Color stability of bleached tooth enamel brushed with different stain-removing toothpastes.

OBJECTIVE: The effects of four toothpastes on the color stability of in-office bleached tooth specimens were determined. MATERIALS AND METHODS: We evaluated an experimental toothpaste (EXP) and three commercially available toothpastes: Colgate Optic White (OPW), Aquafresh White & Protect (AWP), and Crest 3D White (CDW). OPW, AWP, and CDW contained inorganic abrasives, whereas EXP and AWP contained sodium polyphosphate. Forty-eight randomly selected human-extracted maxillary central incisors were bleached and brushed twice daily over 30 days. We analyzed the final color difference (ΔE*ab, ΔE00 , ΔWID ), arithmetic average surface roughness (Ra) of the enamel measured on days 0 and 30, and scanning electron microscopy images of enamel surfaces and toothpastes. ΔE*ab, ΔE00 , ΔWID , and Ra were analyzed using one-way analysis of variance and Tukey's test (α = 0.05). RESULTS: ΔE*ab and ΔE00 values were significantly lower after toothbrushing with EXP, OPW, and CDW than with AWP. OPW induced the greatest positive ΔWID . Ra was significantly increased by OPW and CDW, but slightly increased by AWP, with cube-like particles, and EXP, with no particle-like structures. CONCLUSIONS: Only EXP stabilized the color of bleached teeth without increasing the enamel surface roughness. Sodium polyphosphate with approximately 10 phosphate groups was effective at removing stains. CLINICAL SIGNIFICANCE: The effect of toothpaste on the color stability of bleached teeth depends on the constituting abrasives and chemical components. Polyphosphoric acid has different stain-removal effects depending on its degree of polymerization. Additionally, although certain types of abrasives may be effective for color stability, they also increase the surface roughness of the enamel.

Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes.

SUMMARYAcidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels.

A comprehensive investigation to the fate of phosphorus in full-scale wastewater treatment plants using aluminum salts for enhanced phosphorus removal.

This study investigated the fate of phosphorus (P) in 8 full-scale municipal wastewater treatment plants (WWTPs) in Shanghai, China, in which both biological nutrient removal and aluminum-based chemical phosphorus removal were used. The results showed that 83.8-98.9 % P was transferred to the sludge in the 8 WWTPs by both chemical and biological reactions. P speciation analysis indicated that chemical P precipitates accounted for 84.3 % in the activated sludge, of which crystalline AlPO4 and amorphous iron­phosphorus compounds (FePs) were the main components. Sludge with more water-soluble and weakly adsorbed P was generated in the anaerobic-anoxic-oxic (A/A/O) process than in other processes. Among the 8 WWTPs, the one with the largest flow rate and relatively short sludge retention time (SRT) had the best potential to release P from all types of sludge. The recovery potential of P from thickened sludge can be improved by separately thickening the sludge produced in the high-efficiency sedimentation tank or feeding it into the dewatering process directly. Different P removal chemicals and dosing points changed the amount of chemical precipitate formed but had little effect on the composition of P accumulating organisms (PAOs) at the genus level. Although aluminum-based coagulants were applied in the investigated WWTPs, Fe in wastewater had the most positive effect on the proliferation of PAOs. The synthesis of polyphosphate was also related to the metabolism of PAOs as it affected transmembrane inorganic phosphate (Pi) transport and polyhydroxybutyrate (PHB) synthesis. The in-depth understanding of the fate of P is beneficial to improve P recovery efficiency in WWTPs.

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 PO 3 - ${\rm{PO}}_3^ - $ monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 PO 3 - $\;{\rm{PO}}_3^ - $ monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 PO 3 - ${\rm{PO}}_3^ - $ monomers), trimetaphosphate (trimetaP: 3 PO 3 - ${\rm{PO}}_3^ - $ ring), tripolyphosphate (tripolyP), pyrophosphate (PPi ), and inorganic orthophosphate (o-Pi ) were visualized after separation by an in situ hydrolytic degradation process to o-Pi that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. 31 P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PPi diffusivity by <10%. Linear regression between the Rf peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 PO 3 - ${\rm{PO}}_3^ - $ monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-Pi , but no PPi .

Chemical derivatization as a novel strategy for selective and sensitive determination of intracellular di-and triphosphate anabolites in peripheral blood mononuclear cells.

Quantitation of intracellular tri-phosphate anabolite, GSK1-TP, was required to understand drug efficacy of a proprietary molecule, GSK1; while quantitation of the di-phosphate, GSK1-DP, provided an indicator of potential GSK1-TP instability during sample processing and storage. A novel derivatization approach with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hexylamine was developed to mitigate the challenges associated with the analysis of GSK1-DP and GSK1-TP, rendering them more amendable to reversed-phase LC-MS/MS detection. Extensive effort was spent to minimize the analyte loss and cell counts variability during peripheral blood mononuclear cells (PBMC) collection and best practices were thoroughly discussed. A solution of 30/70/2 (v/v/v) RPMI-1640/methanol/trichloroacetic acid proved to be an effective method of analyte stabilization and recovery. Methods were developed for simultaneous analysis of GSK1-DP and GSK1-TP with a limit of quantitation of 2.0 ng/mL in dog and rat PBMC lysate, with a subsequent improvement to 0.1 ng/mL for the analysis of GSK1-TP in human PBMC lysate. All three assays were found to be robust over the PBMC concentration range of 1-25 × 106 lysed cells/mL. This novel methodology could alleviate some challenges associated with the bioanalysis of intracellular phosphorylated anabolites such as PBMC collection variability, analyte instability, poor chromatographic retention, high carryover, matrix effect and insufficient assay sensitivity.

Integration of Microbial Transformation Mechanism of Polyphosphate Accumulation and Sulfur Cycle in Subtropical Marine Mangrove Ecosystems with Spartina alterniflora Invasion.

Excessive phosphorus can lead to eutrophication in marine and coastal ecosystems. Sulfur metabolism-associated microorganisms stimulate biological phosphorous removal. However, the integrating co-biotransformation mechanism of phosphorus and sulfur in subtropical marine mangrove ecosystems with Spartina alterniflora invasion is poorly understood. In this study, an ecological model of the coupling biotransformation of sulfur and phosphorus is constructed using metagenomic analysis and quantitative polymerase chain reaction strategies. Phylogenetic analysis profiling, a distinctive microbiome with high frequencies of Gammaproteobacteria and Deltaproteobacteria, appears to be an adaptive characteristic of microbial structures in subtropical mangrove ecosystems. Functional analysis reveals that the levels of sulfate reduction, sulfur oxidation, and poly-phosphate (Poly-P) aggregation decrease with increasing depth. However, at depths of 25-50 cm in the mangrove ecosystems with S. alterniflora invasion, the abundance of sulfate reduction genes, sulfur oxidation genes, and polyphosphate kinase (ppk) significantly increased. A strong positive correlation was found among ppk, sulfate reduction, sulfur oxidation, and sulfur metabolizing microorganisms, and the content of sulfide was significantly and positively correlated with the abundance of ppk. Further microbial identification suggested that Desulfobacterales, Anaerolineales, and Chromatiales potentially drove the coupling biotransformation of phosphorus and sulfur cycling. In particular, Desulfobacterales exhibited dominance in the microbial community structure. Our findings provided insights into the simultaneous co-biotransformation of phosphorus and sulfur bioconversions in subtropical marine mangrove ecosystems with S. alterniflora invasion.

Extraction processes reduce polyphosphate ion migration, dispersion and diffusion as detected with gel electrophoresis and 31 P DOSY NMR.

Inorganic polyphosphates (polyPs) have been identified in eukaryotic and prokaryotic cells alike. Various extraction methods have been optimized as a necessary step before identification and measurement of these polymers. Three commercially available sodium polyP glasses were either dissolved or dissolved and extracted by two commonly used polyP extraction techniques - perchloric acid or buffered phenol-chloroform. The products were separated by polyacrylamide gel electrophoresis (PAGE), stained with toluidine blue O, and the migration results quantitatively compared. Both extraction processes reduced the relative migration distances of the peak and leading edges, and the stained band lengths, suggesting reduced polyP migration and dispersion. 31 P diffusion-ordered spectroscopy nuclear magnetic resonance confirmed that polyP extraction by perchloric acid or phenol-chloroform processes reduced polyP diffusion coefficients and suggested hydrolytic degradation with stronger end-chain signals. Reduced polyP diffusivity after extraction makes possible an overestimation of synthetic polyP chain length assignment when compared to unextracted polyP ladders with PAGE. The mechanism(s) for reduced synthetic polyP diffusion after extraction and intracellular chemical environment effects on migration are not known.

New insights into the role of acidocalcisomes in trypanosomatids.

Acidocalcisomes are electron-dense organelles rich in polyphosphate and inorganic and organic cations that are acidified by proton pumps, and possess several channels, pumps, and transporters. They are present in bacteria and eukaryotes and have been studied in greater detail in trypanosomatids. Biogenesis studies of trypanosomatid acidocalcisomes found that they share properties with lysosome-related organelles of animal cells. In addition to their described roles in autophagy, cation and phosphorus storage, osmoregulation, pH homeostasis, and pathogenesis, recent studies have defined the role of these organelles in phosphate utilization, calcium ion (Ca2+ ) signaling, and bioenergetics, and will be the main subject of this review.

Vtc5 Is Localized to the Vacuole Membrane by the Conserved AP-3 Complex to Regulate Polyphosphate Synthesis in Budding Yeast.

Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3 residues to thousands of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound vacuolar transporter chaperone (VTC) complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen, where it is stored at high concentrations. VTC's activity is promoted by an accessory subunit called Vtc5. In this work, we found that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we found that Vtc5 is rerouted to the vacuole lumen by the endosomal sorting complex required for transport (ESCRT), where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes. IMPORTANCE Long polymers of inorganic phosphates called polyphosphates are ubiquitous across biological kingdoms. From bacteria to humans, they have diverse functions related to protein homeostasis, energy metabolism, and cell signaling. In this study, we provide new insights into the intracellular trafficking of the polyphosphate biosynthetic machinery in the budding yeast S. cerevisiae. The critical advances of the work are 2-fold. First, it provides an explanation for decreased polyphosphate levels observed in cells mutated for a conserved intracellular trafficking machine. Second, it defines critical pathways that are highly likely to serve as hubs for polyphosphate regulation in yeast and other species.

Comparing Biochemical and Raman Microscopy Analyses of Starch, Lipids, Polyphosphate, and Guanine Pools during the Cell Cycle of Desmodesmus quadricauda.

Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.

Detection of polyphosphates in seafood and its relevance toward food safety.

Polyphosphates are permitted as food additives (Regulation EC No 1129/2011) but their undeclared utilisation is considered fraudulent. They improve water holding capacity of the seafood, preventing biochemical/physical changes during commercialization. The key objective of this study was the detection of polyphosphate in various seafood categories, by means of high-performance ion-exchange chromatography with suppressed conductometry (HPIEC-SCD) coupled to Q-Exactive Orbitrap high resolution mass spectrometry (HRMS-Orbitrap). Ten frozen cuttlefish samples did not reveal any treatment, while in ten frigate tunas, high concentration of orthophospate was found. Unambiguous hexametaphosphate presence was demonstrated in four prawn samples, while triphosphate was quantified (11.2 ± 4 ug/g) in another four prawn samples that contained orthophosphate (10225 ± 1102 ug/g), as well. Other samples sporadically encompassed polyphosphates profiles that varied according species and processing type. This analytical approach provided sustenance in better understanding regarding utilization of polyphosphates through HRMS fingerprinting of anionic species that would be specific in food safety control.

Living on the edge: Prospects for enhanced biological phosphorus removal at low sludge retention time under different temperature scenarios.

The design of new wastewater treatment plants with the aim of capturing organic matter for energy recovery is a current focus of research. Operating with low sludge residence time (SRT) appears to be a key factor in maximizing organic matter recovery. In these new configurations, it is assumed that phosphorus is chemically removed in a tertiary step, but the integration of enhanced biological phosphorus removal (EBPR) into these short-SRT systems seems to be an alternative worth studying. A key point of this integration is to prevent the washout of polyphosphate accumulating organisms (PAO) despite the low SRT applied. However, the minimum SRT required to avoid PAO washout depends on temperature, due to its effects on reaction kinetics, gas transfer rates, biomass growth and decay rates. This work includes a wide range of short and long-term experiments to understand these interactions and shows which combinations of SRT and temperature are detrimental to PAO growth. For example, an EBPR system operating at 20 °C and SRT = 5 d showed good performance, but EBPR activity was lost at 10 °C. EBPR operated at SRT = 10 d had 86% P removal at 20 °C but decreased to 71% at 15 °C and progressively lost its activity at lower temperature. The temperature coefficient obtained for PAO show a low degree of temperature dependence (θ = 1.047 ± 0.014), and should be considered when designing short-SRT systems with EBPR.

La-based-adsorbents for efficient biological phosphorus treatment of wastewater: Synergistically strengthen of chemical and biological removal.

The present work demonstrated the invention of synergistically strengthen of chemical and biological removal of phosphorus (P) in biological wastewater treatment, which was achieved by exposure the bioreactors to different levels of La-based-adsorbents. We fabricated a high-performance La2O2CO3 micro-adsorbent (H-La2O2CO3) and added it into sequencing batch reactors. When activated sludge was exposed to 40 mg/L H-La2O2CO3 for 40 d, effluent total phosphorus (TP) concentration significantly decreased to approximately 0.18 mg/L, with the steady removal efficiency of 96.4%, which is superior to the biological phosphorus removal (BPR). The effect of H-La2O2CO3 dosages on P removal in biological wastewater treatment was also detailedly investigated. The H-La2O2CO3 adsorbent could not only capture P by chemical bonding itself, but also increased protein (PN) contents of extracellular polymeric substances (EPS) and changed the functional group of EPS to chemically adsorb P. Additionally, the results of 16s rDNA molecular analysis revealed that the species richness and microbial diversity varied with the different dosages of adsorbent. Sequence analyses showed that the appropriate concentration of H-La2O2CO3 addition increased the contents of several polyphosphate accumulating organisms (PAOs) at genus level in sludge.

Effect of chlorine dioxide and phosphates on the quality of tiger frog (Rana tigrina) meat during 4 °C storage.

Tiger frog (Rana tigrina) meat is extremely perishable. This study investigated the antimicrobial efficacy of chlorine dioxide (ClO2 ) on frog meat, optimized the formulation of a phosphate-based enhancement solution by response surface methodology (RSM), and determined the quality parameters (i.e., total aerobic counts [TAC], pH, drip loss, cooking loss, color measurements, shear force, total volatile basic nitrogen [TVB-N], and thiobarbituric acid-reactive substances [TBARS]) of refrigerated frog meat pretreated with ClO2 and the optimized blend of phosphates. Treatments of frog meat with 35 and 70 ppm ClO2 for 3, 5, and 10 min achieved a 0.7-, 0.9- and 0.9-, and 0.8-, 1.4- and 1.6-log CFU/g reduction of TAC, respectively, indicating the antimicrobial efficacy of ClO2 was concentration- and time-dependent with such that higher concentrations and/or longer exposure time achieved greater bacterial reductions. The concentrations of the phosphates, including sodium tripolyphosphate (STPP), sodium pyrophosphate (SPP), and sodium hexametaphosphate (SHMP), were optimized as the formula of 0.3% STPP and 0.45% SPP obtaining the highest water retention of the frog meat. After washed with 70 ppm ClO2 for 10 min and subsequently soaked with 0.3% STPP and 0.45% SPP for 30 min, the frog meat stored at 4 °C shown significantly (P < 0.05) lower TAC (<4.4 log CFU/g) and higher water holding capacity during the whole storage of 12 days, compared to the control. Results indicated that the two-step process may be applicable to slow down deterioration and maintain quality frog meat during refrigeration. PRACTICAL APPLICATION: This research provides a means to slow down deterioration, maintain quality frog meat, and improve stability during refrigeration. Refrigerated frog meat products, which are preferred by consumers with juicier and more tender texture compared to the frozen-thawed meat, could be developed by the frog industry based on the data from this study.

Methods for the Analysis of Polyphosphate in the Life Sciences.

Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function.

Biosynthesis and half-life of MBX-2168-triphosphate in herpes virus-infected cells.

The third generation of methylenecyclopropane nucleoside analogs (MCPNAs) elicit an anti-viral effect against all three sub-classes of herpes viruses without inducing cytotoxicity in vitro. It has been previously established that the mechanism of action of MCPNAs is similar to that of ganciclovir (GCV) or acyclovir (ACV). However, the activation of MBX-2168, a third generation MCPNA, involves additional and unique enzymatic steps and this process has not been examined in virus-infected cells. To that end, herpes virus-infected cells were incubated with MBX-2168, synguanol, GCV, or ACV. Incubation of HCMV-infected cells with five times the EC50 of MBX-2168 (4.0 µM), synguanol (10.5 µM), or GCV (25 µM) resulted in a time-dependent increase in triphosphate accumulation reaching a maximum of 48.1 ± 5.5, 45.5 ± 2.5, and 42.6 ± 3.7 pmol/106 cells at 120 h, respectively. Additionally, half-lives of these compounds were similar in HCMV-infected cells (GCV-TP = 25.5 ± 2.7 h; MBX-2168-TP/synguanol-TP = 23.0 ± 1.4 h). HSV-1-infected cells incubated with five times the EC50 of MBX-2168 (33.5 µM) or ACV (5.0 µM) demonstrated a time-dependent increase in triphosphate levels reaching a maximum of 12.3 ± 1.5 and 11.6 ± 0.7 pmol/106 cells at 24 h, respectively. ACV-TP and MBX-2168-TP also had similar half-lives under these conditions (27.3 ± 4.8 h and 22.2 ± 2.2 h, respectively). We therefore conclude that although MBX-2168 does not follow the classical route of nucleoside analog activation, the metabolic profile of MBX-2168 is similar to other nucleoside analogs such as GCV and ACV that do.

Dictyostelium discoideum as a Model to Study Inositol Polyphosphates and Inorganic Polyphosphate.

The yeast Saccharomyces cerevisiae has given us much information on the metabolism and function of inositol polyphosphates and inorganic polyphosphate. To expand our knowledge of the metabolic as well as functional connections between inositol polyphosphates and inorganic polyphosphate, we have refined and developed techniques to extract and analyze these molecules in a second eukaryotic experimental model, the amoeba Dictyostelium discoideum. This amoeba, possessing a well-defined developmental program, is ideal to study physiological changes in the levels of inositol polyphosphates and inorganic polyphosphate, since levels of both molecules increase at late stages of development. We detail here the methods used to extract inositol polyphosphates using perchloric acid and inorganic polyphosphate using acidic phenol. We also present the postextraction procedures to visualize and quantify these molecules by polyacrylamide gel electrophoresis and by malachite green assay.

Aromatic vs. Aliphatic Hyperbranched Polyphosphoesters as Flame Retardants in Epoxy Resins.

The current trend for future flame retardants (FRs) goes to novel efficient halogen-free materials, due to the ban of several halogenated FRs. Among the most promising alternatives are phosphorus-based FRs, and of those, polymeric materials with complex shape have been recently reported. Herein, we present novel halogen-free aromatic and aliphatic hyperbranched polyphosphoesters (hbPPEs), which were synthesized by olefin metathesis polymerization and investigated them as a FR in epoxy resins. We compare their efficiency (aliphatic vs. aromatic) and further assess the differences between the monomeric compounds and the hbPPEs. The decomposition and vaporizing behavior of a compound is an important factor in its flame-retardant behavior, but also the interaction with the pyrolyzing matrix has a significant influence on the performance. Therefore, the challenge in designing a FR is to optimize the chemical structure and its decomposition pathway to the matrix, with regards to time and temperature. This behavior becomes obvious in this study, and explains the superior gas phase activity of the aliphatic FRs.

Purification of silica-free DNA and characterization of its role in coagulation.

BACKGROUND: Although extracellular DNA has been reported to activate coagulation, its direct effects and consequent interpretations have recently been questioned because of silica and polyphosphate (polyP) contaminations when DNA is isolated using common silica-based kits. OBJECTIVES: To identify and characterize alternative methods of isolating DNA that is free of silica with functionally undetectable levels of polyP. METHODS: DNA was isolated from the whole blood or buffy coat using three different DNA isolation kits: (a) the silica-based QIAGEN QIAMP DNA Blood mini kit (silica-DNA), (b) the non-silica-based QIAGEN PAXgene Blood DNA kit (PAX-DNA), and (c) the non-silica-based QuickGene DNA whole blood kit large (DBL-DNA). The procoagulant properties of DNA were assessed by thrombin generation and plasma clotting assays. A polyP detection assay was used to detect polyP contamination. RESULTS AND CONCLUSIONS: Unlike the isolated DNA, commercially available calf thymus DNA contains thrombinlike amidolytic activity. The PAX-DNA and DBL-DNA did not contain silica nor functionally detectable polyP as contaminants. Both PAX- and DBL-DNA were procoagulant in a dose-dependent manner, which is neutralized with deoxyribonuclease I (DNase I). Thus, we recommend the use of PAX-DNA or DBL-DNA for functional studies to investigate the role of extracellular DNA.

Isolated Saccharomyces cerevisiae vacuoles contain low-molecular-mass transition-metal polyphosphate complexes.

Vacuoles play major roles in the trafficking, storage, and homeostasis of metal ions in fungi and plants. In this study, 29 batches of vacuoles were isolated from Saccharomyces cerevisiae. Flow-through solutions (FTS) obtained by passing vacuolar extracts through a 10 kDa cut-off membrane were characterized for metal content using an anaerobic liquid chromatography system interfaced to an online ICP-MS. Nearly all iron, zinc, and manganese ions in these solutions were present as low-molecular-mass (LMM) complexes. Metal-detected peaks with masses between 500-1700 Da dominated; phosphorus-detected peaks generally comigrated. The distribution of metal:polyphosphate complexes was dominated by particular chain-lengths rather than a broad binomial distribution. Similarly treated synthetic FeIII polyphosphate complexes showed similar peaks. Treatment with a phosphatase disrupted the LMM metal-bound species in vacuolar FTSs. These results indicated metal:polyphosphate complexes 6-20 phosphate units in length and coordinated by 1-3 metals on average per chain. The speciation of iron in FTSs from iron-deficient cells was qualitatively similar, but intensities were lower. Under healthy conditions, nearly all copper ions in vacuolar FTSs were present as 1-2 species with masses between 4800-7800 Da. The absence of these high-mass peaks in vacuolar FTS from cup1Δ cells suggests that they were due to metallothionein, Cup1. Disrupting copper homeostasis increased the amount of LMM copper:polyphosphate complexes in vacuoles (masses between 1500-1700 Da). Potentially dangerous LMM copper species in the cytosol of metallothionein-deficient cells may traffic into vacuoles for sequestration and detoxification.