The NOMEGA gene required for female gametophyte development encodes the putative APC6/CDC16 component of the Anaphase Promoting Complex in Arabidopsis

Development of the female gametophyte involves several rounds of nuclear divisions during which nuclei are rearranged and finally cellularized to form a mature seven-celled embryo sac. During these nuclear divisions, key proteins involved in the cell cycle need to be degraded quickly in order to facilitate both the metaphase-anaphase transition stage and late anaphase. Here, we report the characterization of an Arabidopsis mutant nomega, which results in arrest of the embryo sac development at the two-nucleate stage. The NOMEGA gene product shows high homology to the APC6/cell division cycle (CDC)16 subunit of the Anaphase Promoting Complex/Cyclosome (APC/C). The phenotype of the nomega mutant is quite different from that of the hobbit mutant, which had suggested a role for the plant APC/C in auxin signalling. We show that nomega mutant embryo sacs are unable to degrade Cyclin B, an important APC/C substrate, providing further evidence of a role for the NOMEGA gene product and the plant APC/C in cell cycle progression during gametophyte development.     

Supplemented nutrition decreases helminth burden and increases drug efficacy in a natural host–helminth system

Gastrointestinal (GI) helminths are common parasites of humans, wildlife, and livestock, causing chronic infections. In humans and wildlife, poor nutrition or limited resources can compromise an individual''s immune response, predisposing them to higher helminth burdens. This relationship has been tested in laboratory models by investigating infection outcomes following reductions of specific nutrients. However, much less is known about how diet supplementation can impact susceptibility to infection, acquisition of immunity, and drug efficacy in natural host–helminth systems. We experimentally supplemented the diet of wood mice (Apodemus sylvaticus) with high-quality nutrition and measured resistance to the common GI nematode Heligmosomoides polygyrus. To test whether diet can enhance immunity to reinfection, we also administered anthelmintic treatment in both natural and captive populations. Supplemented wood mice were more resistant to H. polygyrus infection, cleared worms more efficiently after treatment, avoided a post-treatment infection rebound, produced stronger general and parasite-specific antibody responses, and maintained better body condition. In addition, when applied in conjunction with anthelmintic treatment, supplemented nutrition significantly reduced H. polygyrus transmission potential. These results show the rapid and extensive benefits of a well-balanced diet and have important implications for both disease control and wildlife health under changing environmental conditions.     

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6'')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.     

Falcarindiol inhibits LPS-induced inflammation via attenuating MAPK and JAK-STAT signaling pathways in murine macrophage RAW 264.7 cells

The purpose of the study was to investigate the mechanism of total flavone of Desmodium styracifolium (TFDS) in regulating the formation of urinary calculi. Protein levels of KIM-1, LC3-II, p-p38 were measured by Western blot. The effect of different COM concentrations, different TFDS concentrations, SB203580 (specific inhibitor of p38/MAPK), and overexpression of KIM-1 on cell viability were detected by WST-1 assay. The apoptotic cells and FITC positive cells were detected by flow cytometry. HK-2 cell viability decreased with the increase of COM concentration, and the protein levels of KIM-1, LC3-II, and p-p38 increased with the time. Blocking the p38/MAPK pathway or co-cultured with TFDS inhibited the effects of COM on apoptosis and autophagy of HK-2 cells. In addition, blocking the p38/MAPK pathway inhibited the expression of KIM-1. In COM-induced cells, after treated with SB203580, overexpression of KIM-1 could reverse the protection effect of SB203580 on COM-induced cell damage and the inhibition of SB203580 on COM-induced excessive autophagy, suggesting p38/MAPK regulated KIM-1 to regulate COM-induced cell apoptosis and autophagy. Finally, we proved that TFDS inhibited p38/MAPK pathway. And the protection effect of COM-induced cell injury increased with the increase of TFDS concentration, and the adhesion between COM and cells decreased with the increase of TFDS concentration. With the increase of the concentration of TFDS, p38/MAPK pathway was gradually inhibited, and KIM-1 and autophagy related proteins were decreased. TFDS inhibited HK-2 cell apoptosis and autophagy by regulating KIM-1 via p38/MAPK pathway.     

Resistogram typing as an epidemiological tool for Escherichia coli isolates of poultry origin

A rapid method for the detection of corynetoxins, tunicamycin-like antibiotics, is described. Test samples were applied to or grown on an agar medium and overlain with Clavibacter tritici which is highly sensitive to the toxins. The method could detect 50 ng of tunicamycin. Corynetoxins in a range of field and laboratory samples were readily detected.     

Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae)

A strain of T. chilonis, an egg parasitoid of lepidopteran pests tolerant to the most commonly used cyclodiene insecticide--endosulfan was developed in the laboratory. Tolerance to endosulfan was induced by exposing adult parasitoids sequentially from a sub-lethal concentration (0.004%) to the field recommended concentration (0.09%). The strain acquired tolerance to the insecticide after 341 generation of continuous exposure with LC50 values of 1074.96 ppm as compared to LC50 of (70.91 ppm) in susceptible strain. The genetical study showed that F1 crosses exhibited a semi-dominant response to endosulfan with degree of dominance value (D) of 0.58. The resistant factor of tolerant strain was 15.1 folds and of F1 cross were 8.53 folds over susceptible strain. Under net house conditions, the tolerant strain parasitised 56% Helicoverpa armigera eggs on potted cotton plants immediately after an insecticide spray, compared to 3% by the susceptible strain. High percentage survival of the immature stages of the tolerant strain proved their ability to withstand the insecticide load. Breakdown of insecticide tolerance in the strain occurred after four generations in absence of insecticide load. Use of the tolerant strain as a component of bio-intensive IPM in various crops where insecticide use is higher is discussed.     

The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain–CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3''s lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP–CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP–CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.CENTROMERES are essential for chromosome inheritance, because they nucleate kinetochores, the protein complexes on eukaryotic chromosomes that attach to spindle microtubules. Despite the essential requirement for centromeres in chromosome segregation, their DNA sequences and the sequences of kinetochore proteins are highly variable. Kinetochores in Saccharomyces cerevisiae and related budding yeasts assemble on small, unique centromere DNAs (125 bp in S. cerevisiae) (Meraldi et al. 2006). Centromere DNAs in the fission yeast Schizosaccharomyces pombe are larger, consisting of a central core sequence of 4–5 kb, which binds kinetochore proteins, flanked by large inverted repeats whose heterochromatic nature is important for centromere function (the total size of the S. pombe centromere DNA is 35–110 kb). At the other extreme from small yeast centromeres are holocentric organisms, such as Caenorhabditis elegans, in which kinetochore proteins bind along the entire length of mitotic chromosomes (Dernburg 2001). Most plants and animals have extremely large centromere DNA tracts consisting of megabases of simple tandem repeats. The repeat sequence evolves extremely rapidly, and only a small fraction of the repeat array is likely to be bound by kinetochore proteins. Furthermore, kinetochores can be nucleated by noncentromeric DNA sequences in plant and animal cells (Amor and Choo 2002; Nagaki et al. 2004; Nasuda et al. 2005; Heun et al. 2006; Wade et al. 2009). Despite these findings, the maintenance of massive centromere repeat arrays in both animal and plant taxa suggests that repeats are a central feature of centromere biology in these organisms.Although centromere DNAs are extremely diverse, all eukaryote kinetochores contain the centromere-specific histone H3 variant CENH3 (originally described as CENP-A in human) (Henikoff and Dalal 2005; Black and Bassett 2008). CENH3 replaces conventional H3 specifically in a subset of centromere nucleosomes. It is essential for kinetochore function in all eukaryotes where this requirement has been tested. Conventional histones are among the most conserved proteins in eukaryote genomes. In contrast, CENH3 is rapidly evolving. The C-terminal histone-fold domain, which complexes with other histones to form the globular nucleosome core, can be aligned with conventional H3''s but evolves rapidly and shows signatures of adaptive evolution in some residues (Malik and Henikoff 2001; Talbert et al. 2002; Cooper and Henikoff 2004). The N-terminal tail domain of conventional histone H3 protrudes from the nucleosome core and is not resolved in the structure solved by X-ray crystallography (Luger et al. 1997). In CENH3, the tail domain evolves so rapidly that its sequence can barely be aligned between closely related species.Experiments in yeast and in animals have delineated functionally important regions within CENH3. S. cerevisiae kinetochores contain only a single CENH3/Cse4p nucleosome (Furuyama and Biggins 2007). In S. cerevisiae Cse4p, amino acid residues required for normal function are distributed throughout the histone-fold domain (Keith et al. 1999). The N-terminal tail of Cse4p contains an essential region termed the END domain, but overexpression of a Cse4p lacking the tail altogether can rescue a cse4 deletion mutant (Chen et al. 2000; Morey et al. 2004). In Drosophila melanogaster cells, CENH3/Cid from the distantly related D. bipectinata did not localize to kinetochores unless a specific region of the histone-fold domain, loop 1, was swapped with the corresponding region from D. melanogaster CENH3/Cid (Vermaak et al. 2002). In human, the histone-fold domain is important for centromere targeting (Sullivan et al. 1994). The functionally important region within the histone-fold domain was further defined by inserting loop 1 and the α-2 helix from CENH3/CENP-A (termed the CENP-A targeting domain, or CATD) into conventional H3 (Black et al. 2004). H3 containing the CATD acquires several functions of CENP-A when expressed in human cells. It localizes to kinetochores, binds the kinetochore protein CENP-N, has a rigid secondary structure when assembled into nucleosomes, and can restore normal chromosome segregation in cells depleted for CENP-A using RNA interference (RNAi) (Black et al. 2004, 2007a,b; Carroll et al. 2009).Despite these extensive studies, questions about structure–function relationships within CENH3 remain. CENH3 function may differ between small yeast centromeres and the large tandem repeat centromeres of animals and plants, particularly because larger centromere DNAs are likely to contain many more CENH3 nucleosomes and may require a higher level of organization. Experiments in D. melanogaster and in human cells have used RNAi to downregulate the endogenous protein, and a conditional knockout has been made in chicken DT-40 cells (Blower and Karpen 2001; Goshima et al. 2003; Regnier et al. 2005; Black et al. 2007b). These experiments are challenging because CENH3 is very stable. If preexisting CENH3 is partitioned equally between duplicated sister centromeres, its amount will be approximately halved at each cell division. Therefore the protein may persist for many cell divisions after induction of RNAi, as shown by Western blots indicating that ∼10% of endogenous CENH3 remains in human cells subjected to two rounds of RNAi (Black et al. 2007b).We have chosen to study CENH3 in the model plant A. thaliana, which combines facile genetics and transgenesis with centromere DNA structure that is similar to most plants and animals (megabases of tandem repeats with a repeating unit of 178 bp) (Murata et al. 1994; Copenhaver et al. 1999). Although Drosophila and mouse CENH3 knockout mutants have been characterized (Howman et al. 2000; Blower et al. 2006), a large-scale structure–function analysis of CENH3 has not been attempted in these organisms. A cenh3 null mutant in A. thaliana allows us to completely replace the endogenous protein with transgenic variants (Ravi and Chan 2010). Here we report four major conclusions regarding CENH3 function in A. thaliana: (1) CENH3 function requires an N-terminal histone tail domain, although either the CENH3 tail or the H3 tail can support mitotic chromosome segregation. (2) Inserting the CENP-A targeting domain of CENH3 into H3 does not confer CENH3 function. (3) Complementation of cenh3 by heterologous CENH3 requires that the species of origin be closely related to A. thaliana. (4) Localization of a heterologous CENH3 protein to kinetochores in the presence of native CENH3 does not necessarily indicate that it can complement a cenh3 mutant. Overall, our results indicate that requirements for CENH3 function in A. thaliana are more stringent that those obtained in human cells. They underscore the usefulness of comparative studies of centromere function using genetically tractable experimental organisms.     

oiwa,a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase,Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.     

Identification of Pak1 inhibitors using water thermodynamic analysis

Abstract

p21-activated kinases (Paks) play an integral component in various cellular diverse processes. The full activation of Pak is dependent upon several serine residues present in the N-terminal region, a threonine present at the activation loop, and finally the phosphorylation of these residues ensure the complete activation of Pak1. The present study deals with the identification of novel potent candidates of Pak1 using computational methods as anti-cancer compounds. A diverse energy based pharmacophore (e-pharmacophore) was developed using four co-crystal inhibitors of Pak1 having pharmacophore features of 5 (DRDRR), 6 (DRHADR), and 7 (RRARDRP and DRRDADH) hypotheses. These models were used for rigorous screening against e-molecule database. The obtained hits were filtered using ADME/T and molecular docking to identify the high affinity binders. These hits were subjected to hierarchical clustering using dendritic fingerprint inorder to identify structurally diverse molecules. The diverse hits were scored against generated water maps to obtain WM/MM ΔG binding energy. Furthermore, molecular dynamics simulation and density functional theory calculations were performed on the final hits to understand the stability of the complexes. Five structurally diverse novel Pak1 inhibitors (4835785, 32198676, 32407813, 76038049, and 32945545) were obtained from virtual screening, water thermodynamics and WM/MM ΔG binding energy. All hits revealed similar mode of binding pattern with the hinge region residues replacing the unstable water molecules in the binding site. The obtained novel hits could be used as a platform to design potent drugs that could be experimentally tested against cancer patients having increased Pak1 expression.