Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.

The ASK1 and ASK2 genes are essential for Arabidopsis early development.

The requirement of CUL1 for Arabidopsis embryogenesis suggests that Skp1-CUL1-F-box protein (SCF) complexes play important roles during embryo development. Among the 21 Arabidopsis Skp1-like genes (ASKs), it is unknown which ASK gene(s) is essential for embryo development. In this study, we demonstrate a vital role for ASK1 and ASK2 in Arabidopsis embryogenesis and postembryonic development through analysis of the ask1 ask2 double mutant. Our detailed analysis indicates that the double mutations in both ASK1 and ASK2 affect cell division and cell expansion/elongation and cause a developmental delay during embryogenesis and lethality in seedling growth. The expression patterns of ASK1 and ASK2 were examined further and found to be consistent with their roles in embryogenesis and seedling development. We propose that mutations in ASK1 and ASK2 abolish all of the ASK1- and ASK2-based SCF and non-SCF complexes, resulting in alteration of gene expression and leading to defects in growth and development.

The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis.

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.

Origin of double dinitrogen release feature during fast switching between lean and rich cycles for NOx storage reduction catalysts

The performance of NOx storage and reduction over 1.5 wt% Pt/20 wt% KNO3/K2Ti8O17 and 1.5 wt% Pt/K2Ti8O17 catalysts has been investigated using combined fast transient kinetic switching and isotopically labelled (NO)-N-15 at 350 degrees C. The evolution of product N-2 has revealed two significant peaks during 60 s lean/1.3 s rich switches. It also found that the presence of CO2 in the feed affects the release of N-2 in the second peak. Regardless of the presence/absence of water in the feed, only one peak of N-2 was observed in the absence of CO2. Gas-phase NH3 was not observed in any of the experiments. However, in the presence of CO2 the results obtained from in situ DRIFTS-MS analysis showed that isocyanate species are formed and stored during the rich cycles, probably from the reaction between NOx and CO, in which CO was formed via the reverse water-gas shift reaction. 

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells. (C) 2008 by Radiation Research Society.

A new polycythaemia vera-associated SOCS3 SH2 mutant (SOCS3(F136L)) cannot regulate erythropoietin responses

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.

A randomised double-blind placebo-controlled phase II study of AGI004 for control of chemotherapy-induced diarrhoea

Background: AGI004 is a controlled-release transdermal patch preparation of mecamylamine. We conducted a randomised placebo-controlled phase II study of two dose levels of AGI004 in chemotherapy-induced diarrhoea (CID).

Methods: Adult patients receiving chemotherapy who had experienced diarrhoea (NCI grade 1-2) during previous cycles of chemotherapy were eligible. In all, 64 patients were randomised to receive AGI004 4mg then 8mg per 24 h transdermal patch or placebo for two sequential cycles of chemotherapy. Patients' severity of diarrhoea was physician-assessed using NCI grade of diarrhoea and patient-assessed using information recorded in daily diaries of bowel movements.

Results: Overall AGI004 doubled the odds of a response to treatment on the first day of chemotherapy based on physician assessment of NCI grade of diarrhoea compared with placebo (odds ratio = 2.0, 90% confidence interval: 0.9-4.5) and there was a trend to improved response rates for AGI004 for the full treatment cycle although these results were not statistically significant. There was also evidence of significantly improved response rates based on patient assessment of diarrhoea both overall (P = 0.05) and at the 8-mg dose level (P = 0.02) compared with placebo.

Conclusion: AGI004 demonstrated effectiveness in reducing chemotherapy-associated diarrhoea, with results suggesting response across multiple measurements of diarrhoea. Treatment was well tolerated with no drug-related adverse events. Further evaluation of this agent in the management of CID is warranted.

A simply effective double-coating cathode with MnO2 nanosheets/graphene as functionalized interlayer for high performance lithium-sulfur batteries

Herein, we report a facile and effective adsorption strategy to improve the performance of Lithium-Sulfur (Li-S) batteries. MnO₂ nanosheets grown on the surface of highly conductive graphene resulted in a coupled composite bilayer electrode when coated onto a sulfur cathode. In this way, a high initial specific capacity of 1395 mA h g^-1 at a rate of 0.5C, a coulombic efficiency approaching 100% and steady cyclic efficiency with a fade rate of 0.3% per cycle from 10 to 100 cycles has been achieved. This hybrid electrode not only shows enhanced electrochemical performance but can also be easily controlled and scaled thereby aiding future commercialization of high-performance Li-S batteries.

Binary Mixtures of 1-Butyl-1-Methylpyrrolidinium Bis{(trifluoromethyl)Sulfonyl}Imide and Aliphatic Nitrile Solvents As Electrolyte for Electrochemical Double Layer Capacitors (Invited).

Electrochemical double layer capacitors (EDLCs), also known as supercapacitors, are promising energy storage devices, especially when considering high power applications [1]. EDLCs can be charged and discharged within seconds [1], feature high power (10 kW·kg-1) and an excellent cycle life (>500,000 cycles). All these properties are a result of the energy storage process of EDLCs, which relies on storing energy by charge separation instead of chemical redox reactions, as utilized in battery systems. Upon charging, double layers are forming at the electrode/electrolyte interface consisting of the electrolyte’s ions and electric charges at the electrode surface.In state-of-the-art EDLC systems activated carbons (AC) are used as active materials and tetraethylammonium tetrafluoroborate ([Et4N][BF4]) dissolved in organic solvents like propylene carbonate (PC) or acetonitrile (ACN) are commonly used as the electrolyte [2]. These combinations of materials allow operative voltages up to 2.7 V - 2.8 V and an energy in the order of 5 Wh·kg-1[3]. The energy of EDLCs is dependent on the square of the operative voltage, thus increasing the usable operative voltage has a strong effect on the delivered energy of the device [1]. Due to their high electrochemical stability, ionic liquids (ILs) were thoroughly investigated as electrolytes for EDLCs, as well as, batteries, enabling high operating voltages as high as 3.2 V - 3.5 V for the former [2]. While their unique ionic structure allows the usage of neat ILs as electrolyte in EDLCs, ILs suffer from low conductivity and high viscosity increasing the intrinsic resistance and, as a result, a lower power output of the device. In order to overcome this issue, the usage of blends of ionic liquids and organic solvents has been considered a feasible strategy as they combine high usable voltages, while still retaining good transport properties at the same time.In our recent work the ionic liquid 1-butyl-1-methylpyrrolidinium bis{(trifluoromethyl)sulfonyl}imide ([Pyrr14][TFSI]) was combined with two nitrile-based organic solvents, namely butyronitrile (BTN) and adiponitrile (ADN), and the resulting blends were investing regarding their usage in electrochemical double layer capacitors [4,5]. Firstly, the physicochemical properties were investigated, showing good transport properties for both blends, which are similar to the state-of-the-art combination of [Et4N][BF4] in PC. Secondly, the electrochemical properties for EDLC application were studied in depth revealing a high electrochemical stability with a maximum operative voltage as high as 3.7 V. In full cells these high voltage organic solvent based electrolytes have a good performance in terms of capacitance and an acceptable equivalent series resistance at cut-off voltages of 3.2 and 3.5 V. However, long term stability tests by float testing revealed stability issues when using a maximum voltage of 3.5 V for prolonged time, whereas at 3.2 V no such issues are observed (Fig. 1).Considering the obtained results, the usage of ADN and BTN blends with [Pyrr14][TFSI] in EDLCs appears to be an interesting alternative to state-of-the-art organic solvent based electrolytes, allowing the usage of higher maximum operative voltages while having similar transport properties to 1 mol∙dm-3 [Et4N][BF4] in PC at the same time.