SlPPDK modulates sugar and acid metabolism to influence flavor quality during tomato fruit ripening.

Fruit ripening is a highly intricate process, where the dynamic interplay of soluble sugar and organic acid metabolism is crucial for developing the characteristic flavor qualities. Pyruvate orthophosphate dikinase (PPDK) plays a pivotal role in modulating the process of gluconeogenesis during plant development. However, the specific physiological role of PPDK in fruit development has yet to be elucidated. In this study, we investigated the expression pattern, subcellular localization and functional significance of SlPPDK in tomato fruits. Our results reveal that SlPPDK is highly expressed in fruits and flowers, with its expression progressively increasing as the fruit ripens. Subcellular localization analyses demonstrate that SlPPDK is distributed in the cell membrane, cytoplasm, and nucleus. Using CRISPR/Cas9 technology, we generated SlPPDK knockout mutants, which exhibited a marked reduction in enzyme activity, leading to significant alterations in sugar and organic acid metabolism. These findings highlight the critical role of SlPPDK in maintaining the sugar-acid balance essential for tomato flavor quality and provide a foundation for future breeding strategies aimed at enhancing tomato fruit flavor.

Considering a Utility-Centric Framework Based on "Minimum Orthophosphate" Criteria for Mitigation of Elevated Cuprosolvency in Drinking Water.

Gaps in the United States Environmental Protection Agency (US EPA) Lead and Copper Rule (LCR) leave some consumers and their pets vulnerable to high cuprosolvency in drinking water. This study seeks to help proactive utilities who wish to mitigate cuprosolvency problems through the addition of orthophosphate corrosion inhibitors. The minimum doses of orthophosphate necessary to achieve acceptable cuprosolvency in relatively new copper pipe were estimated as a function of alkalinity via linear regressions for the 90th, 95th, and 100th percentile copper tube segments (R2 > 0.98, n = 4). Orthophosphate was very effective at reducing cuprosolvency in the short term but, in some cases, resulted in higher long-term copper concentrations than the corresponding condition without orthophosphate. Alternatives to predicting "long-term" results for copper tubes using simpler bench tests starting with fresh Cu(OH)2 solids showed promise but would require further vetting to overcome limitations such as maintaining water chemistry and orthophosphate residuals and to ensure comparability to results using copper tube.

Highly Efficient Far-Red Emitting Mg21Ca4Na4(PO4)18: Ce3+, Eu3+ Luminescent Glasses: A Novel Approach Towards Plant Cultivation Technologies.

One of the exciting developments in contemporary luminescence research is the development of rare earth triggered luminescent glasses, which are a type of lanthanide activated luminous material. For the first time, Ce3+, Eu3+ activated/co-activated Mg21Ca4Na4(PO4)18 orthophosphate glasses have been synthesized using the proposed work's melt quenching technique. The proposed glass sample's XRD pattern has an amorphous character, although its most prominent peak matches data from the Mg21Ca4Na4(PO4)18 standard ICSD database. FT-IR analysis was used to analyze the proposed glass sample's vibrational characteristics. Co-activated Mg21Ca4Na4(PO4)18 glass exhibits large emission peaks under UV excitations that cover the far red area during a photoluminescence examination. These outcomes demonstrate the proposed sample's value in applications such as WLEDs and plant cultivation.

Effect of a calcium silicate cement and experimental glass ionomer cements containing calcium orthophosphate particles on demineralized dentin.

OBJECTIVE: The study aims to evaluate the effect of a glass ionomer cement (GIC; Fuji 9 Gold Label, GC) with added calcium orthophosphate particles and a calcium silicate cement (CSC; Biodentine, Septodont) regarding ion release, degradation in water, mineral content, and mechanical properties of demineralized dentin samples. METHODS: GIC, GIC + 5% DCPD (dicalcium phosphate dihydrate), GIC + 15% DCPD, GIC + 5% ß-TCP (tricalcium phosphate), GIC + 15% ß-TCP (by mass), and CSC were evaluated for Ca2+/Sr2+/F- release in water for 56 days. Cement mass loss was evaluated after 7-day immersion in water. Partially demineralized dentin disks were kept in contact with materials while immersed in simulated body fluid (SBF) at 37 °C for 56 days. The "mineral-to-matrix ratio" (MMR) was determined by ATR-FTIR spectroscopy. Dentin hardness and elastic modulus were obtained by nanoindentation. Samples were observed under scanning and transmission electron microscopy. Data were analyzed by ANOVA/Tukey test (α = 0.05). RESULTS: Ca2+ release from CSC and GIC (µg/cm2) were 4737.0 ± 735.9 and 13.6 ± 1.6, respectively. In relation to the unmodified GIC, the addition of DCPD or ß-TCP increased ion release (p < 0.001). Only the dentin disks in contact with CSC presented higher MMR (p < 0.05) and mechanical properties than those restored with a resin composite used as control (p < 0.05). Mass loss was similar for GIC and CSC; however, the addition of DCPD or ß-TCP increased GIC degradation (p < 0.05). CONCLUSION: Despite the increase in ion release, the additional Ca2+ sources did not impart remineralizing capability to GIC. Both unmodified GIC and CSC showed similar degradation in water. CLINICAL RELEVANCE: CSC was able to promote dentin remineralization.

Fractionation and Lability of Phosphorus Species in Cottonseed Meal-Derived Biochars as Influenced by Pyrolysis Temperature.

Defatted cottonseed meal (CSM), the residue of cottonseeds after oil extraction, is a major byproduct of the cotton industry. Converting CSM to biochar and utilizing the goods in agricultural and environmental applications may be a value-added, sustainable approach to recycling this byproduct. In this study, raw CSM was transformed into biochar via complete batch slow pyrolysis at 300, 350, 400, 450, 500, 550, and 600 °C. Thermochemical transformation of phosphorus (P) in CSM during pyrolysis was explored. Fractionation, lability, and potential bioavailability of total P (TP) in CSM-derived biochars were evaluated using sequential and batch chemical extraction techniques. The recovery of feed P in biochar was nearly 100% at ≤550 °C and was reduced to <88% at 600 °C. During pyrolysis, the organic P (OP) molecules predominant in CSM were transformed into inorganic P (IP) forms, first to polyphosphates and subsequently to orthophosphates as promoted by a higher pyrolysis temperature. Conversion to biochar greatly reduced the mobility, lability, and bioavailability of TP in CSM. The biochar TP consisted of 9.3-17.9% of readily labile (water-extractable) P, 10.3-24.1% of generally labile (sequentially NaHCO3-extractable) P, 0.5-2.8% of moderately labile (sequentially NaOH-extractable) P, 17.0-53.8% of low labile (sequentially HCl-extractable) P, and 17.8-47.5% of residual (unextractable) P. Mehlich-3 and 1 M HCl were effective batch extraction reagents for estimating the "readily to mid-term" available and the "overall" available P pools of CSM-derived biochars, respectively. The biochar generated at 450 °C exhibited the lowest proportions of readily labile P and residual P compounds, suggesting 450 °C as the optimal pyrolysis temperature to convert CSM to biochar with maximal P bioavailability and minimal runoff risk.

Demonstrating the utility of sugar-phosphate phosphatases in coupled enzyme assays: galactose-1-phosphate uridylyltransferase as proof-of-concept.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase).

Shotgun Metagenomics Reveals Impacts of Copper and Water Heater Anodes on Pathogens and Microbiomes in Hot Water Plumbing Systems.

Hot water building plumbing systems are vulnerable to the proliferation of opportunistic pathogens (OPs), including Legionella pneumophila and Mycobacterium avium. Implementation of copper as a disinfectant could help reduce OPs, but a mechanistic understanding of the effects on the microbial community under real-world plumbing conditions is lacking. Here, we carried out a controlled pilot-scale study of hot water systems and applied shotgun metagenomic sequencing to examine the effects of copper dose (0-2 mg/L), orthophosphate corrosion control agent, and water heater anode materials (aluminum vs magnesium vs powered anode) on the bulk water and biofilm microbiome composition. Metagenomic analysis revealed that, even though a copper dose of 1.2 mg/L was required to reduce Legionella and Mycobacterium numbers, lower doses (e.g., ≤0.6 mg/L) measurably impacted the broader microbial community, indicating that the OP strains colonizing these systems were highly copper tolerant. Orthophosphate addition reduced bioavailability of copper, both to OPs and to the broader microbiome. Functional gene analysis indicated that both membrane damage and interruption of nucleic acid replication are likely at play in copper inactivation mechanisms. This study identifies key factors (e.g., orthophosphate, copper resistance, and anode materials) that can confound the efficacy of copper for controlling OPs in hot water plumbing.

An electrochemiluminescence immunosensor with double co-reaction accelerators based on Ag3PO4@EuPO4-AgNP for detecting squamous cell carcinoma antigen.

This study aimed to design a sandwich electrochemiluminescence (ECL) immunosensor with double co-reaction accelerators for sensitively detecting squamous cell carcinoma antigen (SCCA). First, silver orthophosphate (Ag3PO4) nanoparticles were modified on the surface of EuPO4 nanowires to improve their poor dispersibility/solubility. At the same time, EuPO4 was used as a co-reaction accelerator to catalyze S2O82- to produce more intermediates (SO4•-), significantly enhancing the ECL signal of Ag3PO4. Ag nanoparticles (AgNP) modified on Ag3PO4@EuPO4 composite nanomaterials were used not only as linkers of luminescence groups and biomarkers but also as a co-reaction accelerator to effectively enhance ECL signal. The designed ECL immunosensor displayed several advantages, including good stability and reproducibility. Under the optimal conditions, its linear range in detecting SCCA was 0.0001-50 ng·mL-1, the detection limit was 25 fg·mL-1 (S/N = 3), the recovery was 96.6-100.4%, and the relative standard deviation was less than 4.8%. It was successfully applied to detect SCCA in human serum.

The Use of Bi-Potentiostat as a Simple and Accurate Electrochemical Approach for the Determination of Orthophosphate in Seawater.

Autonomous on-site monitoring of orthophosphate (PO43-), an important nutrient for primary production in natural waters, is urgently needed. Here, we report on the development and validation of an on-site autonomous electrochemical analyzer for PO43- in seawater. The approach is based on the use of flow injection analysis in conjunction with a dual electrochemical cell (i.e., a bi-potentiostat detector (FIA-DECD) that uses two working electrodes sharing the same reference and counter electrode. The two working electrodes are used (molybdate/carbon paste electrode (CPE) and CPE) to correct for matrix effects. Optimization of squarewave voltammetry parameters (including step potential, amplitude, and frequency) was undertaken to enhance analytical sensitivity. Possible interferences from non-ionic surfactants and humic acid were investigated. The limit of quantification in artificial seawater (30 g/L NaCl, pH 0.8) was 0.014 µM for a linear concentration range of 0.02-3 µM. The system used a Python script for operation and data processing. The analyzer was tested for ship-board PO43- determination during a four-day research cruise in the North Sea. The analyzer successfully measured 34 samples and achieved a good correlation (Pearson' R = 0.91) with discretely collected water samples analyzed using a laboratory-based colorimetric reference analyzer.

Recovery of Phosphate(V) Ions from Water and Wastewater Using Chitosan-Based Sorbents Modified-A Literature Review.

Over the past two decades, there has been increasing interest in the use of low-cost and effective sorbents in water treatment. Hybrid chitosan sorbents are potential materials for the adsorptive removal of phosphorus, which occurs in natural waters mainly in the form of orthophosphate(V). Even though there are numerous publications on this topic, the use of such sorbents in industrial water treatment and purification is limited and controversial. However, due to the explosive human population growth, the ever-increasing global demand for food has contributed to the consumption of phosphorus compounds and other biogenic elements (such as nitrogen, potassium, or sodium) in plant cultivation and animal husbandry. Therefore, the recovery and reuse of phosphorus compounds is an important issue to investigate for the development and maintenance of a circular economy. This paper characterizes the problem of the presence of excess phosphorus in water reservoirs and presents methods for the adsorptive removal of phosphate(V) from water matrices using chitosan composites. Additionally, we compare the impact of modifications, structure, and form of chitosan composites on the efficiency of phosphate ion removal and adsorption capacity. The state of knowledge regarding the mechanism of adsorption is detailed, and the results of research on the desorption of phosphates are described.

Risk assessment and identification of factors influencing the historical concentrations of microcystin in Lake Taihu, China.

Understanding the history of microcystins (MCs) pollution in large lakes can help inform future lake management. We collected sediment cores from Lake Taihu to: investigate the long-term record of MCs (MC-LR, MC-YR, and MC-RR), explore the main environmental drivers of MCs, and assess their public health and ecological risks. Results showed that MCs content in all cores increased over time. The core from north Taihu had the highest MC concentrations, with an average total MCs (sum of MC-LR, MC-YR, and MC-RR = TMCs) content of (74.31±328.55) ng/g. The core from eastern Taihu showed the lowest average TMCs content of (2.91±3.95) ng/g. PCA showed that sediment MCs at the three sites were positively correlated with sediment chlorophyll-a. MC-LR and MC-YR in northern and western Taihu negatively correlated with both the sediment total organic carbon/sediment total nitrogen ratio (STOC/STN) and water nitrate (NO3--N) concentration, but three MC congeners at eastern Taihu showed positive correlations with water orthophosphate (PO43--P), NO3--N, and STOC/STN. Generalized additive model analysis at each site revealed that NO3--N was the main TMCs driver in northern and western Taihu where phytoplankton dominated, whereas PO43--P was the main TMCs driver in eastern Taihu where macrophytes dominated. At the whole lake scale, total phosphorus (TP) and PO43--P were the most important environmental drivers influencing MCs; TP explained 47.4%, 44.2%, and 47.6% while orthophosphate explained 34.8%, 31.2%, and 34.7% of the deviance on TMCs, MC-LR, and MC-YR, respectively. NO3--N also showed a strong effect on MCs variation, especially on MC-YR. Risk assessment showed that both ecological and public health risk has increased in recent years. We conclude that while control of phosphorus and nitrogen input should be a major focus for future lake management, lake zone-specific management strategies may also be important.

Spectroscopic study of the 4fn-15dtransitions of LaPO4doped with Pr3+ or co-doped with Pr3+ and Gd3+ in the vacuum ultra violet region.

The energy off-dtransitions depends on the crystalline field in which the lanthanide ion is inserted. Depending on the experimental setup, these transitions could occur at high energy, so several studies regarding theoretical data have been conducted. Here, we present the experimental determination of the energy of interconfigurational 4fn â†’ 4fn-15d (f-d)transitions from Pr3+ions to the lanthanum orthophosphate LaPO4matrix; we have also determined the bandgap value for this host. The experiments were carried out at the Synchrotron setup of the Brazilian LNLS laboratory. Specifically, we synthesized LaPO4:Pr3+and LaPO4:Pr3+/Gd3+by the hydrothermal method under different pH conditions or by spray pyrolysis. The particles resulting from hydrothermal synthesis had different morphologies and the influence of pH value was showed: the reaction medium was controlled along the process, which changed the surface potential. On the basis of Raman spectroscopy and x-ray diffraction analyses, we found that the crystalline phase was monoclinic monazite for all the samples. We studied the 4f5dlevel and bandgap transitions at high energy by absorption analysis in the VUV range. The experimental results were 7.5 eV (LaPO4bandgap) and 5 eV (4fn→ 4fn-15dtransition of the Pr3+ion), which were close to the theoretical values reported in the literature for this ion and this matrix.

Early phase effects of silicate and orthophosphate on lead (Pb) corrosion scale development and Pb release.

Orthophosphate is widely used to control lead (Pb) release in drinking water distribution systems, but phosphorus addition is not sustainable. Alternative corrosion control treatments are needed, and sodium silicate is one possibility. Here, pre-corroded Pb coupons-with and without free chlorine-were used to examine early-phase corrosion scale development after silicate addition, with orthophosphate as a reference corrosion inhibitor. Scale development was evaluated in terms of total Pb release, phase transformation, electrochemical impedance, morphological changes, Pb dissolution kinetics, and short-term Pb-Cu galvanic corrosion. Elevated Pb release occurred for approximately one month after silicate addition, and total Pb release peaked at 1968.1 µg/L and 1176.9 µg/L from systems with and without free chlorine, respectively. In contrast, orthophosphate-treated coupons exhibited fewer, less pronounced spikes in Pb release. By day 354, the median total Pb release from orthophosphate-treated coupons with and without free chlorine had decreased to 3.7 and 5.0 µg/L, respectively, while the median total Pb release from corresponding silicate-treated coupons was much higher, at 44.9 µg/L and 34.3 µg/L. Calcium lead apatite (Ca0.56Pb3.77(PO4)3OH0.67) was identified in orthophosphate-treated scales, with hydroxylpyromorphite (Pb5(PO4)3OH) present in the absence of free chlorine. Plattnerite occurred on coupons in all chlorinated systems. Pb silicate compounds were not detected, but Ca2SiO4 and Na2Ca2(SiO3)3 were identified by X-ray powder diffraction. The charge transfer: film resistance ratio characterizing the orthophosphate-treated coupons decreased slowly while that of the silicate-treated coupons increased after silicate was added. These variations suggest orthophosphate provided better corrosion control than silicate did. Silicate treatment generally caused degradation of the top Pb scale layer, resulting in elevated Pb release, while orthophosphate encouraged the growth of more structured, generally thicker, corrosion scales that were effective in limiting Pb release.

Strategies for enhancing phosphorous removal in Vertical Flow Constructed Wetlands.

Constructed wetlands (CWs) are among the fastest emerging treatment methods for wastewater treatment. Unlike their organics and nitrogen removal capacities, the potential of CWs as a sink for phosphorous is still debatable. In this study, the secondary data from several CWs treating domestic sewage were compiled and assessed. Curves were plotted between orthophosphate-phosphorous (PO43--P) loading and the corresponding removal rates. Other factors affecting PO43--P removal like depth of the CW, surface area, organic loading rate etc. Were also analyzed. Removal rates of PO43--P were conforming to a linear positive relation with the loading rates. Pea gravel as a CW medium performed consistently well (60-80% removal) for a wide range of influent PO43--P loading (0.5-1.5 g/m3-d). The increased depth of the wetland appears to favor phosphate removal. PO43--P removal was found to be correlated with outlet dissolved oxygen, total Kjeldahl nitrogen removal and effluent nitrate. The study suggests that proper design, optimal organic loading and suitable pre-treatment may increase the applicability of CWs for phosphate removal from domestic wastewater. Larger area requirements can also be avoided by increasing their depth while keeping the volume of the filter media the same.

Enhancing resource recovery via cranberry syrup waste at the Wisconsin Rapids WRRF: An experimental and modeling study.

The Wisconsin Rapids Wastewater Treatment Plant (WRWWTP) is faced with a more stringent effluent phosphorus requirement that will drive capital investment between 2020 and 2025. The facility will need to achieve a monthly average value of 0.36 mg L-1 of total phosphorus (TP). While the facility has sufficient influent carbon to drive a conventional enhanced biological phosphorus removal (EBPR) configuration, the existing infrastructure makes the addition of influent selector zones cost prohibitive. Underutilized aeration basin capacity was repurposed for testing return activated sludge (RAS) fermentation. The WRWWTP began pilot testing of RAS fermentation in April 2021. The facility moved through a series of operational setpoints to optimize phosphorus removal in a sidestream RAS (SSR) configuration, including RAS diversion, decrease of DO in aeration basins and chemical dosing shutoff. One of the key implementations was the addition of cranberry syrup waste to provide additional carbon for RAS fermentation, converting the process to a SSR plus carbon (SSRC) configuration. By the end of the testing period, effluent total phosphorus was averaging less than 0.4 mg L-1 with no chemical addition. A model was developed in the SUMO platform and was used to capture orthophosphate trends during the testing period. The model investigated microbial population dynamics and found that the operational changes including RAS diversion, chemical dosing shutoff and cranberry syrup waste addition impacted the enrichment of phosphorus accumulating organisms (PAO). After performing a sensitivity analysis on hydrolysis parameters, the predicted hydrolysis rate around 1.8-1.9 mg COD g VSS-1 hr-1 was found to match the batch rate testing data. This is the first study where cranberry syrup waste was used to successfully enhance EBPR performance, resulting in 90% TP removal. While further research is needed regarding the composition of the waste matrix and the microbial community composition, this expands the routes for resource recovery in the field of wastewater treatment.

Quartz Crystal Microbalance with Dissipation: A New Approach of Examining Corrosion of New Copper Surfaces in Drinking Water.

Corrosion of copper material in drinking water systems causes public health concerns and plumbing failures. This study investigated the early corrosion of new copper surfaces in situ using a novel technique: quartz crystal microbalance with dissipation (QCMD). The QCMD results showed that increasing the water pH from 6.5 to 9.0 and the addition of 6 mg/L orthophosphate at pH 6.5 and 9.0 slowed down the copper surface mass changes as indicated by the reduced changes in frequency (Δf5) at 51-89% and total copper release at 29-72%. The water pH 9.0 without orthophosphate was the most likely to induce localized corrosion relative to other conditions at pH 6.5 and pH 9.0 with orthophosphate. Based on the changes in dissipation values (ΔD5) from QCMD and the morphology, microstructure, and composition of the deposited copper corrosion byproducts, digital microscopy, field-emission scanning electron microscopy with energy dispersive spectroscopy, and X-ray photoelectron spectrometry analyses confirmed that the pH and orthophosphate inhibited copper corrosion with different mechanisms. QCMD provided sensitive, rapid, and continuous responses to mass and surface changes and can be useful for evaluating early water corrosivity to new copper.

High Temperature Ultrasonic Transducers: A Review.

There are many fields such as online monitoring of manufacturing processes, non-destructive testing in nuclear plants, or corrosion rate monitoring techniques of steel pipes in which measurements must be performed at elevated temperatures. For that high temperature ultrasonic transducers are necessary. In the presented paper, a literature review on the main types of such transducers, piezoelectric materials, backings, and the bonding techniques of transducers elements suitable for high temperatures, is presented. In this review, the main focus is on ultrasonic transducers with piezoelectric elements suitable for operation at temperatures higher than of the most commercially available transducers, i.e., 150 °C. The main types of the ultrasonic transducers that are discussed are the transducers with thin protectors, which may serve as matching layers, transducers with high temperature delay lines, wedges, and waveguide type transducers. The piezoelectric materials suitable for high temperature applications such as aluminum nitride, lithium niobate, gallium orthophosphate, bismuth titanate, oxyborate crystals, lead metaniobate, and other piezoceramics are analyzed. Bonding techniques used for joining of the transducer elements such as joining with glue, soldering, brazing, dry contact, and diffusion bonding are discussed. Special attention is paid to efficient diffusion and thermo-sonic diffusion bonding techniques. Various types of backings necessary for improving a bandwidth and to obtain a short pulse response are described.

Gluconeogenesis in Plants: A Key Interface between Organic Acid/Amino Acid/Lipid and Sugar Metabolism.

Gluconeogenesis is a key interface between organic acid/amino acid/lipid and sugar metabolism. The aims of this article are four-fold. First, to provide a concise overview of plant gluconeogenesis. Second, to emphasise the widespread occurrence of gluconeogenesis and its utilisation in diverse processes. Third, to stress the importance of the vacuolar storage and release of Krebs cycle acids/nitrogenous compounds, and of the role of gluconeogenesis and malic enzyme in this process. Fourth, to outline the contribution of fine control of enzyme activity to the coordinate-regulation of gluconeogenesis and malate metabolism, and the importance of cytosolic pH in this.

The acidocalcisome inositol-1,4,5-trisphosphate receptor of Trypanosoma brucei is stimulated by luminal polyphosphate hydrolysis products.

Acidocalcisomes are acidic calcium stores rich in polyphosphate (polyP) and are present in trypanosomes and also in a diverse range of other organisms. Ca2+ is released from these organelles through a channel, inositol 1,4,5-trisphosphate receptor (TbIP3R), which is essential for growth and infectivity of the parasite Trypanosoma brucei However, the mechanism by which TbIP3R controls Ca2+ release is unclear. In this work, we expressed TbIP3R in a chicken B lymphocyte cell line in which the genes for all three vertebrate IP3Rs were stably ablated (DT40-3KO). We show that IP3-mediated Ca2+ release depends on Ca2+ but not on ATP concentration and is inhibited by heparin, caffeine, and 2-aminomethoxydiphenyl borate (2-APB). Excised patch clamp recordings from nuclear membranes of DT40 cells expressing only TbIP3R disclosed that luminal inorganic orthophosphate (Pi) or pyrophosphate (PPi), and neutral or alkaline pH can stimulate IP3-generated currents. In contrast, polyP or acidic pH did not induce these currents, and nuclear membranes obtained from cells expressing rat IP3R were unresponsive to polyP or its hydrolysis products. Our results are consistent with the notion that polyP hydrolysis products within acidocalcisomes or alkalinization of their luminal pH activate TbIP3R and Ca2+ release. We conclude that TbIP3R is well-adapted to its role as the major Ca2+ release channel of acidocalcisomes in T. brucei.

Improving the stability of the malachite green method for the determination of phosphate using Pluronic F68.

The analytical determination of phosphate constitutes a fundamental step for the evaluation of catalytic activities of many enzymes involved in the hydrolysis of phosphate-containing biomolecules. The most sensitive colorimetric methods to quantify phosphate are based on measuring the spectral changes produced by the adsorption of malachite green to 12-molybdophosphoric acid. Malachite green methods are time-consuming because they require the preparation of color reagent on the day of use, due to its low stability. In this work we propose a modification of the malachite green method that overcomes these problems and only requires a one-step, ready-to-use stock solution including perchloric acid and Pluronic F68. The improved reaction mixture allowed the quantification of phosphate with a limit of detection of 0.22 µM, a dynamical range up to 80 µM depending on the optical path-length, and a molar absorption coefficient at 640 nm of 94000 M-1cm-1. Color development reaches a steady level within 30 min and remains constant for at least 2 h. The high stability of the color reagent allows long-term storage for at least 1 year. The optimized procedure is especially useful to measure phosphate-containing biomolecule levels and enzyme activities when low values are critical.