Evaluating vanadium bioavailability to cabbage in rural soils using geochemical and micro-spectroscopic techniques.

Assessing the vanadium (V) fractionation and speciation to predict its bioavailability using a combined approach of geochemical extractions and micro-spectroscopic techniques is still not well studied. Therefore, we aimed to determine the bioavailability of V in rural soils using single extractants, sequential extraction procedure, and the X-ray absorption near edge structure (XANES) spectroscopy. We collected and characterized ninety four samples originated from horizons of seventeen soil profiles in Taiwan. We determined the total content of V and its geochemical fractions using the BCR sequential extraction procedure to predict its potential mobility. We also assessed the bioavailability of V in the soils using four availability indices i.e., CaCl2, HCl, ethylenediaminetetraacetic acid (EDTA), and NaHCO3 and related them to its uptake by Chinese cabbage (Brassica chinensis L.). Additionally, we determined the V speciation by vanadium K-edge XANES spectra. Moreover, we studied the elemental compositions of the soils using Electron Probe Micro Analysis (EPMA). Vanadium was mainly distributed in the residual fraction (81-98% of total V). Among the potential mobile fractions, V was mainly associated with Fe oxides, as identified by the BCR sequential extraction and EMPA. The XANES analysis indicated that V mainly existed in the soils as V(IV) and V(V). The EDTA and NaHCO3 extracted more V than CaCl2 and HCl, and both, particularly NaHCO3 were positively and significantly correlated with the total soil content and plant shoot concentrations of V; therefore NaHCO3 might be recommended as a bioavailability index for soil V. We hypothesize that the NaHCO3 may extract vanadate from soil surfaces and also vanadate transformed from vanadyl at alkaline pH during the extraction. The NaHCO3-extracted V can be predicted by a function of soil total V, CEC, and pH. Our results should be verified using different soils and plants in the future.

Epigenetic regulation affects gene amplification in Drosophila development.

In Drosophila melanogaster, in response to developmental transcription factors, and by repeated initiation of DNA replication of four chorion genes, ovarian follicle cells, form an onion skin-type structure at the replication origins. The DNA replication machinery is conserved from yeast to humans. Subunits of the origin recognition complex (ORC) is comprised of Orc1, Orc2, and Cdc6 genes. While mutations of Orc1 and Orc2 and not Cdc6can be lethal, overexpression of these genes lead to female sterility. Ecdysone, is a steroidal prohormone of the major insect molting hormone 20-hydroxyecdysone that in Drosophila, triggers molting, metamorphosis, and oogenesis. To this end, we identified several ecdysone receptor (EcR) binding sites around gene amplification loci. We also found that H3K4 was trimethylated at chorion gene amplification origins, but not at the act1 locus. Female mutants overexpressing Lsd1 (a dimethyl histone H3K4 demethylase) or Lid (a trimethyl histone H3K4 demethylase), but not a Lid mutant, were sterile. The data suggest that ecdysone signaling determines which origin initiates DNA replication and contributes to the development. Screening strategies using Drosophila offer the opportunity for development of drugs that reduce gene amplification and alter histone modification associated with epigenetic effects.

Comparison of Sample Preparation Methods for Multielements Analysis of Olive Oil by ICP-MS.

Elemental analysis of olive oils by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is challenging because of the high organic load in olive oil samples and the low analyte concentrations. However, conflicting operating procedures in the preparation of oils prior to analysis by ICP-MS have been reported to overcome these difficulties. This study compared three methods of inorganic elements' extraction from olive oils: The two commonly used microwave-assisted, acid digestion, and liquid-liquid, ultrasound-assisted extraction methods; and an optimized method: The combined microwave digestion-evaporation. Overall, microwave digestion-based methods did not compare opportunely, and ultrasound-assisted extraction was found to provide the best accord between simplicity of use, detection limits and precision improvement. The detection limits were in the range of 0.3-160 µg·kg-1, 0.012-190 µg·kg-1 and 0.00061-1.5 µg·kg-1, while repeatabilities were in the range of 5-21%, 5.4-99% and 5.1-40% for the microwave digestion, the combined digestion-evaporation and the ultrasound assisted extraction, respectively. The ultrasound-assisted extraction is therefore recommended as a preparation method for olive oils prior to analysis by ICP-MS. The broader range of elements that can be accurately detected is expected to help increase the discriminatory power and performance of geographical traceability models.

Interregional traceability of Tunisian olive oils to the provenance soil by multielemental fingerprinting and chemometrics.

The aim of this study was to prove the usefulness of multielements as provenance markers of olive oils by evaluating their link with soil composition and their discriminatory power. Eleven elements in twenty-one olive oils and their paired soils from four Tunisian regions were characterized. Chemometrics have been implemented for ICP-MS data processing. Principal component analysis identified the predominant geochemical source of the elements in the oils based on their associations according to Goldschmidt's rule. Although a clear correlation was not proven, correspondence was identified between the discriminating elements for both the soils and olive oils, which included Fe, Rb, Mg, and Pb. Linear discriminant analysis achieved classification and prediction rates of 92.1% and 87.3%, respectively. Our study substantiates the validity of multielements as markers of the olive oils' provenance, and that an elemental fingerprinting approach can be successfully applied in the construction of a database of Tunisian olive oils.

Seabird-affected taluses are denitrification hotspots and potential N2O emitters in the High Arctic.

In High Arctic tundra ecosystems, seabird colonies create nitrogen cycling hotspots because of bird-derived labile organic matter. However, knowledge about the nitrogen cycle in such ornithocoprophilous tundra is limited. Here, we determined denitrification potentials and in-situ nitrous oxide (N2O) emissions of surface soils on plant-covered taluses under piscivorous seabird cliffs at two sites (BL and ST) near Ny-Ålesund, Svalbard, in the European High Arctic. Talus soils at both locations had very high denitrification potentials at 10 °C (2.62-4.88 mg N kg-1 dry soil h-1), near the mean daily maximum air temperature in July in Ny-Ålesund, with positive temperature responses at 20 °C (Q10 values, 1.6-2.3). The talus soils contained abundant denitrification genes, suggesting that they are denitrification hotspots. However, high in-situ N2O emissions, indicating the presence of both active aerobic nitrification and anaerobic denitrification, were observed only at BL (max. 16.6 µg N m-2 h-1). Rapid nitrogen turnover at BL was supported by lower carbon-to-nitrogen ratios, higher nitrate content, and higher δ15N values in the soils at BL compared with those at ST. These are attributed to the 30-fold larger seabird density at BL than at ST, providing the larger organic matter input.

Endoreduplication of the mouse genome in the absence of ORC1.

The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with LoxP sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ORC1 ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.

DNA replication machinery is required for development in Drosophila.

In Drosophila, some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

Chromosome and genetic testing using ChIP assay.

Chromatin immunoprecipitation (ChIP) assay can be used to easily visualize information about proteins, DNA, and RNA on chromosomes and is widely used for analysis of genomes, epigenomes, mRNAs, and non-coding RNAs. The ChIP assay can detect, not only DNA-binding proteins of various organisms, but also the temporal and spatial regulating mechanisms of RNA-binding proteins. Because of these features, demand for ChIP assay is expected to grow. Here, by using yeast and Drosophila as examples, we describe the superiority of the improved ChIP assay that we have developed.

Internal structure of cesium-bearing radioactive microparticles released from Fukushima nuclear power plant.

Microparticles containing substantial amounts of radiocesium collected from the ground in Fukushima were investigated mainly by transmission electron microscopy (TEM) and X-ray microanalysis with scanning TEM (STEM). Particles of around 2 µm in diameter are basically silicate glass containing Fe and Zn as transition metals, Cs, Rb and K as alkali ions, and Sn as substantial elements. These elements are homogeneously distributed in the glass except Cs which has a concentration gradient, increasing from center to surface. Nano-sized crystallites such as copper- zinc- and molybdenum sulfide, and silver telluride were found inside the microparticles, which probably resulted from the segregation of the silicate and sulfide (telluride) during molten-stage. An alkali-depleted layer of ca. 0.2 µm thick exists at the outer side of the particle collected from cedar leaves 8 months after the nuclear accident, suggesting gradual leaching of radiocesium from the microparticles in the natural environment.

Arsenic biotransformation by Streptomyces†sp. isolated from rice rhizosphere.

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As-contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S-adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.

[A case and literature review of ovarian carcinosarcoma with long-term survival after repeated recurrences].

Ovarian carcinosarcoma is a rare and aggressive tumor with a poor prognosis. We report a case of ovarian carcinosarcoma and also review the literature. In 2000, a 63-year-old woman underwent optimal cytoreductive surgery for ovarian carcinosarcoma( International Federation of Gynaecology and Obstetrics[FIGO]stage III c[pT3cN0M0]). She received adjuvant chemotherapy with paclitaxel and carboplatin(TC). In 2005, a recurrent tumor was noted anterior to the sacrum. The patient had a complete response after 6 cycles of TC chemotherapy; however, a year later, the tumor recurred and was resected. In 2013, the tumor recurred adjacent to the right kidney and was surgically removed after a partial response to 3 cycles of TC chemotherapy. The pathologic findings included epithelial and non-epithelial components with histologic variation and differentiation; specifically, a leiomyosarcoma, cartilaginous tissues with cellular atypia, and a rhabdomyosarcoma were identified in specimens obtained during the first, second, and third surgical procedures, respectively. In keeping with the combination theory of histogenesis, the ovarian carcinosarcoma described herein may have originated from a monoclonal stem cell. The long survival of this patient is attributed to optimal cytoreduction during the primary operation, solitary recurrent tumors that were completely resected, and sensitivity to chemotherapy.

An orc1 allele with a mutated APC motif is female sterile with amplification defects.

The origin recognition complex 1 (ORC1) is the largest subunit of the ORC, the heteromeric hexamer. ORC1 is an essential component of the pre-replicative complex (pre-RC) that licenses eukaryote DNA replication origins. The levels of ORC1 fluctuate during the mitotic cell cycle in Drosophila as well as in some human cells. Proteolysis of ORC1 occurs at the end of M phase in Drosophila, which is mediated by the anaphase-promoting complex (APC), and in late S phase in human cells by Skip-Cullin-F box (SCF). Previously we showed that proteolysis of ORC1 by APC is mediated by the ORC1 destruction box (the O-box), an APC motif conserved among species yet distinct from the D-box or KEN-box. Recently we showed that replacing the O-box with the D-box (ORC1O→D) changes the degradation profile of ORC1 during a canonical cell cycle. Here we report further characterization of the ORC1O→D allele that turned out to be a useful tool to examine the function of ORC1 in other modes of DNA replication during oogenesis. In endoreplication stages ORC1O→D does not change any DNA content profiles, consistent with our previous finding that ORC is dispensable for endoreplication. However, in amplification stage replication efficiency of ORC1O→D is drastically reduced, which resulted in amplification defects that led to thin egg shell phenotype. Taken together, our analyses show that orc1 allele newly identified is female sterile and possesses a unique feature of phenotypes that are distinct in different modes of DNA replication.

Function of the origin recognition complex 1 (ORC1) outside DNA replication in Drosophila.

The origin recognition complex (ORC) is an essential component of the pre-replicative complex (pre-RC) that binds to replication origins for licensing. Levels of the largest ORC subunit, ORC1, oscillate during the mitotic cell cycle and regulate origin usage. In Drosophila, ORC1 levels increase at the G(1)/S transition following E2F-dependent transcriptional activation, remain high until the end of M phase and then decrease at the M/G(1) transition when ORC1 is targeted for proteolysis by the anaphase-promoting complex (APC). A function, if any, for Drosophila ORC1 after S phase has not been described. Here, we determined the role of ORC1 at stages outside S phase by generating ORC1 derivatives with a modified ORC1 degradation box (the O-box) and examining the effects in vivo. These modifications either stabilized ORC1 by mutating the O-box (ORC1(Omut)) so that it is no longer targeted by APC or changed its degradation profile by replacing the O-box with the D-box of human cyclin B (ORC1(O→D)), so that degradation would occur earlier. We determined the distribution and tested the function of these ORC1 derivatives in an orc1 mutant background so that only the mutated protein was expressed. Stable version of ORC1, ORC1 (Omut), showed no effects on cell cycle progression; however, ORC1(O→D), which is degraded early at the G(2)/M transition, led to a higher frequency of M-phase cells but not S-phase cells. Taken together, our results indicate the timing of ORC1 degradation is required for timely progression in M phase.

Endoreplication: the advantage to initiating DNA replication without the ORC?

The endocycle is a developmentally specialized cell cycle that lacks M phase and consists of only S and G phases. Endoreplicating cells acquire high ploidies by multiple rounds of replication without cell divisions in order to increase protein synthesis to support growth and development of the organism. Endoreplication occurs in developmentally specialized cell types after they terminally differentiate. These cells include: ovarian nurse cells and follicle cells, most of the larval tissues in flies and the placenta giant trophoblasts and megakaryocytes in mammals. To date, studies of endoreplication have mainly focused on two aspects: Cyclin-dependent kinase (CDK)-mediated controls to license and re-license replication origins in the absence of mitosis, and development or differentiation signaling pathways that mediate the transition from mitotic replication to endoreplication. The replication initiation machinery itself has not been much studied, partly because it has been generally considered to consist of the same set of factors used in mitotic cycle. Recently we reported a loss-of-function analysis of the Drosophila orc1 gene, which revealed that the Origin Recognition Complex (ORC) is dispensable for endoreplication. This finding is surprising and rather provocative as it runs against the accepted dogma and is expected to stimulate discussion and interest in the identification of the molecular mechanisms of the initiation of endoreplication. What follows is a highly speculative view of how endoreplication occurs in the absence of the ORC and what advantage ORC-independent replication brings to the organism.

The origin recognition complex is dispensable for endoreplication in Drosophila.

The origin recognition complex (ORC) is an essential component of the prereplication complex (pre-RC) in mitotic cell cycles. The role of ORC as a foundation to assemble the pre-RC is conserved from yeast to human. Furthermore, in metazoans ORC plays a key role in determining the timing of replication initiation and origin usage. In this report we have produced and analyzed a Drosophila orc1 allele to investigate the roles of ORC1 in three different modes of DNA replication during development. As expected, ORC1 is essential for mitotic replication and proliferation in brains and imaginal discs, as well as for gene amplification in ovarian follicle cells. Surprisingly, however, ORC1 is not required for endoreplication. Decreased cell number in orc1 mutant salivary glands is consistent with the idea that undetectable levels of maternal ORC1 during embryogenesis fail to support further proliferation. Nevertheless, these cells begin endoreplicating normally and reach a final ploidy of >1000C in the absence of zygotic synthesis of ORC1. The dispensability of ORC is further supported by an examination of other ORC members, whereas Double-parked protein/Cdt1 and minichromosome maintenance proteins are apparently essential for endoreplication, implying that some aspects of initiation are shared among the three modes of DNA replication. This study provides insight into the physiologic roles of ORC during metazoan development and proposes that DNA replication initiation is governed differently in mitotic and endocycles.

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle.

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. Here, we report that APC/C Fzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We demonstrate that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, our data suggest that the activity of APC/C Fzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, our data suggest a model in which the cyclic activity of APC/C Fzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. We propose that, as is observed in mitotic cycles, during endocycles, APC/C Fzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression.

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest.

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway.

DNA replication-related element (DRE) and the DRE-binding factor (DREF) play an important role in regulating DNA replication-related genes such as PCNA and DNA polymerase alpha in Drosophila. We have previously reported that overexpression of DREF in developing eye imaginal discs induced ectopic DNA synthesis and apoptosis, which results in rough eyes. To identify genetic interactants with the DREF gene, we have carried out a screen for modifiers of the rough eye phenotype. One of the suppressor genes identified was the Drosophila orc2 gene. A search for known transcription factor recognition sites revealed that the orc2 gene contains three DREs, named DRE1 (+14 to +21), DRE2 (-205 to -198), and DRE3 (-709 to -702). Band mobility shift analysis using Kc cell nuclear extracts detected the specific complex formed between DREF and the DRE1 or DRE2. Specific binding of DREF to genomic region containing the DRE1 or DRE2 was further demonstrated by chromatin immunoprecipitation assays, suggesting that these are the genuine complexes formed in vivo. The luciferase assay in Kc cells indicated that the DRE sites in the orc2 promoter are involved in a transcriptional regulation of the orc2 gene. The results, taken together, demonstrate that the orc2 gene is under the control of DREF pathway.

A novel motif governs APC-dependent degradation of Drosophila ORC1 in vivo.

Regulated degradation plays a key role in setting the level of many factors that govern cell cycle progression. In Drosophila, the largest subunit of the origin recognition complex protein 1 (ORC1) is degraded at the end of M phase and throughout much of G1 by anaphase-promoting complexes (APC) activated by Fzr/Cdh1. We show here that none of the previously identified APC motifs targets ORC1 for degradation. Instead, a novel sequence, the O-box, is necessary and sufficient to direct Fzr/Cdh1-dependent polyubiquitylation in vitro and degradation in vivo. The O-box is similar to but distinct from the well characterized D-box. Finally, we show that O-box motifs in two other proteins, Drosophila Abnormal Spindle and Schizosaccharomyces pombe Cut2, contribute to Cdh1-dependent polyubiquitylation in vitro, suggesting that the O-box may mediate degradation of a variety of cell cycle factors.

Degradation of origin recognition complex large subunit by the anaphase-promoting complex in Drosophila.

The initiation of DNA synthesis is thought to occur at sites bound by a heteromeric origin recognition complex (ORC). Previously, we have shown that in Drosophila, the level of the large subunit, ORC1, is modulated during cell cycle progression and that changes in ORC1 concentration alter origin utilization during development. Here, we investigate the mechanisms underlying cell cycle-dependent degradation of ORC1. We show that signals in the non-conserved N-terminal domain of ORC1 mediate its degradation upon exit from mitosis and in G1 phase by the anaphase-promoting complex (APC) in vivo. Degradation appears to be the result of direct action of the APC, as the N-terminal domain is ubiquitylated by purified APC in vitro. This regulated proteolysis is potent, sufficient to generate a normal temporal distribution of protein even when transcription of ORC1 is driven by strong constitutive promoters. These observations suggest that in Drosophila, ORC1 regulates origin utilization much as does Cdc6 in budding yeast.