The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3–5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators. Loss of function mutations in the p53 pathway frequently arise during cancer development ,2,3. AppMouse double minute 2 (Mdm2), also known as E3 ubiquitin-protein ligase Mdm2 proto-oncogene, and its homolog Mmd4, also known as MdmX, are negative regulators of p53 ,21,22. MPhosphorylation of p53TAD upon cellular stress leads to increased transcription of its target genes and increased association with its coactivator, CREB binding protein (CBP)/p300 . The CBPDuring phosphorylation the phosphate group contains a double negative charge that affects protein conformation mainly due to the electrostatic effects that occur between the phosphate and surrounding charged atoms of the protein ,48. ThesTo study the effects of phosphorylation on protein structure and function phosphomimetic mutations have been used extensively. Phosphomimetic mutations are amino acid substitutions (Ser/Thr to Asp or Glu and Tyr to Glu) that mimic the effect of a phosphorylated residue ,55,56. TIn this present study, we obtain insights into the role of phosphorylation in modulating interactions of p53 with Mdm2, MdmX, and CBP/KIX and the effect of phosphorylation on transient secondary structure. We assessed the effects of single- and double-site phosphomimetic mutations of p53TAD upon binding to the N-terminal domain of Mdm2, MdmX, and the KIX domain of CBP. Due to the location of S15 and T18 in a region of p53TAD containing transient helical secondary structure and the importance of their phosphorylation for regulation, we engineered p53TAD phosphomimetics, S15D and S15D/T18E, to determine if the phosphomimicry of p53TAD at these sites affects protein-protein binding and changes the levels of transient helical secondary structure. Our data shows that multisite phosphomimicry reduces the binding affinity of p53TAD to Mdm2 and MdmX. In contrast to earlier published results on p53 phosphorylation, single and multisite phosphomimetics of p53TAD have equivalent, small effects on binding with KIX ,43,46. W15N-labeled or 15N- and 13C-labeled samples 1 g/L of 15N-labeled ammonium chloride and/or 0.2% (w/v) 13C-labeled glucose were added in the place of nitrogen and carbon sources .All cell growth experiments were performed in M9 media. In order to make Escherichia coli cells grown in M9 medium. These cultures were induced at an OD600 of 0.6 with 1 mM Isopropyl-β-d-thiogalactopyranoside and grown for 18 h at 15 °C. Cultures were centrifuged at 11,000× g and frozen at −80 °C. Pellets were resuspended in glutathione S-transferase (GST) binding buffer , 0.02% NaN3, 2 mM dithiothreitol (DTT), pH 7.4) containing protease inhibitors  per 2 L of culture and lysed with a French Press pressure cell using a minimum pressure of 20,000 pounds per square inch (psi). The lysate was centrifuged at 38,720× g for 1 h. The supernatant was filtered and then applied to a column containing 25 mL Glutathione Sepharose 4 Fast Flow resin. Protein fractions were eluted with three column volumes of 25 mM Tris Base, 25 mM Tris-HCl, 300 mM NaCl, 2.5 mM EDTA, 0.02% NaN3, 2 mM DTT, pH 7.4 and 10 mM reduced glutathione. Fractions were analyzed using polyacrylamide gel electrophoresis (PAGE) and those fractions containing the protein were combined and dialyzed into 25 mM Tris Base, 25 mM Tris-HCl, 300 mM NaCl, 2.5 mM EDTA, 0.02% NaN3, 2 mM DTT, pH 7.4, and the GST tag was cleaved using a 1:100 ratio of Human Rhinovirus 3C (HRV3C) protease. Samples were then applied to a column containing 25 mL Glutathione Sepharose 4 Fast Flow resin. Fractions were analyzed using PAGE and those fractions containing the protein were combined and dialyzed into gel filtration buffer  containing 2 mM DTT. The constructs were then loaded onto a GE HiLoad 16/60 Superdex 75 column. The column was equilibrated and the protein eluted with gel filtration buffer at a flow rate of 1.5 mL/min. Protein purity was verified using PAGE analysis.Samples of human Mdm2 and MdmX, corresponding to residues 17–125 and 23–111, respectively, were expressed using pGEX-6p-2 vectors in BL21 (DE3) Escherichia coli cells from New England Biolabs  for expression using the heat-shock method then plated on agar plates that contained kanamycin for expression. Single colonies from this transformation were used to inoculate 50 mL cultures of M9 media that were grown overnight. The overnight cultures were then re-inoculated into 2 L of M9 media at an OD600 of 0.04. These cultures were induced at an OD600 of 0.6 with 1 mM Isopropyl-β-d-thiogalactopyranoside and grown for 22 h at 15 °C. Cultures were centrifuged at 11.000× g and frozen at −80 °C. After expression, pellet was suspended in 25 mL of lysis buffer  containing protease inhibitors  per 2 L of culture and lysed with a French Press pressure cell using a minimum pressure of 20,000 psi. The soluble fraction was isolated by centrifugation at 38,720 g for 1 h. The supernatant was filtered and added to a column containing 30 mL of Ni-NTA Superflow resin . All buffers used on the NiNTA column were run at a flow rate of 3 mL/min. The column was washed with two column volumes of lysis buffer and the p53 eluted with three column volumes of elution buffer . Fractions were analyzed using PAGE and those fractions containing the protein were combined and dialyzed into gel filtration buffer  using 3500 Da MWCO dialysis tubing . The p53 protein was then concentrated in an Amicon Ultra-15 3K centrifugal filter device and the HIS-tag was removed by cleaving for 3 h at room temperature for the p53TAD WT (p53TAD) and overnight at room temperature for the other constructs with the Sigma-Aldrich Thrombin CleanCleave Kit (RECOMT) . The completion of the cleavage reaction was verified using PAGE. The cleaved p53 constructs were dialyzed in lysis buffer and then further purified by chromatography on Ni-NTA resin. Fractions were analyzed using PAGE and those fractions containing the protein were combined and dialyzed into gel filtration buffer and the constructs were then loaded onto a GE HiLoad 16/60 Superdex 75 column. The column was equilibrated and the protein eluted with gel filtration buffer at a flow rate of 1.5 mL/min. Protein purity was verified using PAGE analysis.The KIX (586–672) construct was expressed as N-terminal fusions with a 7-His tag. The plasmid was transformed into BL21 (DE3) Escherichia coli cells from New England Biolabs using the heat-shock method then plated on agar plates that contained kanamycin for expression. Single colonies from this transformation were used to inoculate 60 mL cultures of M9 media that were grown overnight. The overnight cultures were then re-inoculated into 2 L of M9 media at an OD600 of 0.04. These cultures were induced at an OD600 of 0.6 with 1 mM Isopropyl-β-d-thiogalactopyranoside and grown for 5 h at 37 °C. Cultures were centrifuged at 11,000× g and frozen at −80°C. After expression, pellets containing unlabeled and double-labeled  p53TAD peptides were suspended in 25 mL of lysis buffer  containing protease inhibitors (Sigma Aldrich) per 2 L of culture and lysed with a French Press pressure cell using a minimum pressure of 20,000 psi. The soluble fraction was isolated by centrifugation at 38,720 g for 1 h. The supernatant was filtered and added to a column containing 30 mL of Ni-NTA Superflow resin . All buffers used on the NiNTA column were run at a flow rate of 3 mL/min. The column was washed with two column volumes of lysis buffer and the p53 eluted with three column volumes of elution buffer . Fractions were analyzed using PAGE and those fractions containing the protein were combined and dialyzed into gel filtration buffer  using 3500Da MWCO dialysis tubing . The p53 protein was then concentrated in an Amicon Ultra-15 3K centrifugal filter device and the HIS-tag was removed by cleaving for 4 h at room temperature for the p53TAD and overnight at room temperature for the other constructs with the Sigma-Aldrich Thrombin CleanCleave Kit (RECOMT) . The completion of the cleavage reaction was verified using PAGE. The cleaved p53 constructs were dialyzed in lysis buffer and then further purified by chromatography on Ni-NTA resin. Fractions were analyzed using PAGE and those fractions containing the protein were combined and dialyzed into gel filtration buffer and the constructs were then loaded onto a GE HiLoad 16/60 Superdex 75 column. The column was equilibrated and the protein eluted with gel filtration buffer at a flow rate of 1.5 mL/min. Protein purity was verified using PAGE analysis.Constructs for S15D (residues 1–73), and S15D/T18E (residues 1–73) were generated by Polymerase chain reaction (PCR) site-directed mutagenesis QuickChange II  using the following primers for S15D d(GTCGAGCCCCCTCTGGATCAGGAAACATTTTC) and d(GAAAATGTTTCCTGATCCAGAGGGGGCTCGAC) and for S15D/T18E d(CGAGCCCCCTCTGGATCAGGAAGAATTTTCAGACCTATGG) and d(CCATAGGTCTGAAAATTCTTCCTGATCCAGAGGGGGCTCG). All p53TAD constructs were expressed as N-terminal fusions with a 7-HIS tag. The samples were then cleaved with Fisher Bioreagents OPTIMIZED  Dpn1, then were transformed into XL1-Blue cells from Agilent Technologies using agar plates that contained kanamycin. The plasmids from the bacterial colonies were isolated using the Thermo Scientific GeneJET Plasmid Miniprep kit. The plasmid samples were sequenced at Eurofins Genomics and then transformed into BL21 (DE3) 2PO4, 150 mM NaCl, 1 mM EDTA, 0.02% NaN3, 8 mM β-mercaptoethanol, pH 6.8. Binding experiments involving KIX were dialyzed against 50 mM Tris, 50 mM NaCl, pH 7.0. Experiments were performed at 25 °C. The typical concentration of p53 constructs (syringe) ranged from 50–500 μM and for Mdm2, MdmX and KIX (cell) 5–50 μM. Peptide concentrations were determined by absorbance at 280 nm. A typical ITC experiment consisted of one injection of 5 μL, followed by 29 injections of 10 μL up to a 2.5-fold molar excess of titrant. Data were analyzed with the Origin70 ITC software from MicroCal. Averages and standard deviations from three different ITC experiments are shown. Integrated ITC data were fit with single-site binding models and the stoichiometry ranged from 0.8 to 1.2. Errors in Kd were calculated from triplicate measurements.Isothermal titration calorimetry (ITC) experiments were performed using a GE MicroCal VP-ITC instrument. Proteins were dialyzed against 50 mM NaH15N- and 13C-labeled samples at 50 μM, at 25 °C on a Varian VNMRS 800 MHz spectrometer equipped with a triple-resonance pulse field Z-axis gradient cold probe. To make the amide 1H and 15N as well as 13Cα, 13Cβ, and 13CO resonance assignments, sensitivity-enhanced 1H–15N heteronuclear single quantum coherence (HSQC) and three-dimensional HNCACB and HNCO experiments were performed on the uniformly 15N- and 13C-labeled samples in 90% H2O/8% D2O, 50 mM NaH2PO4, 50 mM NaCl, 1 mM EDTA, and 0.02% NaN3, pH 6.8. For the HNCO the Varian VNMRS 600 MHz spectrometer with a triple resonance pulse field Z-axis gradient cold probe was used. The sweep widths were 9689.9 (t3) Hz × 3770.1 (t2) Hz × 1944.5 (t1) Hz, and complex data points were 1024 (t3) Hz × 64 (t2) Hz × 32 (t1) Hz. For p53TAD the HSQC and the HNCACB were performed on the 600 MHz spectrometer. The sweep widths and complex points for the HSQC were 7225.4 (t2) Hz × 1500 (t1) Hz and 1024 (t2) Hz × 128 (t1) Hz, respectively. The HNCACB experiment, data were acquired in the 1H, 13C, and 15N dimensions using 7225.4 (t3) Hz × 12063.8 (t2) Hz × 1500 (t1) Hz sweep widths and 1024 (t3) Hz × 128 (t2) Hz × 32 (t1) Hz complex data points. For S15D the HSQC was performed on the 600 MHz spectrometer and the HNCACB was performed on the 800 MHz spectrometer. The sweep widths and complex points for the HSQC were 7266 (t2) Hz × 1943.2 (t1) Hz and 1024 (t2) Hz × 128 (t1) Hz, respectively. The HNCACB experiment, data were acquired in the 1H, 13C, and 15N dimensions using 9689.9 (t3) Hz × 14074.9 (t2) Hz × 1944.3 (t1) Hz sweep widths and 1024 (t3) Hz × 128 (t2) Hz × 32 (t1) Hz complex data points. For S15D/T18E the HSQC and the HNCACB were performed on the 800 MHz spectrometer. The sweep widths and complex points for the HSQC were 9689.9 (t2) Hz × 1944.4 (t1) Hz and 1024 (t2) Hz × 128 (t1) Hz, respectively. The HNCACB experiment, data were acquired in the 1H, 13C, and 15N dimensions using 9689.9 (t3) Hz × 14074.9 (t2) Hz × 1944.3 (t1) Hz sweep widths and 1024 (t3) Hz × 128 (t2) Hz × 32 (t1) Hz complex data points. All NMR spectra were processed with NMRFxProcessor and analyzed using NMRView J software  . Th. Th62]. Secondary chemical shift values were calculated using the prediction of temperature, neighbor and pH-corrected chemical shifts for intrinsically disordered proteins (POTENCI) software . This sod using data from triplicate measurements. Our results showed that p53TAD and S15D bound Mdm2/MdmX with similar affinities, whereas S15D/T18E displayed a 2.5–4.5-fold reduction with Mdm2 and a 5-fold reduction with MdmX  to determine the effect of the phosphomimetic mutations on p53TAD binding to Mdm2 (residues 17–125), MdmX (residues 23–111), excluding the N-terminal “lid” and KIX (residues 586–672)  70,71,7,771,72. ith MdmX A,B. Bindith MdmX . Howeverith MdmX . InteresUsing phosphomimetic mutations we investigated the effect of phosphorylation on the binding of p53 with Mdm2, MdmX, and CBP/KIX and the transient secondary structure of p53TAD. By increasing negative charge of neighboring residues at the positive end (N-terminus) of the helix formed by p53 in the bound state with Mdm2, we expected a stabilization of the helical dipole in accordance with what is seen in antiparallel helix interactions . Our resBinding affinities of the p53TAD phosphomimetics for the Mdm2, MdmX and KIX were determined using ITC . For botd of 11 μM, similar to values found in previous studies [Binding affinity of p53 to KIX was not significantly altered between p53TAD and the phosphomimetics. We found p53TAD binds to KIX with a K studies ,33,46. I studies ,33,43,84


The relevance of subclinical hyperthyroidism in the elderly has not been clearly defined. We studied whether the reported association of low TSH with frailty is an indicator of subclinical hyperthyroidism as assessed by free T3 levels. In a retrospective chart review of patients seen between January 2017 and December 2018 at the Phoenix VA Medical Center, we identified 100 patients aged ≥60 years with at least 2 low TSH measurements (<0.5 µIU/ml) and a free T3 measurement within 6 months of the measured TSH and 50 sex- and age-matched controls (TSH 0.5-5.0 µIU/ml). Patients with exogenous or clinical hyperthyroidism were excluded. We used a deficit accumulation approach evaluating 31 factors, to create a frailty index between 0 and 1 for each patient. The higher the FI, the more likely (p<0.001) it was that patients had expired in the interim. Patients with low (0.31±0.11 µIU/mL) vs. normal (1.84±0.84 µIU/mL) TSH had higher mean FI compared to controls . TSH significantly predicted frailty score (p<0.0001) independent of age. However, lower TSH was not associated with higher free T3 or free T4 levels. There was a nonsignificant inverse association of free T3 levels with FI (P = 0.09), which disappeared when adjusted for age. Similar to prior studies, low TSH predicted frailty. However, neither free T3 nor free T4 predicted low TSH or frailty index, suggesting that the association of low TSH with frailty is not due to subclinical hyperthyroidism, but perhaps to effects of comorbidities on TSH secretion.


Ganoderma fungus due to its low toxicity. As part of our ongoing search for antioxidative constituents from the fruiting bodies of Ganoderma lucidum, the chemical constituents were investigated and seven secondary metabolites, including one new lanostane triterpene (1), two known aromatic meroterpenoids (6–7), and four known triterpenes (2–5), were isolated by a series of chromatographic methods. The structures of the seven compounds were elucidated by spectroscopic techniques. The isolated compounds were tested in vitro for antioxidant potencies and neuroprotective activities against H2O2 and aged Aβ-induced cell death in SH-SY5Y cells. As a result, compounds 1, 6, and 7 exhibited potent antioxidant and neuroprotective activities. Additionally, all isolated compounds were tested for radical scavenging activities. Compounds 6 and 7 showed the comparable free radical scavenging activities with the standard drug in both ABTS ) and ORAC  experiments. The results from this study suggested that G. lucidum and its metabolites  may be potential functional food ingredients for the antioxidation and prevention of neurogenerative diseases.Reactive oxygen/nitrogen species generated in the human body can cause oxidative damage associated with many degenerative diseases such as atherosclerosis, dementia, coronary heart diseases, aging, and cancer. There is a great interest in developing new antioxidants from  Reactive oxygen species (ROS) can cause extensive damage to DNA, proteins, and lipids. This could be the fundamental cause of aging and many other important diseases such as cancer, cardiovascular diseases, and neurogeneration . DepositGanoderma lucidum is known as the “mushroom of immortality” and considered to be a panacea to cure all kinds of diseases in Chinese folklore. As demonstrated by the numerous publications, polysaccharides are important contributors to the antioxidant properties reported for Ganoderma species + at m/z 571.3241  in the high resolution electrospray ionization mass spectroscopy (HRESIMS). The 1H nuclear magnetic resonance (NMR) spectrum , 0.95 (s), 1.04 (s), 1.26 (s), 1.27 (s), and two doublet methyl signals at δ 0.89  and 1.19 . The 13C NMR and distortionless enhancement by polarization transfer (DEPT) spectra . These NMR characteristics were similar to those of methyl ganoderate G  to give four main fractions (Fr.1–Fr.4). Fr.1 (400 g) was subjected to silica gel column chromatography with CH2Cl2/MeOH  and divided into four fractions. The 50:1 fraction was applied to a chromatography column for isolation of 3 (5 mg). The 20:1 fraction was treated by Sephadex LH-20 (MeOH) and divided into three subfractions . Fr.A was applied to a chromatography column for isolation of 2 (8.8 mg). Fr.B was subjected to semipreparative HPLC  and compound 7 ). The 5:1 fraction was also subjected to silica gel column chromatography and three subfractions were obtained . Compound 4 (6.5 mg) and 5 (8.5 mg) were isolated from the 2:1 fraction by repeated silica gel column chromatography (CH2Cl2/MeOH). Reverse phase silica gel  was used to treat the 1:2 fraction to give 1 (20.1 mg).The dried fruiting bodies of 1): [α]25D −90 ; UV (MeOH); λmax (log ε): 253 (4.05) nm. IR (KBr) vmax 3425, 2972, 1720, 1629, 1461 cm-1; 1H and 13C-DEPT data: see m/z 571.3241 .Methyl ganoderate G1 , which was calculated from the log–dose inhibition curve.ABTS radical cation scavenging activity was assayed according to instructions by the Beyotime Institute of Biotechnology. The stock solutions included ABTS solution and oxidant solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 16 h at room temperature in the dark. The solution was then diluted by mixing 1 mL working solution with 90 ml 80% ethanol in order to obtain an absorbance of 0.7 ± 0.05 at 734 nm. A fresh ABTS solution was prepared for each assay. Samples (10 μL) with a concentration range of 0.05–3.00 mM were mixed with 200 μL of fresh ABTS solution and the mixture was left at room temperature for 6 min. The absorbance was then measured at 734 nm. Trolox was used as a reference compound. The radical scavenging activity of each sample was expressed in terms of the ECThe ORAC assay was carried out based on the previously described procedure with slight modification . In brieThe human neuroblastoma cell line (SH-SY5Y) was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. SH-SY5Y cells were cultured in MEM/F12 (1:1) medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2. 1–7 were dissolved in DMSO for use (DMSO in each well was less than 0.1%). Before treatment, 100 μL SH-SY5Y cells were seeded on a 96-well plate at the density of 1 × 105 cells/mL and cultured for 24 h. The medium was changed to 99 μL fresh medium containing 200 μM H2O2, and then cells were treated with 1 μL different concentrations of compounds 1–7 for 12 h. CCK-8 solution (10 μL) was added to every well, and coincubated for 2 h at 37 °C. The absorbance was measured at 450 nm using a TECAN microplate reader.Compounds 1, 6, and 7 were dissolved in DMSO for use (DMSO in each well was less than 0.1%). Before treatment, 100 μL SH-SY5Y cells were seeded on a 96-well plate at the density of 1 × 105 cells/mL and cultured for 24 h. The medium was changed to 99 μL fresh medium containing 25 μM aged Aβ25–35, and then cells were treated with 1 μL different concentrations of compounds 1, 6, and 7 for 24 h. CCK-8 solution (10 μL) was added to every well, and coincubated for 2 h at 37 °C. The absorbance was measured at 450 nm using a Bio-Rad microplate reader.Compounds 4 cells per well). Drug treatment, H2O2 stimulation, and Aβ25–35 stimulation were carried out as described in The generation of intracellular reactive oxygen species (ROS) was measured using the DCFH-DA method . Brieflyp-value <0.05.Data analysis was performed using GraphPad Prism 6.0 software . All results are presented as the mean ± S.D. and a two-tailed test or a one-way analysis of variance (ANOVA) was used to determine the statistical significance. Differences were considered to be significant for


Specifically, a layered type delta manganese dioxide (δ-MnO2) allows reversible insertion/extraction of Zn2+ ions and exhibits high storage capacity of Zn2+ ions. However, a poor conductivity of δ-MnO2, as well as other crystallographic forms, limits its potential applications. This study focuses on δ-MnO2 with nanoflower structure supported on graphite flake, namely MNG, for use as an intercalation host material of rechargeable aqueous ZIBs. Pristine δ-MnO2 nanoflowers and MNG were synthesized and examined using X-ray diffraction, electron spectroscopy, and electrochemical techniques. Also, performances of the batteries with the pristine δ-MnO2 nanoflowers and MNG cathodes were studied in CR2032 coin cells. MNG exhibits a fast insertion/extraction of Zn2+ ions with diffusion scheme and pseudocapacitive behavior. The battery using MNG cathode exhibited a high initial discharge capacity of 235 mAh/g at 200 mA/g specific current density compared to 130 mAh/g which is displayed by the pristine δ-MnO2 cathode at the same specific current density. MNG demonstrated superior electrical conductivity compared to the pristine δ-MnO2. The results obtained pave the way for improving the electrical conductivity of MnO2 by using graphite flake support. The graphite flake support significantly improved performances of ZIBs and made them attractive for use in a wide variety of energy applications.Manganese oxide (MnO MnO2 were previously studied and applied for a variety of energy storage devices7. Also, it was applied in different metal-ion batteries including Li-ion battery (LIB)8, Mg-ion battery (MIB)9 and Zn-ion battery (ZIB)11. MnO2 cathodes are inexpensive and exhibit a high theoretical capacity. Recently, aqueous Zn/MnO2 batteries are considered as promising alternative energy devices due to their high safety and the abundance of both Zn, MnO2 and the electrolyte14.Manganese dioxide , carbon nanofibers (CNFs), graphene and graphite, have been integrated with MnO2. MnO2/graphene nanoflowers were synthesized in the form of sandwich-structured nanoflowers which exhibited excellent super capacitive properties effectively making a very conductive electrode material for high-performance super capacitors22. However, it is significant that MnO2 supported on graphite has not been reported previously in ZIBs application. The crystal structure of graphite consists of parallel planes of carbon atoms which is conductive primarily along its planes24. In this way, graphite is classified as a semimetal due to its high electrical conductivity25. Therefore, MnO2 supported on graphite is considered as the candidate due to an improvement in electronic conductivity and an increase in the stability of the electrode materials for ZIBs.However, MnO2 nanoflower/graphite as a cathode host material for rechargeable aqueous ZIBs. The δ-MnO2 nanoflower supported on graphite not only increases the electrical conductivity and discharge capacity of the battery but also improves the insertion/extraction acceleration by increasing the active area of the δ-MnO2 nanoflower. Accordingly, the electrochemical properties and performances of the batteries which use the MNG as host material cathode are examined and discussed.The present study reports on δ-MnO4), manganese sulfate monohydrate (MnSO4.H2O), zinc sulfate (ZnSO4), sulfuric acid (H2SO4), and cellulose acetate were purchased from Ajax Finechem. Nickel foam  was purchased from Qijing Trading Co., Ltd. Whatman filter paper No.1 was purchased from Sigma-Aldrich. Graphite foil was purchased from Shenzhen 3KS Electronic Material Co. Ltd. Zn sheet (99.99%) was purchased from Sirikul Engineering Ltd., Part.Reagent grade chemicals were obtained and used without further purification unless noted otherwise. The graphite powder was purchased from Aldrich Company. Potassium permanganate (KMnO2 nanoparticles were synthesized by dissolving KMnO4 (1.98 g) in 60 mL of deionized (DI) water. Then, MnSO4.H2O (0.336 g) was dissolved in 20 mL of DI water. Next, the MnSO4.H2O solution was added dropwise to the KMnO4 solution, and continuous stirring followed for 30 min. Afterward, the mixture was transferred into a 100 mL Teflon autoclave and kept at 160 °C for 24 hr in an oil bath. The product was collected and washed with DI water several times. Then, it was dried at 80 °C for 12 hr. The MNG synthesis was similar to the method reported by Liu et al.22 with some modifications. Graphite (1.0 g) was mixed in DI water (500 mL) with KMnO4 (10.0 g). The mixture of graphite and KMnO4 was stirred for 18 hr. Then, 98% of H2SO4 (5 mL) was added dropwise into the mixture. The solution was continuously stirred for 1 hr and heated to and maintained at 80 °C for 1 hr. After that, the solution was diluted in 1 L of DI water and allowed to stand at room temperature. The solution was filtered using cellulose filter paper (pore size 11 µm). Then, the MNG precipitates were collected and washed by DI water several times until the violet color disappeared. Subsequently, the precipitates were dried at 80 °C overnight.The pristine δ-MnOX-ray Diffraction  of the powder samples was carried out with Cu Kα radiation at a scanning range of 5–80°. Field Emission Scanning Electron Microscope  was used to take the morphology image and nanoflower size of MNG.2 was prepared by mixing together 70% wt. of the pristine δ-MnO2, 20% wt. of carbon black (CB), and 10% wt. of cellulose acetate binder. Acetone was used to adjust the viscosity of the slurries. Each mixed slurry was coated on graphite foil using a lab coating machine  and dried at 70 °C under vacuum. The thickness of the cathode material deposited was 25 µm. The zinc anode was prepared by electrodeposition of zinc from ZnSO4 (0.5 M) aqueous solution onto Ni-foam using zinc sheet as a counter electrode at the current density of 60 mA/cm2. The amount of zinc deposited was 20 mg/cm2. Both cathode and anode were punched into a 15 mm diameter disk. The filter paper was punched into a 19 mm disk and used as the separator. Then, 0.3 mL of ZnSO4 (1 M) was added to the cell. The testing cells were fabricated as a coin cell (CR2032).The cathode using MNG was prepared by mixing together 70% wt. of MNG, 20% wt. of carbon black (CB), and 10% wt. of cellulose acetate binder. Alternatively, the cathode using pristine δ-MnO2+/Zn. A battery testing system  was used to investigate the performance of the battery. The charge-transfer resistance, as illustrated by the Nyquist plots for the cathode, was carried out using an Electrochemical Impedance Spectroscopy (EIS) technique using an amplitude of 10 mV in the frequency range of 1–100,000 Hz.Electrochemical measurements were carried out using a CR2032 coin cell. Cyclic Voltammetry (CV) was performed by Potentiostat  at a scan rate of 0.5 mV/s in the voltage range 1.0–1.8 V versus Zn2 nanoflower/graphite (MNG) was prepared by a modified method of Liu et al.22. In Fig. 2 and graphite are shown. Thus, it can be seen that both patterns, i.e. δ-MnO2 (JSPDS card no. 80–1098)27 and graphite (JSPDS card no. 41–1487)28 match well. In the XRD pattern of MNG. The diffraction peaks (2θ) at 12.2°, 26.5°, 36.6°, and 65.6° correspond to the (001), (002), (−111) and (−312) crystal planes of δ-MnO2, respectively29. The high-intensity diffraction peak of MNG at 26.5° indicates the high crystallinity of graphite. δ-MnO2 possesses a planar layered-structure as illustrated by the inset of Fig. 27. Further, the XRD analysis confirmed that the delta (δ) phase was presented in the pristine δ-MnO2 sample  solvated species are formed and lastly (3) Zn2+ ions are reduced to Zn and deposited back on the zinc anode, respectively. The electrochemical reaction may be expressed as in Eq.  state and layered type δ-ZnxMnO2 with Mn(II) state.In Fig. 2+/Zn were measured using CR2032 coin cells. As shown in Fig. 2/activated carbon composite for supercapacitors32. It is noted that the MNG electrode does not present the capacitive behavior of the electrode34. The capacitive effect is characterized by analyzing the cyclic voltammetry data at different sweep rates as in Eq.  and br (reduction process) of the MNG cathode are 0.58 and 0.62, respectively. Since the average b values are close to 0.5, it may imply that the redox reactions on the MNG cathode are controlled by the diffusion process. MnO2 is a transition metal oxide that typically displays the pseudocapacitance behavior37. The capacitive-controlled process occurs only on the surface. However, in the case of MNG cathode, the characteristic of Zn2+ ions insertion/extraction deviates from capacitive-controlled process towards the diffusion-controlled process. That is, the insertion/extraction of Zn2+ ions occur not only on the surface but also the pores inside. The result shows good agreement of a fast Zn2+ ion insertion/extraction or high rate property for the battery35.To investigate the kinetics of the MNG electrode, cyclic voltammograms using sweep rates of 0.5 to 32 mV/s in the voltage range 1.0–1.8 V versus Zn2 is used as a comparable cathode. Figure 2 and MNG electrodes at a scan rate of 0.5 mV/s over the potential range 1.0–1.8 V for the initial three cycles. During the first cycle, two distinct peaks are observed at 1.20 and 1.57 V for the pristine δ-MnO2 electrode and at 1.18 and 1.54 V for MNG. The peaks in the low potential region appear at 1.20 and 1.18 V which can be attributed to Zn2+ ion insertion into the δ-MnO2 host structure. In the higher potential region, two oxidation peaks can be seen at 1.57 and 1.54 V for δ-MnO2 and MNG, respectively, which correspond to the extraction of Zn2+ ions from the δ-MnO2 host structure. The results suggest that, the oxidation state of Mn is reduced to Mn3+ states and is oxidized back to Mn4+ states, during Zn2+ ion insertion and extraction, respectively38. On subsequent cycling, two distinct peaks appear at 1.37 and 1.21 V for Zn2+ ion insertion into δ-MnO2 whereas the peaks at 1.38 and 1.20 V were observed for MNG in the low voltage region. In the high voltage region, the peak at 1.57 and shoulder at 1.62 V can be clearly seen for the pristine δ-MnO2. Likewise, the peak at 1.53 V and shoulder at 1.60 V can be clearly seen for the MNG electrodes. The cyclic voltammogram, having two peaks during discharge and having a peak with shoulder during charge, exhibits typical characteristics of the electrochemical insertion/extraction of Zn2+ ions in MnO2 structure41. These results with two peaks during discharge may be more clearly described by the two-step reaction of Zn2+ ion insertion in electrochemical reaction38 and spinel-type ZnMn2O4 transformation33. In the following scan cycles, the peaks at 1.37 V for δ-MnO2 and at 1.38 for MNG increase gradually during discharge indicating an activation process38. The CV curve of MNG exhibits a higher peak intensity and a larger enclosed area when compared with the pristine δ-MnO2 indicating improved electrochemical performance and fast Zn2+ ion insertion/extraction42.In order to compare the improved electrochemical properties of MNG as the cathode for ZIB, the pristine δ-MnO2 and MNG cathode in a coin cell battery when cycled at a specific current density of 200 mA/g in the potential range of 1.0–1.8 V. The battery fabricated with the pristine δ-MnO2 and MNG host cathode material under open air condition displays an open-circuit voltage (OCV) of about 1.4 V. The first discharge capacity for MNG is 235 mAh/g whereas the pristine δ-MnO2 registers only 130 mAh/g. Compared to todorokite-type MnO214 and δ-MnO2 nano-flake13, the initial discharge capacity can deliver only 98 mAh/g (at 50 mA/g) and 122 mAh/g (at 83 mA/g), respectively. It is clear that δ-MnO2 supported on graphite samples can accommodate more numbers of Zn2+ ions than the unsupported δ-MnO2 (pristine δ-MnO2). It appears that the nanoflower δ-MnO2 in the structure of MNG tend to enhance the electrode/electrolyte contact area, thereby favoring Zn2+ ion insertion41. In addition, MNG shows a longer horizontal discharge curve than that of δ-MnO2, suggesting a more stable Zn2+ ion insertion into the MNG than in the pristine δ-MnO2. On the subsequent cycle, during the continuous discharge, the voltage profiles present two distinct plateaus at 1.45 and 1.25 V for MNG and 1.4 and 1.2 V for δ-MnO2. These characteristics were also observed for MnO2 electrodes in aqueous ZIB systems43. It can be implied that Zn2+ ions can insert into the layered δ-MnO239 which is in agreement with the two distinct peaks during discharge, as shown in Fig. Figure 2 and MNG electrodes, under the specific current density of 400 mA/g. At the 100th cycle, the discharge and charge capacities registered by δ-MnO2 were 63.3 and 63.8 mAh/g, respectively while that of MNG were 113.4 and 114.2 mAh/g, respectively. The lower capacity retention of δ-MnO2 may result from low intrinsic electronic conductivity because of the appearance of unstable Mn3+ states during zinc-ion insertion10. The higher capacity retention of MNG may indicate that the electronic conductivity of MNG is improved. In the initial cycles, gradual capacity fade was observed for the pristine δ-MnO2 electrode, but fast capacity fade was observed for the MNG electrode. The formation of ZnMn2O4 with the Jahn-Teller Mn3+ ion may contribute to electrode degradation and hence lead to the fast capacity fade13. However, MNG demonstrates higher capacity than the pristine δ-MnO2. Over long-term cycling, the coulombic efficiency of both host material electrodes is maintained at around 100%. Thus, this clearly demonstrates that no irreversible capacity losses occurred41.Figure 2 and MNG host material cathodes are shown. Cycling takes place at various specific current densities of 200, 400, 800 and 1600 mA/g, namely 5 times for each rate. The rate performance of MNG is significantly higher than those of the pristine δ-MnO2. It is indicated that nanoscale morphology of δ-MnO2 nanoflowers on graphite increases the contact area between the electrode and the electrolyte and provides more electrochemically active sites for ion-insertion13. Graphite not only improves the electronic conductivity of the MNG electrode but also tends to disperse the δ-MnO2 nanoflower sites. The MNG cathode can be charged and discharged at different rates; a high rate of 1600 mA/g leads to a discharge and charge capacity of 76 and 64 mAh/g, respectively. When cycled at a specific current density of 200 mA/g, the MNG cathode can deliver a discharge and charge capacity of 181 and 179 mAh/g, respectively. This behavior indicates that the MNG cathode can well be considered for the Zn2+ ion storage material10. It is clear therefore that MNG can improve not only the cycling performance but also the rate performance for ZIBs.In Fig. 2 and MNG host cathodes was examined using EIS. The curves of the pristine δ-MnO2 and MNG host cathodes consist of depressed semicircles and diffusion drift which can be perfectly fitted using Randles equivalent circuit . The result indicates that the electrical conductivity of the MNG sample is improved by constructing a conductive support using the graphite. The relationship between real impedance (Z′) and angular frequency (ω) in the low frequency region can be expressed accordingly by Eq.  indicating that the diffusion coefficient of MNG is higher than that of the pristine δ-MnO2, as in Eq. . After the 100th cycle, the discharge capacity registered by the δ-MnO2 was only 63.3 mAh/g whereas the MNG delivered a discharge capacity of 113.4 mAh/g. Moreover, the conductivity of the supporting graphite improved the conductivity by reducing the charge transfer resistance of the cathode materials. The present results concluded that the supporting graphite not only improved the electrical conductivity but also enhanced the specific capacity and the cycling performance of the pristine δ-MnO2. Thus, MNG is one of promising candidate cathode materials for ZIBs.In summary, δ-MnOSupplementary Information


Correction to: BMC Microbiolhttps://doi.org/10.1186/s12866-016-0629-3Following publication of the original article , the autIn the published version panels 5 and 6 of the Fig. 6G are similar, but the nuclease assays contained two different nucleotide cofactors. During assembly and handling of the images for this figure, the same image, albeit with different contrast, was used unintentionally for panels 5 and 6. This image corresponded to panel 6.The assay addressed the effect of ADP  and ATPγS  on the nuclease activity of HpMutS2 on Holliday junction substrate. In absence of cofactors , HpMutS2 degrades Holliday junction at 26.49 ± 3.95 pM min-1 (Table 3). The addition of ADP and ATPγS reduced the nuclease activity of HpMutS2 by ~ 1.5-fold, resulting in the cleavage rates of 18.85 ± 1.9 pM min-1 and 17.44 ± 0.84 pM min-1, respectively.The quantification of the assays shown in Fig. 6D were derived from the correct gel images and therefore Fig. 6D and DNA cleavage rates presented in Table 3 remains unchanged.Corrected Figure ^6 is presented below:


V2O5 nanofibers are synthesized via colloidal route and characterized by XRD, SEM, EDX, and TEM techniques. In order to prepare PDMS-g-PUR, linear aliphatic PUR having one –COOH functional group (PUR-COOH) is synthesized and grafted onto aminopropyl functionalized PDMS by EDC/HCl coupling reaction. PUR-COOH and PDMS-g-PUR are characterized by 1H NMR, FTIR. PDMS-g-PUR/V2O5 nanofiber composites are prepared and characterized by DSC/TGA, FTIR, and tensile tests. The self-healing ability of PDMS-graft-PUR and composites are determined by mechanical tests and optical microscope. Tensile strength data obtained from mechanical tests show that healing efficiencies of PDMS-g-PUR increase with healing time and reach 85.4 ± 1.2 % after waiting 120 min at 50 °C. The addition of V2O5 nanofibers enhances the mechanical properties and healing efficiency of the PDMS-g-PUR. An increase of healing efficiency and max tensile strength from 85.4 ± 1.2% to 95.3 ± 0.4% and 113.08 ± 5.24 kPa to 1443.40 ± 8.96 kPa is observed after the addition of 10 wt % V2O5 nanofiber into the polymer.The self-healing ability can be imparted to the polymers by different mechanisms. In this study, self-healing polydimethylsiloxane-graft-polyurethane (PDMS- In recent years, self-healing polydimethylsiloxane (PDMS) elastomers became one of the most appealing polymers as biomedical devices, flexible electronics, and actuators due to their good chemical stability, biocompatibility, and flexibility ,5,6,7,8.In supramolecular systems; the non-covalent interactions such as π-π interaction ,14,15,163O4 particles at different amounts, were incorporated into the elastomer by Skov and coworkers. Due to the presence of magnetic Fe3O4 particles, inductive heat was generated with an application of alternative magnetic field, which improved the self-healing properties of the copolymer significantly without any change in the mechanical properties  (AP-PDMS) was obtained from Gelest . All chemicals except PEG were used as received. PEG was purified by azeotropic distillation in toluene and stored under vacuum at 50 °C before each polymer synthesis. Hexamethylene diisocyanate (HMDI), polyethylene glycol (PEG) (2O5) nanofibers were synthesized by a colloidal solution method as described in the literature [2O5 colloidal solution was prepared by dissolving 1 g of ammonium metavanadate in 200 mL deionized water and adding 10 g of acidic ion-exchange resin  into the solution. The colloidal solution was stirred for 10 min at 80 °C and cooled slowly (≈0.5 °C/min) to room temperature. After the resin was filtered, the solution was dried at room temperature. Subsequently, the dried nanofiber cluster was calcined at 150 °C in an air atmosphere under current humidity conditions to form a stable V2O5 phase.Vanadium pentoxide (Vterature . BrieflyIn order the synthesize polyurethane having one –COOH end group (PUR-COOH), linear aliphatic polyurethane having one –OH group (PUR-OH) was synthesized by step growth polymerization using DBTDL as a catalyst and then a –OH functional group was converted to a –COOH group using citric acid as illustrated in 2O  and 66.7 mmol APPDMS was dissolved in THF at room temperature, separately. Then, the solutions were mixed and stirred in a three-necked round bottom flask under reflux at room temperature for 1 h. Then, 0.3 mmol PUR-COOH was added into the reaction mixture. The mixture was stirred continuously at 60 °C until the polymer precipitated at the bottom of the flask. The gel like polymer was separated from the solution and washed with THF. The synthesized graft copolymer (PDMS-g-PUR) was dried in a vacuum oven at 40 °C for 48 h. The grafting of PUR-COOH onto AP-PDMS was accomplished through carbodiimide coupling reaction using water soluble EDC. HCl as shown in g-PUR/V2O5 composites, 5 g PDMS-g-PUR was dissolved in 100 mL DMSO at 60 °C for 48 h. V2O5 nanofibers were dispersed in 100 mL of DMSO  were poured into a Petri dish and then dried in a vacuum oven at 60 °C for 24 h. The dried samples were purified with acetone in a Soxhlet apparatus for 2 h, to remove the residual DMSO in the PDMS-g-PUR/V2O5 composites. All PDMS-g-PUR/V2O5 composites were dried at 40 °C for 12 h under vacuum. Afterwards, the PDMS-g-PUR/V2O5 supramolecular polymer composites were stored in a desiccator under argon until further characterization.To prepare PDMS- of DMSO  using a 2O5 nanofibers and residual phase such as VO and amorphous phases were investigated by XRD analysis. The evaluation of phases was compared for V2O5 nanofibers dried at room temperature and calcined at 150 °C. Powdered specimens were scanned from 2q = 10–90°, at a scanning rate of 2°/min, using a Rigaku DMax 2200  diffractometer  at 40 kV and 40 mA. SEM and EDX analysis were performed to the calcined V2O5 nanofibers. A Phillips XL30 SFEG  scanning electron microscope was used for microstructural examination of samples with Secondary Electron Images, SEI, used predominantly, and distribution of elements identified with an energy dispersive X-ray spectrometer, EDX . Representative SEM images and spectrum were presented. Calcined V2O5 nanofibers were dispersed in methanol and a drop of the mixture was placed onto carbon coated copper grids to analyze the formation by TEM  analyses. The EDX detector of the TEM analyzer was used to verify the presence of elements.The V1H NMR (a Varian NMRS-500 nuclear magnetic resonance instrument (500 MHz), West Sussex, UK) were used for chemical analyses of PUR-COOH and PDMS-g-PUR. FTIR analysis was performed between 4000–400 cm−1 at room temperature with an ATR set-up. 1H NMR spectra were recorded using deuterated DMSO or chloroform (CDCl3) as a solvent. Differential scanning calorimetric (DSC) analyses were carried out to obtain glass transition temperatures (Tg) of pristine polymer and polymer composites. A DSC-TA instrument Q200  was used under a nitrogen environment and the samples (each ~5 mg) were scanned from −80 °C to 100 °C with 10 °C/min ramp rate. Thermogravimetric analysis (TGA) were performed with a TGA-TA instrument 50  under elevated temperature and nitrogen environment with a flow rate of 40 mL/min. An Instron  5567 model universal electromechanical test machine was used to measure the mechanical properties of the pristine polymer and polymer composites as well as those that had been damaged and subsequently healed. The crosshead speed was 5 mm/min and a 100 N load cell was used. Six dog-bone shape specimens were tested based on ASTM 638 Type V standard for each composition. Mean values obtained from tested six specimens were reported.Gel Permeation Chromatography (GPC) was used to determine the molecular weight of PUR-COOH. GPC experiments were carried out in THF at room temperature with 1 mL/min flow rate using an Agilent 1100 GPC . The number average molecular weight and polydispersity index of PUR-COOH was 6011 g/mol and 2.39 respectively. Fourier-transform infrared spectroscopy  and pristineσ and healedσ are the tensile strength of pristine and healed samples at the rupture. In addition, the healing behavior of the damaged regions was visualized via optical microscope  imaging at 50 °C (achieved with the use of a heating apparatus) as they underwent self-healing at 50 °C (with heating performed using a heating apparatus).Healing experiments were carried out at 50 °C. The samples were cut through the center with a sharp zirconia blade and then the pieces were brought together. This process was repeated three times and the change in the percentage of healing was examined after each time. In addition, the change of strength of healed samples was monitored as a function of time. After performing a tensile test on pristine and healed samples, the healing efficiency was calculated, which was defined as follows:2O5 nanofibers. The obtained V2O5 nanofibers were characterized by XRD, SEM, and TEM analyses. The presence of V2O5 phases at different temperatures was studied by XRD technique. 2O5 nanofibers as a function of temperature. No considerable diffraction peak is observed in the pattern of V2O5 dried at room temperature  was synthesized by a step polymerization reaction using DBTDL as a catalyst in THF. 1H NMR spectrum of PUR-OH. The –OH functional group was then converted to –COOH group using citric acid to enable the synthesis of copolymer through EDC coupling reaction [In order to prepare the PDMS-reaction . 1H NMR analyses were performed. −1 and 3120 cm−1 are due to O–H and N–H stretching and can be attributed to urethane linkage [−1 and 2866 cm−1, C=O stretching at 1698 cm−1 and 1626 cm−1 vibrational bands also confirm the PUR formation [To investigate the chemical composition of PUR-COOH in detail, FTIR and  linkage . In addiormation ,33,47. 1H NMR spectrum of PUR-COOH and confirms that the PUR chains have a –COOH end group. This analysis was carried out in deuterated DMSO to observe the –NH linkage more clearly. Both 1H NMR peaks obtained from the DMSO and residual H2O inside the DMSO can be seen from the spectra. The peak at 7.06 (c) ppm can be assigned to –NH which belongs to a urethane linkage. In addition, the peaks at 4.01 (e) ppm, 3.56 (e) ppm and 3.47 (e) ppm can be attributed to the polyethylene glycol monomer. The peak at 12.30 ppm shown with g corresponds to COOH groups. Thus, the results obtained from NMR spectra show consistency with FTIR results and indicate that the PUR-COOH synthesis was successful.g-PUR, the PUR-COOH was grafted onto aminopropyl functionalized PDMS by an EDC coupling reaction. To show the success of the grafting, the comparison between the FTIR and 1H NMR spectra of PDMS and PDMS-g-PUR are given in −1 corresponding to an increase in N–H linkage in the polymeric system is seen after grafting. Three peaks between 1500–1700 cm−1 due to C=O and N–H stretching also appeared after graft polymer formation. The 1H NMR spectra of PDMS and PDMS-g-PUR are also compared, the peak at around 3.5 ppm, which is a characteristic peak corresponding to PEG groups in polyurethanes structure is observed in PDMS-g-PUR‘s spectrum. The peak near 0 ppm can be assigned to the H atoms in the –Si–O–CH3 group in the grafted polymer.To synthesize the PDMS-g-PUR polymer was reinforced with V2O5 nanofibers to enhance the mechanical properties, and the effect of V2O5 nanofibers on the healing efficiency was investigated. Changes in Tg via DSC curves, thermal stability via TG analysis, mechanical properties, and healing properties via tensile test and the optical microscope were determined. g-PUR/V2O5 composites. Results of PDMS-g-PUR/V2O5 composites were compared with the result of PDMS-g-PUR to determine the effect of V2O5 on the polymers’ glass transition temperature (Tg). The Tg values of neat polymer and polymer composites are given in Tgs were detected at −53.1 °C and −12.5 °C for the PDMS-g-PUR polymer. It is observed that with a 10 wt % V2O5 nanofiber addition to the polymeric system, the Tg at −12.5 °C disappeared and Tg at −53.2 °C increased to −31.4 °C with a 41% increment. When the percentage of reinforcement further increased, the change in the Tg was slowed down and this can be explained by the maximum loading capacity of PDMS-g-PUR of V2O5 [PDMS- of V2O5 . g-PUR and PDMS-g-PUR/V2O5 composites are given in 2O5 addition, polymer composite decomposition temperature increases to 150 °C and 20 wt % of the polymer composite degrades at 400 °C. The thermal stability of the polymer increases after 10 wt % V2O5 addition due to the restriction of chain mobility [2O5 nanofibers increases, improvement in the thermal stability of PDMS-g-PUR/V2O5 polymer composites is observed. In the case of 50 wt % V2O5 nanofiber reinforced polymer composites, no considerable change in decomposition temperature is observed as compared to the polymer composite containing 30 wt % V2O5, which can be ascribed to the maximum loading capacity of polymer composite and 8.3 wt % weight lost is seen at 400 °C, which is consistent with DSC results. Above 400 °C decomposition was increased enormously for both neat polymer and polymer composites. Thermal properties obtained from TGA curves for PDMS-mobility . When thg-PUR and PDMS-g-PUR/V2O5 polymer composites are characterized via tensile test. First, the rectangular specimens of the pristine PDMS-g-PUR polymer were cut into two pieces. Subsequently, the cut pieces were brought and held together for different times  at 50 °C to determine the healing efficiency through tensile strength obtained from the tensile test. Healing efficiency was calculated from the tensile strength ratio of healed specimens to the pristine polymer or polymer composites. g-PUR and healed PDMS-g-PUR for different healing times. The maximum tensile strength of the pristine polymer is found to be 113.08 ± 5.24 kPa, and the healing efficiency is 31.8 ± 2.7% for the specimens held for 15 min with a 34.51 ± 2.36 kPa tensile strength. When the cut pieces are held together for a longer time, an increase in their maximum stress values and the healing efficiencies are observed. The maximum tensile strength and healing efficiency are found to be 96.52 ± 4.54 kPa and 85.4 ± 1.2%, respectively, for 120 min holding time.The mechanical behavior and healing efficiencies of PDMS-g-PUR polymer were determined, V2O5 nanofibers were incorporated into the PDMS-g-PUR to improve both mechanical properties and healing efficiencies via formation of the PDMS-g-PUR /V2O5 polymer composites. All the specimens for polymer composites were brought together at 50 °C for 120 min after the cut to observe the healing process. g-PUR with polymer composites loaded with different amounts of V2O5 nanofibers up to 70 wt %. The maximum stress increases to 1443.40 ± 8.96 kPa with an addition of 10 wt % V2O5 nanofibers whereas strain value of PDMS-g-PUR/V2O5 composites at maximum stress decreases from 246.82 ± 16.3% to 148.37 ± 4.78% due to the restriction of chain mobility after the addition of 10 wt % V2O5 nanofibers [2O5 addition increases to the 30 wt % and 50 wt %, the tensile strength of composites slightly increases up to 1479.21 ± 17.22 kPa and 1584.38 ± 18.12 kPa, respectively. Nevertheless, with an addition of 70 wt % V2O5 nanofibers, the strength of composite decreases to 1384.00 ± 21.4 kPa and this can be due to the agglomeration of an excessive amount of V2O5 nanofibers inside the polymer matrix. Tensile strain value decreases to 13.05 ± 1.2% after the addition of 30 wt % V2O5 nanofibers, and with a further increase in the amount of nanofibers, no considerable change in tensile values is observed due to maximum loading capacity as detected in the DSC and TGA results. After the mechanical properties and healing efficiency of the PDMS-2O5 nanofiber addition into PDMS-g-PUR and V2O5 nanofiber amount on healing efficiencies, pristine polymer reinforced with 10, 30, and 50 wt % V2O5 nanofibers cut into two pieces were brought together and held for 120 min at 50 °C, and this process was repeated one, two, and three times, respectively. After each cycle of reattachment, specimens were subjected to the tensile test and their first, second, and third healing efficiencies were determined. g-PUR /10V are shown before and after healing cycles. Healing efficiency of PDMS-g-PUR/10V is found to be 95.3 ± 0.4% after the first self-healing cycle. In the supramolecular systems, the healing is mostly generated with the reformation of unbounded hydrogen bonds moieties [In order to see the effect of Vmoieties ,19,20,212O5 fibers [2O5 nanofiber addition, an increase in the healing efficiency of PDMS-g-PUR /V10 polymer composite compared to the PDMS-g-PUR . The PDMS-g-PUR has the maximum tensile strength of 113.08 ± 5.24 kPa and heals itself after waiting 2 h at 50 °C with a healing efficiency of 85.4 ± 1.2%. To improve the mechanical properties and healing efficiency of the PDMS-g-PUR, polymer composites incorporating V2O5 nanofibers which have the ability to make hydrogen bonds are prepared. The mechanical properties and healing efficiencies of PDMS-g-PUR are enhanced with 10 wt % V2O5 nanofiber addition. A decrease in total healing efficiencies is observed when the V2O5 nanofibers content is increased to 30 wt % and 50 wt % but they were still found to be greater than the healing efficiency of the neat PDMS-g-PUR. Addition of V2O5 nanofibers increases the hydrogen bonding capability of the polymer and reduces the contact surface required for the hydrogen bonding, leading to increased healing efficiencies compared to neat polymer. A self-healable supramolecular polymer has been successfully prepared by grafting an aliphatic polyurethane to polydimethylsiloxane (PDMS-


The second DH domain, isolated from the first module in the fluvirucin B1 PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3–β11 and β7–α2. From the catalytic Asp located in α3 to a conserved Pro in β11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,β-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a The online version of this article (10.1007/s10295-019-02189-z) contains supplementary material, which is available to authorized users. The collinear biosynthetic logic of type I modular polyketide synthases (PKSs) make them a promising synthetic biology platform for the production of existing and novel compounds. The dehydratase (DH) domain within type 1 modular PKSs is responsible for the dehydration of specific C-3 hydroxyacyl-acyl carrier protein (ACP) intermediates, resulting in a corresponding enoyl-ACP . These uubstrate , which iubstrate .3) PKS, and the macrolactam-producing fluvirucin B1 (6) PKS  at 1.80 Å and 2.01 Å, respectively. The BorA DH M3 is specific towards trans-cyclopentane-carboxylate-containing polyketide substrate 1, while the FluA DH M1 accepts amide-containing dipeptide polyketide intermediate 4  [Structural and biochemical data suggest the DH substrate selection is based on DH/ACP protein–protein interactions, stereospecificity of the 3-hydroxyl group and substrate specificity  , 25, 30.. S4C–E) . These rsyn-coplanar elimination of water. In addition to the catalytic His and Asp, both BorA DH M3 and FluA DH M1 contain conserved Leu and Tyr residues that outline the catalytic region, the latter orienting a water molecule . The catalytic His is located at the beginning of β3 and is on average less than 3.8 Å away from the Asp, located within α3. Recent findings in the fostriecin DH domain suggest DH domains utilize a single-base mechanism, where the active site His residue acts as the base to deprotonate C-2, subsequently protonating the C-3 hydroxyl group to promote C–O bond-cleavage and elimination of water [The catalytic region of the DH domain contains the catalytic His and Asp dyad responsible for the dehydration reaction via a of water . The carof water .The acyl intermediate-binding region can be defined as the residues outlining the bottom of the substrate-binding cavity, outside of the catalytic region. These residues bind to the chemical moiety past the C-3 hydroxyacyl group of acyl-ACP intermediate. The polyketide intermediate past the C-3 hydroxyacyl group can vary in chemical structure, length, and atom heterogeneity, and is dependent on both the upstream PKS module enzymatic architecture and the diverse starter-units incorporated in the nascent polyketide intermediate. The BorA DH M3 can select for a cyclopentane-carboxylate-containing intermediate Fig. a, while trans-cyclopentane-dicarboxylic acid and β-alanine, in both the free acid and the N-acetyl-cysteamine thioester (SNAC)-coupled form. However, no electron density for the corresponding substrates was observed. The lack of substrate binding may be due to high specificity of both BorA DH M3 and FluA DH M1 for their native PPant-tethered polyketide intermediates 1, 4  in the PPant-binding region of the DH were in close contact with the PPant moiety of 4. Within the catalytic region, the C-3 hydroxyl group of 4 is in proximity and oriented toward the catalytic Asp 231. The orientation of the C-3 hydroxyl towards the Asp 231 positioned the C-2 of 4 next to the catalytic His 69, supporting the mechanism of deprotonation of C-2 by the single catalytic base His 69 [In silico docking of the alanine-protected dipeptide PPant intermediate e His 69 .Fig. 4In4 , on the distal side of the variable loop region between α3 and β1, interacted with the dipeptide moiety of 4. Residues between Ser 185 and the conserved Tyr 189 were in close contact with 4. Surprisingly, the PPant-tethered fluvirucin intermediate 4 does not accommodate the entire catalytic region of the acyl intermediate-binding region. On average, there is a 9.5-Å distance gap between the terminal amino moiety of 4 and the bottom of the substrate-binding cavity. The bottom of the cavity contains several variant polar residues (Glu 241 and Gln 242). The gap identified between these polar residues with 4, the high B-factors associated within the invariant loop and the lack of modeled residues for similar type I DH domains suggest this structural region may adopt a distinct conformational change upon binding to the native substrate. This is further supported by the small structural conformational changes observed between the variable loop regions α3–β11 of the apo FluA DH M1 crystal structure and the energy minimized docked PPant intermediate 4-FluA DH M1 complex structure . Cα superposition results in RMSD values of 2.10 Å within the 18 residues of the α3–β11 loop region.Several residues in the acyl intermediate-binding region were in the vicinity of the nitrogen-containing dipeptide moiety of H) at each position  displayed low H values . 330 DH domain sequences derived from modular type I PKSs were exported from the ClusterCAD database . The secion Fig. a. Overalues Fig. a. SimilaH values. These higher H values were present in the loop regions between β7–α2 and α3–β11  or crotonyl-CoA ((PDB ID: 5I0K). Comparison of the α3–β11 and β7–α2 regions in both structures displays key differences. More residues were modeled in the α3–β11 of PpsC DH structure in the presence of the longer trans-dodec-2-enoyl-CoA substrate, suggesting higher stability of the α3–β11 region upon binding of a substrate that closely resembles the native acyl-ACP intermediate.To further improve our current understanding of DH selectivity towards different acyl-ACP intermediates past the C-3 hydroxyacyl position, we have performed structural analysis on two chemically diverse DH domains. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a –β8 Fig. . These s–β8 Fig. , 5. StruE. coli during protein purification, and low efficiency of the BorA2 PKS to produce adipic acid. This assay may not be optimal for testing chimeric DH activity. Nonetheless, generating soluble and stable chimeric DH domains is a first step towards DH engineering. Further efforts in investigating the activity of chimeric DH domains can be simplified by testing standalone chimeric DH domains containing the α3–β11 loop swaps with more naturally relevant acyl-ACP, acyl-PPant or acyl-SNAC substrates [The identification of both the α3–β11 and β7–α2 regions involved in substrate selection may provide insights into rational engineering of DH domains. In our efforts to improve DH-mediated dehydration in adipic acid production using the engineered BorA2 PKS , we testbstrates .Our structures of the BorA DH M3 and FluA DH M1 provide further structural evidence of substrate selection by DH domains within modular type I PKSs. We present both a sequence-structural analysis between DH domains and identify significant similarities and key differences pertaining to substrate selection. The work presented here in combination with existing DH domain studies should facilitate further engineering efforts of modified PKSs that can process non-natural substrates with improved catalytic properties.https://public-registry.jbei.org/folders/412) [Vector backbone template originated from JPUB_008800 plasmid (Table S1). The genes encoding for BorA DH M3 and FluA DH M1 were PCR amplified from the pDVA00936 and pTL-A01 plasmids, and cloned into a pET28a vector using Gibson assembly methodology . The j5 ers/412) .E. coli cells (Novagen). Cells containing the pET28-DH plasmids were grown to OD600 = 0.8 at 37 °C in TB medium containing 50 µg/mL kanamycin. The cell cultures were cooled to 18 °C and expression was induced using 1 mM IPTG. The cell cultures were incubated for an additional 16 h at 18 °C and harvested by centrifugation at 5525 r.c.f. for 10 min. The cell pellets were resuspended in 50 mM Hepes, pH 7.6, 10% glycerol, 10 mM imidazole, 300 mM NaCl. Resuspended cells were cooled on ice for 30 min and the cells were disrupted using sonication. The cell debris was cleared by centrifugation at 21,036 r.c.f. for 1 h. The supernatant was collected and batch bound to HisPur TM Cobalt Resin (Thermo Scientific) for 1 h at 4 °C. BorA DH M3 and FluA DH M1 were purified according to the manufacturer’s instructions using an imidazole step-gradient. Fractions containing pure protein were determined by SDS-PAGE and fractions containing either BorA DH M3 or FluA DH M1. Removal of the N-terminal 6 × His-tag was conducted by incubating the fractions containing the proteins at 14 °C for 30 h with thrombin from bovine plasma (Sigma-Aldrich) at a concentration of 2 U/mg of recombinant protein and 3.5 mM CaCl2. After thrombin digestion and assessment of N-terminal 6 × His-tag cleavage via SDS-PAGE, we dialyzed the DH domains against 50 mM Hepes, pH 7.6, 5% glycerol. Removal of thrombin and further purification of BorA DH M3 and FluA DH M1 were conducted by anion-exchange chromatography using HiTrap Q FF  according to the manufacturer’s instructions. Purified BorA DH M3 and FluA DH M1 were dialyzed against crystallization buffer, which consisted of 25 mM Hepes, pH 7.6, and 1 mM dithiothreitol . Both BorA DH M3 and FluA DH M1 were concentrated to 12 mg/mL for crystallographic studies.The recombinant BorA DH M3 and FluA DH M1 containing an N-terminal 6 × His-tag were produced in BL21 (DE3) 2, 0.10 M Tris, pH 8.5, and 29% (w/v) PEG 4000. For FluA DH M1, 10 mg/mL protein was crystallized by sitting drop vapor diffusion in drops containing a 1.5:1 ratio of protein solution and 0.40 M NaCl, 0.10 M Tris, pH 8.5, and 29% (w/v) PEG 3350. For both proteins, a final concentration of 15% glycerol was added before flash freezing in liquid nitrogen.Crystallization screening was carried out on a Phoenix robot  using a sparse matrix screening method . BorA DHσ in the Fo-Fc map) and distance from a potential hydrogen bonding partner (< 3.5 Å). TLS refinement [The X-ray data sets for BorA DH M3 and FluA DH M1 were collected at the Berkeley Center for Structural Biology on beamlines 8.2.1 and 8.2.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. Diffraction data were recorded using ADSC Q315R detectors . Processing of image data was performed using the HKL2000 suite of programs . For botfinement  using tefinement , was use4 and the FluA DH M1. The natural PPant-tethered substrate 4 was initially drawn in Chemdraw (PerkinElmer) and transferred in SMILES format to the PHENIX software suite, eLBOW ligand and constraints generator [4 and a constraints (.cif) file. Both files were used to dock 4 into the FluA DH M1. As a reference for substrate binding, we used the co-crystal structure of PpsC DH complexed with trans-dodec-2-enoyl-CoA (PDB ID: 5NJI) to position substrate 4 into the FluA DH M1. We utilized the program Coot to overlay the PpsC DH with chain A of the FluA DH M1 and model in substrate 4 [4 complex was then energy minimized using the program UCSF CHIMERA [We conducted in silico substrate docking between strate 4 . The ent CHIMERA . The lacAll of the protein structure analysis and figures were generated using UCSF CHIMERA  and PyMOThe Shannon entropy of aligned DH sequences was calculated at each position within a refined multiple sequence alignment (MSA). Briefly, 330 sequences derived from PKS non-loading modules containing AT, DH, KR, and ACP domains with active KR and DH domains were exported from the ClusterCAD database . SequencSupplementary material 1 (PDF 1776 kb)Below is the link to the electronic supplementary material.


P. falciparum malaria cases has been documented in the past years, the number of Plasmodium falciparum malaria cases has rapidly decreased passing from 19.638 in 2012 to 4.073 cases in 2016. Concomitantly, the spread of artemisinin resistance markers is raising concern on the future efficacy of the ACTs. An evaluation of the clinical impact of the artemisinin resistance markers is therefore of interest.In Vietnam, a rapid decline of The clinical effectiveness of dihydroartemisinin-piperaquine therapy (DHA-PPQ) has been evaluated in three districts characterized by different rates of ART resistance markers: K13(C580Y) mutation and delayed parasite clearance on day 3 (DPC3). Patients were stratified in 3 groups a) no markers, b) one marker (suspected resistance), c) co-presence of both markers (confirmed resistance). In the studied areas, the clinical effectiveness of DHA-PPQ has been estimated as malaria recrudescence within 60 days.The rate of K13(C580Y) ranged from 75.8% in Krong Pa to 1.2% in Huong Hoa district. DPC3 prevalence was higher in Krong Pa than in Huong Hoa (86.2% vs 39.3%). In the two districts, the prevalence of confirmed resistance was found in 69.0% and 1.2% of patients, respectively. In Thuan Bac district, we found intermediate prevalence of confirmed resistance. Treatment failure was not evidenced in any district. PPQ resistance was not evidenced. Confirmed resistance was associated to the persistence of parasites on day 28 and to 3.4-fold higher parasite density at diagnosis. The effectiveness of malaria control strategies was very high in the studied districts.No treatment failure has been observed in presence of high prevalence of ART resistance and in absence of PPQ resistance. K13(C580Y) was strongly associated to higher parasitemia at admission, on days 3 and 28. Slower parasite clearance was also observed in younger patients. Artemisinin (ART) resistance was first described in Cambodia –4 and, sfalciparum K13 propeller gene) was found to be associated with DPC AGGGCCTCACCAC[BHQ1]-3′), 150 nM MUT probe (5′-[HEX]AGGGCCTTACC[BHQ1]-3′), and 1 U of Platinum Taq DNA (Invitrogen). Amplification was performed under the following conditions: 95°C for 3 min, then 45 cycles at 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s.Hb E detection (Ref. Seq: NG_000007.3) was performed using a mixture of 1 mM dNTPs, 3 mM MgCl2, each primer at 400 nM (forward: 5′-CCAGGACCACATTGTTG-3′ and reverse: 5′-ACCCAAGGAGCCCATTCT-3′),175 nM WT probe (5′-[HEX]ATTTCAACACCTTGACCTGA [BHQ1]-3′), 275 nM MUT probe (5′-[Cy5]ATTTCAACATCTTGACCTGA[BHQ2]-3′), and 1 U of Platinum Taq DNA (Invitrogen). Amplification was performed under the following conditions: 95°C for 3 min, then 45 cycles at 95°C for 15 s, 57°C for 20 s, and 72°C for 20 s.G6PD D. detection (Ref. Seq: NG_009015.1) was performed using 1 mM dNTPs, 4 mM MgCl(Ref. Seq: NG_000006.1) was performed using 10 μL of 2× Taqman (Applied Biosystem) universal master mix, each primer at 400 nM (forward: 5′-GACAAGTTCCTGGCTTCT -3′ and reverse: 5′-ATAGAGAGAACCCAGGCA -3′), 300 nM WT probe (5′-[FAM]GGCTCCAGCTTAACGGTATTTGG[BHQ1]- 3′), and 100 nM MUT probe (5′-[Tx Red]GGCTCCAGCTTGACGGTATTTGG[BHQ1]- 3′) (Invitrogen). Amplification was performed under the following conditions: 50°C for 2 min, 95°C for 10 min, then 45 cycles at 95°C for 15 s, 57°C for 30 s, and 60°C for 30 s. For each protocol, detection and analysis were performed using CFX96 Touch Real-Time PCR Detection System.Hb CS detection t-test (Graph Pad Prism 7.3). A two-tailed p-value of less than 0.05 was considered statistically significant.Comparison between categorical variables was performed using a Pearson's chi-square or Fisher exact test, where appropriate (STATA software version 13). Comparison between quantitative variables was performed using Student’s P. falciparum malaria cases from 2012 to 2016 in the three studied districts. Those data are in agreement with the national data . To exclude the interference that may be exerted by piperaquine resistance, we measured the presence of a specific and sensitive marker of piperaquine resistance (multicopies of PfPM2 gene). In the three districts, DPC3 and K13 mutation rates were measured in a group of ssitemia . The qPCAccording to a WHO classification  we dividWe analyzed the interference of some potential confounding factors on the rate of clearance of parasites. The prevalence of some common human mutations selected by malaria such as G6PD deficiency variant Viangchan, Haemoglobin E and Haemoglobin Constant Spring were measured in the general population and in malaria patients. P. falciparum malaria cases has been observed in Vietnam. On the other hand, the increasing rates of ART resistance markers, the appearance of piperaquine resistance at the Cambodian border [In the past years a sustained decline in the number of n border ,38, the n border , the sign border ,39–46 ann border ,16,20–25n border ,45 to idIn the two districts displaying the highest and the lowest prevalence of the K13 mutation, we observed markedly different rates of DPC3 (86.2% vs 39.3%) confirming the association between the two markers ,20,24. IThe use of quantitative measurements provided additional information: i) the co-occurrence of both ART resistance markers was associated to a 3.4 fold higher parasite density at admission in comparison to patients displaying no ART resistance markers; ii) patients displaying a slower clearance of parasites on day 3  were significantly younger than patients displaying faster clearance. Those observations are in accordance with reports indicating that immunity is a major determinant of the parasite clearance rate –49 that,In conclusion ART resistance markers are not predictive of clinical failure ,25. ThisS1 Fig(PDF)Click here for additional data file.S1 Row Data(RAR)Click here for additional data file.