Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus

Eucalyptus trees are among the most important species for industrial forestry worldwide. However, as with most forest trees, flowering does not begin for one to several years after planting which can limit the rate of conventional and molecular breeding. To speed flowering, we transformed a Eucalyptus grandis × urophylla hybrid (SP7) with a variety of constructs that enable overexpression of FLOWERING LOCUS T (FT). We found that FT expression led to very early flowering, with events showing floral buds within 1–5 months of transplanting to the glasshouse. The most rapid flowering was observed when the cauliflower mosaic virus 35S promoter was used to drive the Arabidopsis thaliana FT gene (AtFT). Early flowering was also observed with AtFT overexpression from a 409S ubiquitin promoter and under heat induction conditions with Populus trichocarpa FT1 (PtFT1) under control of a heat‐shock promoter. Early flowering trees grew robustly, but exhibited a highly branched phenotype compared to the strong apical dominance of nonflowering transgenic and control trees. AtFT‐induced flowers were morphologically normal and produced viable pollen grains and viable self‐ and cross‐pollinated seeds. Many self‐seedlings inherited AtFT and flowered early. FT overexpression‐induced flowering in Eucalyptus may be a valuable means for accelerating breeding and genetic studies as the transgene can be easily segregated away in progeny, restoring normal growth and form.     

Gene expression and the evolution of phenotypic diversity in social wasps

Background  

Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects (ants, social bees, and social wasps), where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution.     

Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork

Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.     

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24, 39-41). Encoded by the dnaN gene, the β clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The β clamp is a head-to-tail, ring-shaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the β clamp is proliferating cell nuclear antigen (PCNA) (15, 28, 40, 41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40, 41). Although the primary structures of the β clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the β clamp is not limited to its well-defined role in genome replication. The Escherichia coli β clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19, 37, 43). This allows the β clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the β clamp binds YabA, a negative regulator of DNA replication initiation (12, 29, 52). It has also been suggested that the B. subtilis β clamp sequesters DnaA from the replication origin during the cell cycle through the binding of DnaA to YabA and the binding of YabA to the β clamp (70). Thus, it is hypothesized that in E. coli and B. subtilis, the β clamp influences the frequency of replication initiation through interactions with Hda and YabA, respectively.The E. coli and B. subtilis β clamp has an important role in translesion DNA synthesis during the replicative bypass of noncoding bases by specialized DNA polymerases belonging to the Y family (20, 33). The roles of the E. coli β clamp in translesion synthesis are well established (5, 8, 30, 31). Binding sites on the E. coli β clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD₂′C) have been identified, and the consequence of disrupting their association with the β clamp has illustrated the critical importance of the β clamp to the activity of both of these polymerases (4, 5, 8, 26, 30, 31, 48, 49).In addition to the involvement of the β clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis β clamp also functions in DNA mismatch repair (MMR) (45, 46, 64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60). In both E. coli and B. subtilis, deletion of the genes mutS and mutL increases the spontaneous mutation frequency several hundredfold (13, 25, 63). In E. coli, MutS recognizes and binds mismatches, while MutL functions as a “matchmaker,” coordinating the actions of other proteins in the MMR pathway, allowing the removal of the mismatch and resynthesis of the resulting gap (reviewed in references 42 and 60). MutS and MutL of E. coli and B. subtilis physically interact with the β clamp (45, 46, 51, 64). Interaction between the B. subtilis β clamp and MutS is important for efficient MMR and organization of MutS-green fluorescent protein (GFP) into foci in response to replication errors, while the function of MutL binding to the β clamp is unknown (64).These studies show that the β clamp is critical for several aspects of DNA metabolism in E. coli and B. subtilis. In E. coli, many dnaN alleles have been examined and used to define the mechanistic roles of the β clamp in vivo (5, 18, 24, 30, 31, 48, 49, 73). A limitation in studying the mechanistic roles of the B. subtilis β clamp is that only two dnaN alleles (β clamp) are available, dnaN5 and dnaN34 (36) (www.bgsc.org/), and both of these alleles do not support growth at temperatures above 49°C, suggesting that they may cause similar defects (36) (www.bgsc.org/). Of these two dnaN alleles, only dnaN5 has been investigated in any detail (36, 53, 64). The mutant β clamp encoded by dnaN5 contains a G73R substitution [dnaN5(G73R)] in a surface-exposed residue located on the outside rim of the β clamp (53, 64). Our previous studies with this allele showed that dnaN5(G73R) confers an increase in mutation frequency at 30°C and 37°C (64). Further characterization of dnaN5(G73R) showed that the increased mutation frequency is caused by a partial defect in MMR (64). Additionally, dnaN5(G73R)-containing cells have a reduced ability to support MutS-GFP focus formation in response to mismatches (64). These results support the hypothesis that G73R in the β clamp causes a defect in DNA replication at 49°C (36) and impaired MMR manifested by a defect in establishing the assembly of MutS-GFP foci in response to replication errors (64).To understand the roles of the B. subtilis β clamp in MMR and DNA replication, we examined the dnaN5 and dnaN34 alleles. We found that the nucleotide sequences of dnaN5 and dnaN34 and the phenotypes they produce were identical, both producing the G73R missense mutation. We analyzed in vivo β clamp^G73R protein levels and found that the β clamp^G73R protein accumulated to wild-type levels at elevated temperatures. To identify amino acid residues that would restore DNA replication at elevated temperatures, we isolated three intragenic suppressors of dnaN5(G73R) that conferred growth of B. subtilis cells at 49°C. Epistasis analysis and determination of the mutation spectrum showed that each dnaN allele isolated in this study caused an MMR-dependent increase in mutation frequency. Additionally, we found that the β clamp binding protein YabA can reduce the efficiency of MMR in vivo when yabA expression is induced. Thus, we have identified residues in the β clamp that are critical for DNA replication and MMR in B. subtilis. We also found that a β clamp binding protein, YabA, can reduce the efficiency of MMR in vivo.     

A randomised, blinded, placebo-controlled trial in dementia patients continuing or stopping neuroleptics (the DART-AD trial)

Background

There have been increasing concerns regarding the safety and efficacy of neuroleptics in people with dementia, but there are very few long-term trials to inform clinical practice. The aim of this study was to determine the impact of long-term treatment with neuroleptic agents upon global cognitive decline and neuropsychiatric symptoms in patients with Alzheimer disease.

Methods and Findings

Design: Randomised, blinded, placebo-controlled parallel two-group treatment discontinuation trial.Setting: Oxfordshire, Newcastle and Gateshead, London and Edinburgh, United Kingdom.Participants: Patients currently prescribed the neuroleptics thioridazine, chlorpromazine, haloperidol trifluoperazine or risperidone for behavioural or psychiatric disturbance in dementia for at least 3 mo.Interventions: Continue neuroleptic treatment for 12 mo or switch to an identical placebo.Outcome measures: Primary outcome was total Severe Impairment Battery (SIB) score. Neuropsychiatric symptoms were evaluated with the Neuropsychiatric Inventory (NPI).Results: 165 patients were randomised (83 to continue treatment and 82 to placebo, i.e., discontinue treatment), of whom 128 (78%) commenced treatment (64 continue/64 placebo). Of those, 26 were lost to follow-up (13 per arm), resulting in 51 patients per arm analysed for the primary outcome. There was no significant difference between the continue treatment and placebo groups in the estimated mean change in SIB scores between baseline and 6 mo; estimated mean difference in deterioration (favouring placebo) −0.4 (95% confidence interval [CI] −6.4 to 5.5), adjusted for baseline value (p = 0.9). For neuropsychiatric symptoms, there was no significant difference between the continue treatment and placebo groups (n = 56 and 53, respectively) in the estimated mean change in NPI scores between baseline and 6 mo; estimated mean difference in deterioration (favouring continue treatment) −2.4 (95% CI −8.2 to 3.5), adjusted for baseline value (p = 0.4). Both results became more pronounced at 12 mo. There was some evidence to suggest that those patients with initial NPI ≥ 15 benefited on neuropsychiatric symptoms from continuing treatment.

Conclusions

For most patients with AD, withdrawal of neuroleptics had no overall detrimental effect on functional and cognitive status. Neuroleptics may have some value in the maintenance treatment of more severe neuropsychiatric symptoms, but this benefit must be weighed against the side effects of therapy.Trial registration: Cochrane Central Registry of Controlled Trials/National Research Register (#ISRCTN33368770).     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Recruitment of PLANT U-BOX13 and the PI4Kβ1/β2 Phosphatidylinositol-4 Kinases by the Small GTPase RabA4B Plays Important Roles during Salicylic Acid-Mediated Plant Defense Signaling in Arabidopsis

Protection against microbial pathogens involves the activation of cellular immune responses in eukaryotes, and this cellular immunity likely involves changes in subcellular membrane trafficking. In eukaryotes, members of the Rab GTPase family of small monomeric regulatory GTPases play prominent roles in the regulation of membrane trafficking. We previously showed that RabA4B is recruited to vesicles that emerge from trans-Golgi network (TGN) compartments and regulates polarized membrane trafficking in plant cells. As part of this regulation, RabA4B recruits the closely related phosphatidylinositol 4-kinase (PI4K) PI4Kβ1 and PI4Kβ2 lipid kinases. Here, we identify a second Arabidopsis thaliana RabA4B-interacting protein, PLANT U-BOX13 (PUB13), which has recently been identified to play important roles in salicylic acid (SA)-mediated defense signaling. We show that PUB13 interacts with RabA4B through N-terminal domains and with phosphatidylinositol 4-phosphate (PI-4P) through a C-terminal armadillo domain. Furthermore, we demonstrate that a functional fluorescent PUB13 fusion protein (YFP-PUB13) localizes to TGN and Golgi compartments and that PUB13, PI4Kβ1, and PI4Kβ2 are negative regulators of SA-mediated induction of pathogenesis-related gene expression. Taken together, these results highlight a role for RabA4B and PI-4P in SA-dependent defense responses.