CDC2B loci and their 40-kb flanking sequences. Collinear orthologous relationships are represented by gray bands. Fusarium oxysporum shares a high sequence identity with F. verticillioides in this region. (B) Intron positions in the CDC2A and CDC2B genes. Dashed lines mark the homologous region. Fg, Fusarium graminearum; Fo, F. oxysporum; Fv, F. verticillioides; Fs, F. solani; Tr, Trichoderma reesei.(A) Synteny of the (PDF)Click here for additional data file.S4 Fighph) cassette. Probe A and probe B are PCR products amplified with primers 5F/6R and H852/H850, respectively. (B) Southern blots of restriction enzyme-digested genomic DNA of PH-1 (WT) and mutants hybridized with probe A (left) and probe B (right). The restriction enzyme used was marked.(A) The split-marker approach used to replace the target gene with the hygromycin phosphotransferase ((PDF)Click here for additional data file.S5 Figcdc2A and cdc2B mutants to oxidative, hyperosmotic, membrane, and cell wall stresses. Four-day old colonies formed by the wild type (PH-1) and cdc2A (C2A1) or cdc2B (C2B1) deletion mutants on PDA with 0.05% H2O2, 0.7 M NaCl, 0.01% SDS, or 200 mg/ml Congo Red. (B) Hyphal morphology of PH-1 and the cdc2A and cdc2B mutants in trichothecene biosynthesis induction (TBI) liquid culture after 72 h of incubation at 25°C.(A) Responses of the (PDF)Click here for additional data file.S6 FigCDC2A-GFP (C2A-N4) and CDC2B-GFP (C2B-N2) transformants were examined by differential interference contrast (DIC) and confocal microscopy after staining with Hoechst. Bar = 20 μm.Germlings (8 h) of the (PDF)Click here for additional data file.S7 FigPCL8/PCL10-like cyclin FGSG_07737. The phylogenetic tree was constructed with the protein sequences of conserved cyclin domains. Only p-values for the approximate likelihood ratios (SH-aLRT) of >0.5 (50%) are indicated. Scale bars correspond to 0.5 amino acid substitutions per site. Gene name or ID number is indicated. ANID, Aspergillus nidulans; FGSG, Fusarium graminearum; MGG, Magnaporthe oryzae; NCU, Neurospora crassa; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; SS1G, Sclerotinia sclerotiorum. (B) Expression profile of these four cyclins during sexual reproduction (FG5) and infection of barley spikes (FG1). The expression data of RMA treatment were downloaded from Plant Expression Database (PLEXdb) (www.plexdb.org). Mean and standard deviation were calculated from data of three biological replicates. hpf, hours post-fertilization; hpi, hours post-inoculation.(A) The maximum likelihood tree of orthologs of three putative Cdc2-cyclins FGSG_01291, FGSG_07132, and FGSG_00941 and one extra (PDF)Click here for additional data file.S8 FigHomo sapiens; Fg, Fusarium graminearum; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.(A) A phylogenetic tree of fungal Aurora kinases constructed with the kinase domain sequences. Each branch was marked with the p-values of >0.5 of approximate likelihood ratios (SH-aLRT). Scale bar corresponds to 0.2 amino acid substitutions per site. (B) Multiple sequence alignment shows the amino acid replacement at the position equivalent to G198 of human Aurora-A (red box). Hs, (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.