Phosphate Release Research Articles

Metal oxide loaded biochars derived from Chinese bai jiu distillers' grains used for the adsorption and controlled release of phosphate

The resource utilization of distillers' grains (DGs) is an essential issue for the sustainable development of Chinese bai jiu industry. In this study, metal oxide (MxOy, M=Mg, Al and Fe) loaded biochar (MBC) was prepared by using DGs for the production of novel adsorption/release materials for phosphate (P). The results indicated that the P adsorption capacity of Mg-BC, Al-BC and Fe-BC were as high as 14.74, 8.06 and 14.51 mg P/g, respectively. XPS and FTIR analysis demonstrated that the adsorption mechanism of MBC for P included electrostatic attraction, hydrogen bonding, ion exchange, precipitation and ligand exchange. The release of P from M-BC+P could be controlled by low molecular weight organic acids (LMWOAs), which were affected by the loaded metal oxides on the biochar. The release of P from Mg-BC+P was higher than 70 % after 7 days, which was about 60 % if Al-BC+P was used. Thus, M-BC+P can be used as a high-performance controlled release phosphate fertilizer, providing a promising value-added application approach for the bai jiu distillers' grains.

Formation of hydroxyapatite nanoprecursors by the addition of bioactive particles in resin-modified glass ionomer cements

The objective of this study was to evaluate the impact of adding bioactive glasses 45S5 and niobophosphate (NbG) in different concentrations (5, 10 and 20%) of two resin modified glass ionomer cements (RMGIC). Two RMGIC were used: Vitro Fil LC (DFL) and Resiglass F (Biodinâmica) they were modified by the addition of two bioactive glasses - 45S5 or NbG. Different concentrations of bioactive glasses (5, 10 and 20 wt%) were mixed with the powder of the RMGICs. The properties evaluated were: alkalinization activity (pH), fluoride (F−), calcium (Ca2+) and phosphate (PO43−) release, flexural strength, flexural modulus, Knoop microhardness and bioactivity. The analyses of the surfaces were examined using SEM and FTIR after storage at 37 °C for 28 days in simulated body fluid (SBF). The data were analyzed using ANOVA One-way (multiple comparison vs control) and Holm-Sidak test (α = 0.05). The addition of 45S5 increased the pH while the addition of NbG neutralized the pH of the Vitro Fil RMGIC. The RMGIC Resiglass maintained its acidic pH condition in almost all conditions. The addition of the 45S5 and NbG in high concentration increased the release of F−, Ca2+ and PO43− ions. Resiglass did not reduce its mechanical properties with the addition of bioactive glasses (p > 0.05). NbG glass did not alter the mechanical properties of the Vitro Fil (p > 0.05). The addition of the bioactive glasses 45S5 and NbG contributed to the formation of nanoprecursors of hydroxyapatite. The obtaining of bioactive RMGIC is dependent on the composition of the material. The NbG glass provides a neutralization of the pH, an increase of the ions release and the amount of precipitates without jeopardizing the mechanical properties of the RMGIC.

Enhanced Phytase Production by Bacillus subtilis subsp. subtilis in Solid State Fermentation and its Utility in Improving Food Nutrition

Phytic acid acts as anti-nutritional factor in food and feed ingredients for monogastric animals as they lack phytases. Phytase production by Bacillus subtilis subsp. subtilis JJBS250 was studied in solid-state fermentation and its applicability in dephytinization of food. Bacterial culture was grown in solid state fermentation using wheat bran and various culture conditions were optimized using 'One variable at a time' (OVAT) approach. Effects of different substrates (wheat bran, wheat straw, sugarcane bagasse), incubation time (24, 48, 72 and 96 h), incubation temperatures (25, 30, 35 and 40°C), pH (4.0, 5.0, 6.0, 7.0 and 8.0) and moisture content (1:1.5, 1:2.0, 1:2.5 and 1:3) were studied on phytase production. Bacterial phytase was used in dephytinization of food samples. Optimization of phytase production was studied in solid state fermentation (SSF) using 'One variable at a time' (OVAT) approach. Bacillus subtilis subsp. subtilis JJBS250 grew well in various agroresidues in SSF and secreted high enzyme titres using wheat bran at 30°C and pH 5.0 after incubation time of 48 h with substrate to moisture ratio of 1:3. Glucose and ammonium sulphate supplementation to wheat bran further enhanced phytase production in SSF. Optimization of phytase production resulted in 2.4-fold improvement in phytase production in solid state fermentation. The enzyme resulted in dephytinization of wheat and rice flours with concomitant release of inorganic phosphate, reducing sugar and soluble protein. Optimization resulted in 2.34-fold enhancement in phytase production by bacterial culture that showed dephytinization of food ingredients with concomitant release of nutritional components. Therefore, phytase of B. subtilis subsp. subtilis JJBS250 could find application in improving nutritional quality of food and feed of monogastric animals.

Removal and potential mechanisms of Cr(Ⅵ) contamination in phosphate mining wasteland by isolated Bacillus megatherium PMW-03

Inappropriate mining strategies have led to the development of an increasing number of phosphate mining wastelands containing a large amount of rock phosphate (RP) contaminated with hexavalent chromium (Cr (Ⅵ)). Although bioremediation has been investigated as an effective strategy for Cr (Ⅵ) removal, few studies have focused on microbial remediation coupled with RP. In this study, a strain of Bacillus megatherium named PMW-03 isolated from a phosphate mining wasteland was used to remediate Cr (Ⅵ) contamination. The liquid medium experiment showed that strain PMW-03 exhibited significant insoluble phosphate solubilization and Cr (Ⅵ) resistance. The maximum release of soluble phosphate and removal rate of Cr (Ⅵ) reached 319.941 mg/L and 94.8%, respectively. The activities of antioxidation enzymes and chromate reductase reached 145.08, 153.71 and 40.07 U/min/gm, which indicated that the bioreduction process participated in the Cr (Ⅵ) removal of strain PMW-03. Furthermore, FTIR and XRD studies showed that Cr (Ⅵ) removal in RP coupled with PMW-03 was also attributed to the biosorption of functional groups on the surface of the strain and the formation of Cr-phosphate precipitates. Finally, the evolution of microbial community structures and functional gene abundance were analyzed to reveal the reduction of Cr (Ⅵ) by microbial remediation. These findings provide some reference and new insights for microbial remediation of Cr (Ⅵ) contamination in phosphate mining wastelands.

The redox chemistry of phosphate complexed green rusts: Limited oxidative transformation and phosphate release

The reductive dissolution of ferric (hydr)oxide-phosphate (P) complex is believed to be responsible for P release under anoxic conditions. Although it has been observed that P release may not correlate to the oxygenation concentrations of bottom waters, the evidence and related mechanism are still missing. In this work, the release of P from green rust (GR)-P complex under low/high pO2 levels and in the presence of high-valent metal oxidants [Cr(VI)] was studied. It was found that high oxidant concentrations led to a fast release rate and a high released amount of P from the GR-P complex. A high amount of adsorbed P retards the oxidation of GR, which inhibits the P release process. A higher Fe(II)/Fe(III) ratio in GR leads to stronger P fixation ability. GR in GR-P complex was transformed to ferrihydrite and then to lepidocrocite and goethite, depending on the oxidation conditions. Our results demonstrate that the increased redox potential introduced by oxidants leads to the release of P from the GR-P complex, which deepens our understanding of the relationship between redox potential and the P fixation by iron-bearing minerals.

Phosphate loaded layered double hydroxides for active corrosion protection of carbon steel

ABSTRACT Layered double hydroxides (LDHs) can serve as corrosion inhibitor reservoirs. In the present work, hydrotalcite-like LDHs were synthesized and employed as host matrixes for the release of phosphate. The morphological and structural properties of the as-prepared LDH-PO4 were characterised by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). The PO4 3- adsorption uptake was successfully confirmed by the XRD results. The corrosion resistance of the coating with LDH-PO4 was investigated by electrochemical impedance spectroscopy (EIS), which was approximately 1 order of magnitude higher than that of the blank coating after immersion for 168 h. Results of the scanning Kelvin probe technique revealed that the potential of LDH-PO4 coating was higher and smoother than that of the blank coating after exposure to 0.5 M NaCl solution. This effect is attributed to the release of PO4 3− that forms a phosphate protective film in the scratched region.

Recent insights into the relative timing of myosin's powerstroke and release of phosphate.

Myosin is a motor enzyme that converts the chemical energy in ATP into mechanical work to drive a myriad of intracellular processes, from muscle contraction to vesicular transport. Key steps in the transduction of energy are the force-generating powerstroke, and the release of phosphate (Pi ) from the nucleotide-binding site. Both events occur rapidly after binding to actin, making it difficult to determine which event occurs first. Early efforts suggested that these events occur simultaneously; however, recent findings indicate that they are separate and distinct events that occur at different rates. High-resolution crystal structures of myosin captured in intermediate states of the ATPase cycle suggest that when Pi is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that Pi -release precedes the powerstroke. However, advances in functional assays, enabling sub-millisecond temporal and nanometer spatial resolution, are challenging this hypothesis. For example, Föster Resonance Energy Transfer (FRET) based assays, as well as single molecule laser trap assays, suggest the opposite; that the powerstroke occurs prior to the release of Pi from myosin's active site. This review provides some historical context and then highlights recent reports that reveal exciting new insight into this fundamental mechanism of energy transduction by this prototypical motor enzyme.

Beyond the single index: Investigating ecological mechanisms underpinning ecosystem multifunctionality with network analysis.

Ecosystems simultaneously deliver multiple functions that relate to both the activities of resident species and environmental conditions. One of the biggest challenges in multifunctionality assessment is balancing analytical simplicity with ecosystem complexity. As an alternative to index‐based approaches, we introduce a multivariate network analysis that uses network theory to assess multifunctionality in terms of the relationships between species' functional traits, environmental characteristics, and functions. We tested our approach in a complex and heterogeneous ecosystem, marine intertidal sandflats. We considered eight ecosystem function, five macrofaunal functional trait groups derived from 36 species, and four environmental characteristics. The indicators of ecosystem functions included the standing stock of primary producers, oxygen production, benthic oxygen consumption, DIN (ammonium and NOx efflux) and phosphate release from the sediments, denitrification, and organic matter degradation at the sediment surface. Trait clusters included functional groups of species that shared combinations of biological traits that affect ecosystem function: small mobile top 2 cm dwellers, suspension feeders, deep‐dwelling worms, hard‐bodied surface dwellers, and tube‐forming worms. Environmental characteristics included sediment organic matter, %mud, %shell hash, and %sediment water content. Our results visualize and quantify how multiple ecosystem elements are connected and contribute to the provision of functions. Small mobile top 2 cm dwellers (among trait clusters) and %mud (among environmental characteristics) were the best predictor for multiple functions. Detailed knowledge of multifunctionality relationships can significantly increase our understanding of the real‐world complexity of natural ecosystems. Multivariate network analysis, as a standalone method or applied alongside already existing single index multifunctionality methods, provides means to advance our understanding of how environmental change and biodiversity loss can influence ecosystem performance across multiple dimensions of functionality. Embedding such a detailed yet holistic multifunctionality assessment in environmental decision‐making will support the assessment of multiple ecosystem services and social‐ecological values.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Turnover of lake sediments treated with sediment microbial fuel cells: a long-term study in a eutrophic lake

Sediment microbial fuel cells (SMFCs) have previously been successfully used to reduce phosphate release from the sediments of eutrophic lakes. In this study, we investigate the risk that SMFCs stimulate sediment decomposition with the unwanted side effect being the release of legacy pollutants stored in sediments. Electrode pairs (16 m2 each) were installed in a eutrophic lake in Denmark and the electricity production was monitored over more than a year at three electrode fields. Equations were derived that allow calculation of the substrate turnover by the SMFCs from the working potential, the open circuit potential, and the external resistance of the SMFCs. The resulting turnover data suggest that the decomposition of the sediment is only slightly expedited by the SMFCs, and that the decomposition process is not significantly stimulated by the type of SMFCs installed in the lake. The measured maximum power density with stainless steel electrodes in the lake sediment was 0.9 mW/m2, which was sufficient to reduce P outflux from sediment. At this power density, the decomposition half-life of the lake sediment (top 5 cm) is calculated to be 277 years, which is only about 10% of natural lake sediment decomposition half-lives. Higher power densities are not necessary for P fixation but inadvertently increase the risk that legacy pollutants buried in the sediment are released.

Abstract 2407: Serine and threonine phosphorylation marks proteins for degradation by ClpXP

Abstract The mitochondrial serine protease ClpXP regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in multiple malignancies and inhibiting or hyperactivating it kills malignant cells in vitro and in vivo. In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phosphor-arginine for degradation. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation. To determine how phosphorylated amino acids influence ClpXP-mediated protein degradation, we incubated recombinant ClpXP with its unnatural substrate FITC-casein and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phosphor-tryrosine (pTry) in a cell-free assay and measured release of fluorogenic FITC. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP while pTyr, pArg, and the dephosphorylated amino acids had no effect on ClpXP activity. Likewise, ApSA and ApTA peptides inhibited ClpXP enzyme activity, while the non-phosphorylated version (ASA and ATA) and no effect. Next, we tested whether the phosphorylation state of full length proteins would influence their degradation by ClpXP. Using gel-based cell-free assays, the phosphorylation enriched α-casein and β-casein were degraded by recombinant ClpXP. In contrast, the κ-casein with low phosphorylation level and dephosphorylated α-casein were not cleaved by ClpXP. In addition, we treated α-casein with lambda-phosphatases to dephosphorylate the protein and confirmed the release of free phosphate by the Malachite green phosphate assay. Phosphatase treated α-casein was resistant to ClpXP cleavage. As ClpXP is an AAA ATPase, we asked if pSer and pThr acted on the ATPase of the enzyme. pSer and pThr did not inhibit the ATPase activity of ClpX, as measured by the malachite green ATPase assay. We also measured the effect of pSer and pThr on the peptidase activity of ClpP alone without its regulatory subunit ClpX. Neither pSer or pThr inhibited ClpP peptidase activity. We investigated if pSer and pThr could bind to ClpX. Using thermal shift binding assays, we demonstrated that pSer and pThr but not pTyr and pArg bind ClpX and none of the phosphorylated amino acids bind ClpP. Finally, we characterized the total and phopho- mitochondrial proteome of T-REX HEK293 wild type and ClpP knockout cells by mass spectrometry. We identified 402 unique mitochondrial proteins and 38 phosphorylated mitochondrial proteins in wild type cells. 20 phosphorylated mitochondrial proteins increased in the ClpP knockout cells compared to wild type with a cut-off of 2 fold. In summary, we discovered that ClpX binds pSer and pThr and phosphorylation of these amino acids mark proteins for degradation by the ClpXP mitochondrial protease. This work highlights a new strategy to develop inhibitors of ClpXP for the treatment of cancer. Citation Format: Yue Feng, Yulia Jitkova, Aaron D. Schimmer. Serine and threonine phosphorylation marks proteins for degradation by ClpXP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2407.

Effects of elevated sulfate in eutrophic waters on the internal phosphate release under oxic conditions across the sediment-water interface

Eutrophication in freshwater environments may be enhanced by the elevation of sulfate in waters, through the release of internal phosphorus (P) from anoxic sediments. However, the influence of increasing but modest sulfate concentrations (less than 3000 μM) on P release under oxic conditions across the sediment-water interface (SWI) in eutrophic freshwater is poorly understood. In this study, the profiles of P, iron (Fe), sulfur (S) and physicochemical parameters were measured in a simulated lacustrine system with varying concentrations of sulfate (970–2600 μM) in overlying water. The results indicated that elevated concentrations of sulfate increased the soluble reactive P in overlying waters under oxic conditions across the SWI. A 100 μM increase of sulfate was found to induce a 0.128 mgm−2d−1 increase of P flux from surface sediments into overlying waters under oxic conditions. Higher sulfate concentrations in the overlying waters increased the concentrations of labile S(-II) in the deep sediments, due to sulfate penetration and subsequent reduction to S(-II). We also found the fluxes of labile Fe (10.34 to 18.33 mgm−2d−1) and P (2.70 to 1.33 mgm−2d−1) from deep to surface sediment were both positive and greater than the corresponding fluxes (Fe, 2.2 to 3.51 and P, 2.6 to 0.39 mgm−2d−1, respectively) from surface sediment to the overlying water, suggesting that reduction of P-bearing Fe(III)(oxyhydr)oxides in deep anoxic sediment acted as a major source of internal P release. In addition, the upward flux of Fe(II) was significantly lower under higher sulfate conditions, indicating that the Fe(II) flux could be mitigated by formation of Fe(II) sulfides in the deep sediment. Under these conditions, less Fe(II) from deep sediments could be re-oxidized and combine with P in the surface, oxic sediment, thereby reducing the retention capacity for P and leading to higher release of internal P to the water column.

A Novel Assay for Phosphoserine Phosphatase Exploiting Serine Acetyltransferase as the Coupling Enzyme.

Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis—the hydrolysis of phosphoserine to serine and inorganic phosphate—in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman’s reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP.

Surface form analysis on complex freeform organic structures – measuring erosive wear on human teeth in vitro

Natural human enamel (NHE) is a complex freeform surface which has presented significant difficulties in measuring surface form change using non-contacting laser profilometry (NCLP). Measuring surface form change on NHE is a metrology proxy for measuring dental tooth structure loss, and characterising this using non-ISO parameters (volume, surface area, and normalised lesion depth) has been seldom studied due surface complexity and undetermined measurement errors. This study determines NCLP measurement errors (instrument repeatability and method reproducibility) for non-ISO parameters, characterises change in surface form on NHE following a dietary pH-cycling model. NHE (n = 1) was scanned consecutively twenty-times using NCLP with/without sample replacement producing consecutive surface profile data. Residual data was created after subtracting consecutive filtered profile data (80 μm, Gaussian filter), and mean (SD) volume, surface area, and normalised lesion depth was determined within a 1.5 mm circular region of interest (ROI). Volume error (expressed as height variation across ROI surface area) was 0.022 μm (instrument repeatability) and 0.149 μm (method reproducibility), whilst surface area error (expressed as percentage change of the surface area deviation across the entire surface area) was 0.034% (repeatability error) and 0.081% (reproducibility error). Sixty-four natural enamel surfaces taped with polyvinyl-chloride tape leaving 1.5 mm exposed ROI underwent dietary erosion cycling (three 5-min cycles, 0.3% citric acid w/v, pH 3.2) generating artificial erosion lesions. Samples were scanned with NCLP before/after each erosion cycle, scans filtered for microtexture, and after-erosion scans were subtracted from before-erosion scans. NCLP results show mean (SD) volume, surface area, normalised depth, and 3D step-height of the eroded area increased significantly after each erosion cycle, with no significant difference in calcium and phosphate release after each cycle. We demonstrate a robust and valid dental model with analysis workflow to measure surface form change in NHE using NCLP, improving understanding of measuring surface form change in complex freeform surfaces.

Modification of Hardwood Derived Biochar to Improve Phosphorus Adsorption

The excessive application of phosphorus in agricultural lands leads to serious environmental issues. Efficient application is beneficial from an economic and environmental perspectives. Biochar can be used as a carrier for slow release of phosphate. However, its adsorption capacity is limited. In this work, biochar was prepared at different pyrolysis temperatures (350–550 °C). The biochar prepared at 550 °C had the highest adsorption capacity and was selected for modification by magnesium impregnation. Magnesium modification enhanced the adsorption capacity by 34% to a theoretical max adsorption capacity of 463.5 mg·g−1. The adsorbed phosphate can be desorbed. The desorption was bi-phasic with fast- and slow-release fractions. The distribution of the phosphate fractions was pH dependent with slow release being most prominent in neutral conditions. Mg modified biochar can be used to recover phosphate and then used as a carrier for slow release of phosphate. The bi-phasic desorption behaviour is useful as the fast release fraction can provide the immediate phosphate needed during plant establishment, while the slow-release fraction maintains steady supply over extended periods.

Copper-Doped Biphasic Calcium Phosphate Powders: Dopant Release, Cytotoxicity and Antibacterial Properties.

Cytotoxicity and antibacterial properties associated with the dopant release of Cu-doped Biphasic Calcium Phosphate (BCP) powders, mainly composed of hydroxyapatite mixed with β-tricalcium phosphate powders, were investigated. Twelve BCP ceramics were synthesized at three different sintering temperatures (600 °C, 900 °C and 1200 °C) and four copper doping rates (x = 0.0, 0.05, 0.10 and 0.20, corresponding to the stoichiometric amount of copper in Ca10Cux(PO4)6(OH)2-2xO2x). Cytotoxicity assessments of Cu-doped BCP powders, using MTT assay with human-Mesenchymal Stem Cells (h-MSCs), indicated no cytotoxicity and the release of less than 12 ppm of copper into the biological medium. The antibacterial activity of the powders was determined against both Gram-positive (methicillin-sensitive (MS) and methicillin resistant (MR) Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The Cu-doped biomaterials exhibited a strong antibacterial activity against MSSA, MRSA and E. coli, releasing approximatively 2.5 ppm after 24 h, whereas 10 ppm were required to induce an antibacterial effect against P. aeruginosa. This study also demonstrated that the culture medium used during experiments can directly impact the antibacterial effect observed; only 4 ppm of Cu2+ were effective for killing all the bacteria in a 1:500 diluted TS medium, whereas 20 ppm were necessary to achieve the same result in a rich, non-diluted standard marrow cell culture medium.

19 DIET-INDUCED OBESITY UNIQUELY REGULATES GLUCOSE AND NACL ABSORPTION IN SPRAGUE DAWLEY RAT SMALL INTESTINAL EPITHELIAL CELLS

Background Traditional coupled NaCl absorption occurs via the dual operation of Na:H (NHE3) and Cl:HCO3 (DRA/PAT1) exchange in the brush border membrane of villus cells in the mammalian small intestine. In genetic models of obesity (Zucker rat and Tallyho mouse) and humans, coupled NaCl absorption was not affected. However, in these animal models, a novel coupling of Na-glucose co-transport (SGLT1) and Cl:HCO3 exchange was increased, potentially important in the pathogenesis of diabetes and hypertension associated with obesity. Human obesity is both genetic and environmental (e.g. diet mediated). But it is not known whether this novel coupled NaCl absorptive pathway is affected in diet-induced obesity (DIO). Hypothesis Novel coupled NaCl absorption mediated by SGLT1 and DRA/PAT1 is stimulated in DIO. Aim Determine the mechanisms of regulation of SGLT1 and DRA/PAT1 in DIO. Methods Sprague Dawley rats were fed with a high-fat diet (HFD; 60% fat calories) from 12-18 wks of age to induce obesity. Control rats were fed with standard chow. BBM vesicles (BBMV) were prepared from villus cells by Mg precipitation. Phlorizine sensitive, 3H-OMG uptake for SGLT1; amiloride-sensitive, 22Na uptake for NHE3 and DIDS sensitive 36Cl uptake for Cl:HCO3 exchange was performed in villus cell BBMV. Na/KATPase activity was determined as inorganic phosphate release. Western blot studies were performed using rat specific antibodies. Results In intact rat villus cells, SGLT1 uptake was significantly increased in HFD rats (2.4±0.2 nmol/mg protein×2 min in control and 6.4±0.6 in HFD; n=4, p Conclusions In DIO, BBM SGLT1 activity was stimulated in villus cells from HFD rats. This is not due to the altered Na extruding capacity of the cell. Cl:HCO3 exchange was also stimulated in DIO while NHE3 remained unaffected. These data indicate that in DIO, while the traditional coupled NaCl absorption is unaffected, a novel coupled NaCl absorptive pathway, via SGLT1 and DRA/PAT1 is stimulated resulting in enhanced glucose and NaCl absorption which is likely important in the pathophysiology of diabetes and hypertension of obesity.

The Enzymatic Activities of Nucleoside Triphosphate Diphosphohydrolases Towards Tenofovir Diphosphate and Their Spatial Distributions in Human Colorectal Tissue

Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug) must be phosphorylated to form a competitive inhibitor of HIV, TFV-diphosphate (TFV-DP). Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from human participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Therefore, we hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this, we incubated recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, and 8), individually, with TFV-DP to determine the abilities of these enzymes to dephosphorylate TFV-DP. Following incubation, TFV-DP levels were measured using ultra-high performance liquid chromatography tandem mass spectrometry. We observed TFV-DP dephosphorylation in the presence of NTPDase 1 or 3 enzymes. Further, a malachite green phosphate assay that measures the release of inorganic phosphate was employed to further confirm the activities of NTPDase 1 and 3 towards TFV-DP. Immunoblotting revealed that NTPDase 1 and 3 proteins are indeed expressed in human colorectal tissue lysates. To visualize the distributions of NTPDases across human colorectal tissue, we employed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI). Initially, trypsin digestions on a range of recombinant NTPDases including 1 and 3 were carried out to detect and identify their tryptic peptides that could be targeted for detection in MALDI MSI. From these digestions, NTPDase 1 yielded peptides, SQHQETPVYLGATAGMR, SLSNYPFDFQGAR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 exhibited the following peptides: YGIVLDAGSSR, GSGISSYGNNPQDVPR, and MFTVLTR. Five tryptic peptides corresponding to NTPDase 5 (which did not exhibit enzymatic activity towards TFV-DP) were identified: LYTHSYLGFGLK, LHQPEEVQR, GSFYAFSYYYDR, QGAETVQGLLEVAK, and LATLGALETEGTDGHTFR. Next, the abovementioned MALDI MS methodology was applied to directly visualize tryptic peptides of NTPDase 1, 3 and 5 in human colorectal tissue sections. In these studies, human colorectal biopsies were collected and sectioned to 20-micron thickness. Tissue sections were subjected to serial washing steps using ethanol. Trypsin and matrix (α-cyano-4-hydroxycinnamic acid) were deposited onto the tissue using a robotic sprayer. We detected tryptic peptides, SQHQETPVYLGATAGMR, and LATLGALETEGTDGHTFR corresponding to NTPDase 1 and 5, respectively. The distribution profiles of NTPDase 1 and 5 were generated using the m/z values of above peptides. The identities of the detected peptides were validated using mass spectrometry-based proteomics analyses. Thus, in this work, we established a MALDI MSI methodology for simultaneous visualization of tryptic peptides corresponding to NTPDases 1 and 5. Results from our in vitro studies suggest that NTPDase 1 and 3 may contribute to the regulation of TFV-DP levels.

Myosin's powerstroke occurs prior to the release of phosphate from the active site.

Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (Pi ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining Pi in its active site, by either elevating Pi in solution or by introducing a mutation in myosin's active site (S217A) to slow Pi -release from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s-1 ) and without a detectable delay, both in the absence and presence of 30 mM Pi . The elevated levels of Pi did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that Pi rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower Pi release rate. These findings provide direct evidence that myosin Va generates a powerstroke with Pi still in its active site. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of Pi from the active site.

Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin

Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.