Phosphate Release Research Articles

HspA1A, a 70‐kDa heat‐shock protein, contains several distinct lipid‐binding sites

HspA1A, a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cell survival. In addition to its functions in protein homeostasis, HspA1A also functions at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although the interactions between HspA1A and lipids have important biological consequences, their mechanistic details remain elusive and unexplored because the amino acids responsible for the lipid binding remain largely uncharacterized and the relationship between the chaperone and lipid‐binding functions remains unknown. To clarify the mechanism of the HspA1A‐lipid interactions, we first characterized the properties of a mutation from Tryptophan to Phenylalanine (W90F), known to affect binding of HspA1A to BMP and a Lysine to Alanine (K71A) mutation, which results in complete loss of the chaperone function. Experiments using the lipid vesicle sedimentation (LVS) method and Surface Plasmon Resonance revealed that although the W90F‐HspA1A binding to BMP was different from the WT, this mutation did not affect the binding to PS. Furthermore, our results revealed that the K71A mutation did not significantly change the binding of HspA1A to lipids under any of the conditions tested. These results allowed us to make two predictions: first, the lipid‐binding sites for PS and BMP are lipid‐specific, and second, the chaperone and lipid‐binding functions are distinct. To support the first prediction, we used a combination of structural superimpositions, sequence alignments, and literature observations and predicted three putative lipid‐binding regions on the HspA1A molecule. We tested these predictions using the LVS assay for 20 single or double mutations spanning two of these regions. Our results revealed that only two of these mutations affected the binding to PS, while a third one altered the binding to BMP. To test the second prediction, we performed a series of experiments to determine the effect of lipid binding to the secondary structure and chaperone functions of HspA1A. We used Circular dichroism spectrometry, measured the release of inorganic phosphate, and determined the rate of refolding of chemically denatured beta‐galactosidase in the presence or absence of particular lipid. These experiments revealed that lipid binding did not alter the secondary structure of the protein and affected neither the rate of ATP hydrolysis nor the rate of protein refolding. Together our findings provide further evidence that HspA1A binds to PS and BMP using different amino acid sites and support the notion that the chaperone and lipid‐binding functions of HspA1A do not overlap. These findings provide the basis for future experiments to test the effects of these mutations on the membrane‐localized functions of HspA1A in cancer and stressed cells.Support or Funding InformationThis project was supported by funds from NIH

Recommendations for tumor lysis syndrome management

The tumor lysis syndrome represents a potentially lethal complication caused by the massive release of nucleic acids, potassium and phosphate into the circulation as a result of the lysis of neoplastic cells, which are characterized by a rapid proliferation capacity and high sensitivity to drugs. This may occur spontaneously prior to the start of treatment, becoming worse after the initiation of chemotherapy. It presents a high mortality; its prevention continues being the most important therapeutic measure. The clinical picture is characterized by the existence of hydroelectrolytic metabolism disorders, in particular hyperkalemia, hyperphosphatemia and hyperuricemia and by the appearance of an acute renal lesion. Adequate therapeutic intervention involves intravenous hydration and measures to prevent or correct metabolic alterations. This article proposes guidelines to follow both in the diagnostic stage and in the treatment of this complication.

Nucleoside Triphosphate Diphosphohydrolases Dephosphorylate the Pharmacologically Active Metabolite of Tenofovir

Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug), an acyclic nucleotide analog, inhibits human immunodeficiency virus (HIV) replication and is currently used for both HIV treatment and prevention (as pre‐exposure prophylaxis). The pharmacologically active form of TFV is TFV‐diphosphate (TFV‐DP). Previously, we reported the heterogeneous distribution of TFV and TFV‐DP in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV‐containing enema. Although the observed heterogeneity in TFV‐DP distribution in colorectal tissue is multifactorial, it could be envisioned that dephosphorylation plays a role in regulating TFV‐DP levels since this molecule is structurally similar to ATP, which is a substrate for nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases). Therefore, in order to gain increased understanding of TFV‐DP disposition, the ability of recombinant human NTPDase 1, 3, 4, 5, and 8 to dephosphorylate TFV‐DP was investigated by incubating these enzymes with TFV‐DP, individually. TFV‐DP levels were measured using an ultra‐high‐performance liquid chromatography tandem mass spectrometry approach. From these incubations, TFV‐DP substrate depletion, a measure of dephosphorylation, was observed in the presence of NTPDase 1 or 3 enzymes. The activities of NTPDase 1 and 3 towards TFV‐DP were further confirmed using a malachite green phosphate assay that measures the release of inorganic phosphate. Further, using immunoblotting, we tested whether NTPDase 1 and 3 are expressed in colorectal tissue. These studies revealed the presence of both proteins in colorectal tissue lysate. In order to gain an understanding of the spatial distribution patterns of NTPDase 1 and 3 in colorectal tissue, Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) was employed. As a first step, we developed a MALDI MSI method to directly detect and identify peptides of NTPDase 1 and 3 following trypsin digestion. Trypsin digestions on NTPDases were carried out using a concentration of 0.05 μg/μl, and α‐cyano‐4‐hydroxycinnamic acid was used as the MALDI matrix. Following trypsin digestions and MALDI MS analysis, peptides corresponding to NTPDase 1 and 3 were detected and identified. From these, NTPDase 1 exhibited peptides, SLSNYPFDFQGAR, SQHQETPVYLGATAGMR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 yielded following peptides: GSGISSYGNNPQDVPR, YGIVLDAGSSR, FTEETWPQIHFEK, and MFTVLTR. Thus, in this study, we established a MALDI MS methodology for detection and identification of peptides corresponding to NTPDase 1 and 3. Furthermore, the above developed method can be used to generate spatial distribution profiles (including cell‐type and region‐specific localization patterns) of NTPDase 1 and 3 in colorectal and other tissues of interest that are susceptible to HIV infection. Data from in vitro incubations indicate that NTPDase 1 and 3 may contribute to the regulation of TFV‐DP levels.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781.

Genome wide underpinning of antagonistic and plant beneficial attributes of Bacillus sp. SBA12

The plant beneficial microorganisms in rhizosphere are promising candidates to counter plant biotic and abiotic stresses. In the present study, Bacillus sp. SBA12 antagonistic to plant pathogenic fungi such as Phytophthora infestans and Sclerotinia sclerotiorum was explored. The complete genome of SBA12 comprises 5,297,566 bp size with GC content 49% and a total of 5698 genes. The genomic organization revealed presence of several biosynthetic clusters for antibiotics, siderophore and various other bioactive compounds such as lanthipeptide, LAP, bacteriocin, siderophore, NRP and terpenes. The gene cluster for fengycin known for its role as elicitors in inducing plant immunity is also reported. Besides this, lytic enzymes chitinases, β-glucanases and proteases involved in pathogen suppression, acid and alkaline phosphatases, deaminases responsible for the phosphate solubilization and release of ammonia are also reported. The genomic organization of SBA12 revealed several hallmarks for plant growth promotion and pathogen suppression activities.

Moderate Alcohol Consumption Uniquely Regulates Sodium-Dependent Glucose Co-Transport in Rat Intestinal Epithelial Cells In Vitro and In Vivo

Chronic alcohol use often leads to malnutrition. However, how the intestinal absorption of nutrients such as glucose may be affected during moderate ethanol use has not been investigated. Glucose is absorbed via sodium (Na)-dependent glucose co-transport (SGLT1; SLC5A1) along the brush border membrane (BBM) of intestinal absorptive villus cells. The aim of this study was to investigate how moderate alcohol consumption affects the absorption of glucose via SGLT1. Intestinal epithelial cells (IEC-18; rat) were exposed to 8.64mM ethanol over 1, 3, 6, and 12 h. Rats (16-wk-old, male, Sprague-Dawley) were administered 2g/kg ethanol over 1, 3, and 6 h. Na-dependent 3H-O-methyl-d-glucose uptake was measured to assess SGLT1 activity. Na-K-ATPase activity was measured as a function of inorganic phosphate release. Protein expression was analyzed by Western blot analysis and immunohistochemical staining. Ethanol significantly decreased Na-dependent glucose absorption in enterocytes in vitro (ethanol treatment: 48.4% of controls at 1 h; P<0.01) and in vivo (ethanol treatment: 60.0% of controls at 1 h; P<0.01). Na-K-ATPase activity was significantly inhibited in vitro (ethanol treatment: 36.9% of controls at 1 h; P<0.01) and in vivo (ethanol treatment: 42.1% of controls at 1 h; P<0.01). Kinetic studies showed that the mechanism of inhibition of Na-glucose co-transport was secondary to a decrease in the affinity (1/Km) of the co-transporter for glucose both in vitro and in vivo. Western blots and immunohistochemistry further demonstrated unaltered amounts of SGLT1 after ethanol treatment. Moderate ethanol significantly decreases glucose absorption in IEC-18 cells and in villus cells of Sprague-Dawley rats. The inhibition of SGLT1 is secondary to an altered Na gradient at the cellular level and secondary to diminished affinity of the co-transporter for glucose at the protein level in the BBM. These observations may, at least in part, explain 1 possible mechanism of the onset of malnutrition associated with alcohol consumption.

Proteolytic activity, degradation, and dissolution of primary and permanent teeth.

Primary and permanent teeth composition may influence dissolution and degradation rates. To compare the dissolution and degradation of primary and permanent teeth. Enamel and dentin powders were obtained from primary molars and premolars and incubated within different pH buffers. Calcium and inorganic phosphate release was quantified in the buffers by atomic absorption and light spectrophotometry. A colorimetric assay was used to assess the MMP activity of primary dentin (PrD) and permanent dentin (PeD). Collagen degradation was assessed by dry mass loss, change in elastic modulus (E), and ICTP and CTX release. Data were submitted to ANOVA and Tukey's tests (α=0.05). Similar dissolution was found between PrD and PeD after 256hours. At pH 4.5, enamel released more minerals than dentin whereas at pH 5.5 the inverse result was observed. MMP activity was similar for both substrates. PrD showed higher dry mass loss after 1week. In general, greater reduction in E was recorded for PrD. Higher quantities of ICTP and CTX were released from PrD after 1week. Primary and permanent teeth presented similar demineralization rates. Collagen degradation, however, was faster and more substantial for PrD.

Biotransformation of struvite by Aspergillus niger: phosphate release and magnesium biomineralization as glushinskite.

Struvite (magnesium ammonium phosphate-MgNH4 PO4 ·6H2 O), which can extensively crystallize in wastewater treatments, is a potential source of N and P as fertilizer, as well as a means of P conservation. However, little is known of microbial interactions with struvite which would result in element release. In this work, the geoactive fungus Aspergillus niger was investigated for struvite transformation on solid and in liquid media. Aspergillus niger was capable of solubilizing natural (fragments and powder) and synthetic struvite when incorporated into solid medium, with accompanying acidification of the media, and extensive precipitation of magnesium oxalate dihydrate (glushinskite, Mg(C2 O4 ).2H2 O) occurring under growing colonies. In liquid media, A. niger was able to solubilize natural and synthetic struvite releasing mobile phosphate (PO4 3- ) and magnesium (Mg2+ ), the latter reacting with excreted oxalate resulting in precipitation of magnesium oxalate dihydrate which also accumulated within the mycelial pellets. Struvite was also found to influence the morphology of A. niger mycelial pellets. These findings contribute further understanding of struvite solubilization, element release and secondary oxalate formation, relevant to the biogeochemical cycling of phosphate minerals, and further directions utilizing these mechanisms in environmental biotechnologies such as element biorecovery and biofertilizer applications.

Phosphorylated PEG-emulsifier: Powerful tool for development of zeta potential changing self-emulsifying drug delivery systems (SEDDS)

AimIt was the aim of this study to synthesize a phosphorylated emulsifier possessing a PEG-linker for establishment of a potent zeta potential changing system in self-emulsifying drug delivery systems (SEDDS). MethodsN,N'-Bis(polyoxyethylene)oleylamine (POA) was phosphorylated utilizing pyrophosphoric acid. Successful synthesis of POA bisphosphate (POAP) was confirmed by NMR and HR CS MAS. After incorporation of 1% POAP into SEDDS (Kolliphor RH 40, Capmul PG-8, Labrafac Lipophile WL 1349, Labrafac PG; 30/20/20/30, v/v), according emulsions were incubated with intestinal alkaline phosphatase (IAP) and the zeta potential was measured. Additionally, the amount of released phosphate upon incubation with IAP or on Caco-2 cells was quantified by malachite green assay. Finally, cell viability studies on Caco-2 cells were performed and mucus permeation properties with and without IAP preincubation were assessed. ResultsPOAP was synthesized as brown viscous liquid with a yield of 36% and could be incorporated into SEDDS. By incubation with IAP a zeta potential shift from −15.1 to 6.5 mV was observed. A corresponding phosphate release in presence of isolated IAP as well as on Caco-2 cells was found. Assessment of the cytotoxic potential revealed no significant alteration in the safety profile of SEDDS by incorporation of POAP. Mucus permeation studies exposed a 2-fold higher permeation of fluorescein diacetate (FDA) having been embedded in SEDDS loaded with POAP in comparison to blank formulation and 3-fold higher permeability than for emulsions having been preincubated with phosphatase. ConclusionThe novel phosphorylated surfactant exhibiting a PEG-linker facilitated a potent zeta potential change of SEDDS.

The Study of Phosphate Release from Artificial Sediment into Water Body Using Diffusive Gradient in Thin Film (DGT) Device in Oxic Condition

The phenomenon of phosphate release in sediments into water bodies under oxic environment has been investigated using the Diffusive Gradient in Thin Film (DGT) technique. This research consists of several stages: polymer synthesis and DGT probe assembly, sediment sampling, DGT deployment in oxic conditions, and phosphate analysis from DGT adsorption results. Acrylamide polymer was successfully synthesized with a composition 15% acrylamide; N-N'-methylenebisacrylamide 0.1% and ferrihydrite as binding gels. DGT probes were assembly by placing a 16 x 3.2 cm polyacrylamide gel, binding gels and filter membranes on the DGT probes. The sediment sample was taken from the Bogor Botanical Gardens at the coordinates 6°36’00.6” S; 106°47’51.0” E. The DGT probe was placed in sediment samples for 1, 3 and 7 days in oxic conditions. After the prescribed time, the binding gel was removed and cut every 1 cm depth, then eluted using 0.25 M H2SO4 and the phosphate concentration was measured using spectrophotometry method. The results showed that the phosphate concentration tends to be higher with the increasing incubation time and depth. Maximum CDGT phosphate released on day 1, day 3 and day 7 were 1.00 µg/L at a depth of 14 cm, 6.61 µg/L at a depth of 14 cm, and 20.92 µg/L at a depth of 11 cm respectively. This ensures that the phosphate in water bodies comes from biogeochemical processes that occur in sediments and is successfully measured through DGT techniques.

Cell surface-expression of the phosphate-binding protein PstS: System development, characterization, and evaluation for phosphorus removal and recovery

Simultaneous overabundance and scarcity of inorganic phosphate (Pi) is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication, but also recover Pi for agricultural reuse. Here, a cell-surface expressed high-affinity phosphate binding protein (PstS) system was developed, and its Pi capture and release potential was evaluated. E. coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein (INP) as an anchoring motif. Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and outer membrane separation analyses. Cell surface characterization was investigated through acid-base titration, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups (i.e., pKa values). Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature, pH, and ionic strength on phosphate capture and release. The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells. Higher temperatures up to 40°C, basic pH conditions (pH = 10.5), and higher ionic strength up to 1.0 mol/L KCl promoted 20%–50% higher phosphate release.

Effect of anisotropy on solute transport in degraded fen peat soils

AbstractPeat soils are heterogeneous, anisotropic porous media. Compared to mineral soils, there is still limited understanding of physical and solute transport properties of fen peat soils. In this study, we aimed to explore the effect of soil anisotropy on solute transport in degraded fen peat. Undisturbed soil cores, taken in vertical and horizontal direction, were collected from one drained and one restored fen peatland both in a comparable state of soil degradation. Saturated hydraulic conductivity (K s) and chemical properties of peat were determined for all soil cores. Miscible displacement experiments were conducted under saturated steady state conditions using potassium bromide as a conservative tracer. The results showed that (1) the K s in vertical direction (K sv) was significantly higher than that in horizontal direction (Ksh), indicating that K s of degraded fen peat behaves anisotropically; (2) pronounced preferential flow occurred in vertical direction with a higher immobile water fraction and a higher pore water velocity; (3) the 5% arrival time (a proxy for the strength of preferential flow) was affected by soil anisotropy as well as study site. A strong correlation was found between 5% arrival time and dispersivity, K s and mobile water fraction; (4) phosphate release was observed from drained peat only. The impact of soil heterogeneity on phosphate leaching was more pronounced than soil anisotropy. The soil core with the strongest preferential flow released the highest amount of phosphate. We conclude that soil anisotropy is crucial in peatland hydrology but additional research is required to fully understand anisotropy effects on solute transport.

Structural Effects and Functional Implications of Phalloidin and Jasplakinolide Binding to Actin Filaments

Actin undergoes structural transitions during polymerization, ATP hydrolysis, and subsequent release of inorganic phosphate. Several actin-binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here, we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin-binding proteins. Furthermore, high-resolution cryoelectron microscopy structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.

Model-Based Analysis of Reactive Transport Processes Governing Fluoride and Phosphate Release and Attenuation during Managed Aquifer Recharge.

In water-scarce areas, the reclamation of wastewater through advanced water treatment and subsequent reinjection into depleted aquifers is an increasingly attractive water management option. However, such injection can trigger a range of water-sediment interactions which need to be well understood and quantified to ensure sustainable operations. In this study, reactive transport modeling was used to analyze and quantify the interacting hydrogeochemical processes controlling the mobilization of fluoride and phosphate during injection of highly treated recycled water into a siliciclastic aquifer. The reactive transport model explained the field-observed fluoride and phosphate transport behavior as a result of the incongruent dissolution of carbonate-rich fluorapatite where (i) a rapid proton exchange reaction primarily released fluoride and calcium, and (ii) equilibrium with a mineral-water interface layer of hydrated dibasic calcium phosphate released phosphate. The modeling results illustrated that net exchange of calcium on cation exchange sites in the sediments postbreakthrough of the injectant was responsible for incongruent mineral dissolution and the associated fluoride and phosphate release. Accordingly, amending calcium chloride into the injectant could potentially reduce fluoride and phosphate mobilization at the study site. Insights from this study are broadly applicable to understanding and preventing geogenic fluoride mobilization from fluoride-bearing apatite minerals in many other aquifers worldwide.

FRET and Optical Trapping Measurements Reveal Relationship between Phosphate Release and the Power Stroke in Myosin V

Myosin V is able to processively walk along actin filaments by coordinating its mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. Our previous FRET measurements of the power stroke in myosin V containing a single IQ domain (MV 1IQ) revealed a fast rotation of the lever arm prior to the phosphate release step and a slower rotation of the lever that occurs during the transition between actomyosin.ADP states. Here we introduce a mutation in the active site (S217A) that is proposed to slow entry of phosphate into the exit tunnel. The mutation causes both fast lever arm rotation and phosphate release to occur with the same rate constant (slowing the maximum rate 10-fold), suggesting entry of phosphate into the exit tunnel gates the lever arm swing. In single molecule laser trapping studies we found the step size of S217A was similar to WT (4nm). Furthermore, raising the Pi concentration in the laser trap assay, which should increase the population of the actomyosin.ADP.Pi state, also did not affect the step size of S217A MV 1IQ. Interestingly, the attached time increased dramatically in the presence of 15 mM Pi. We also found that Pi can inhibit actin gliding in the vitro motility assay in both S217A and WT MV 1IQ in a dose-dependent manner, which is consistent with the optical trapping studies, while Pi did not impact steady-state ATPase activity. Our results suggest a model in which attachment to actin and the power stroke (lever arm swing) occur rapidly before phosphate is released in myosin V. In addition, Pi rebinding to the actomyosin.ADP state may be capable of reversing the power stroke without altering the actin binding interface.

Exploring the State of the F1-ATPase after ATP Binding and before ADP Release: Effects of Conformational Changes on Phosphate Displacement

Single-molecule imaging experiments provide information that is not available from ensemble experiments. We are interested in the interpretation of dynamical studies imaging and manipulation in F₁-ATPase single-molecules. One key question that has arisen in single molecule stalling experiments is the erratic behavior a rotor angle of 55° between the binding and hydrolysis dwell angles of 0 and 80°, respectively. In our previous theoretical work, we used the elastic property of the rotor-stator structure to treat the experiments on controlled rotation. Our modeling suggests that there has to be a change in the bonding network, for example, of hydrogen bonds, as the system transitions between the two dwell points, perhaps at 55°, as indicated by an unusual stalling behavior around that angle. Therefore, in order to get further insights on these events, we have performed full-atomistic molecular dynamics (MD) simulations on the F₁-ATPase to explore the relationship between the conformational changes and the phosphate displacement. The pK_a values for phosphoric acid are 2.2, 7.2, and 12.3; although the effective pK_a for H₂PO₄⁻ within the binding site could be different. Hence, both protonation states (i.e., H₂PO₄⁻ and HPO₄²⁻) were assessed, performing molecular dynamics for 650 ns at 298.15K (Nose-Hoover thermostat) using the CHARMM36 force field. After the analysis of five MD trajectories, we found that the displacement of the phosphate was, to some extent, correlated with the dynamics of the protein. Moreover, one of the MD trajectories led to the complete release of the diprotic phosphate.

Timing and Load-dependence of the Powerstroke and Pi-release in Skeletal Muscle Myosin

Muscle myosin converts chemical energy from ATP into mechanical work to power contraction, however it is still unclear how this transduction occurs at the molecular level. A key unanswered question is whether phosphate release precedes the powerstroke or if the powerstroke occurs first. This has been a challenging question to answer because these events occur faster than the time resolution of most instruments. Therefore, we used an ultrafast single molecule laser trap assay, with sub-millisecond time resolution, to directly visualize the timing of the powerstorke in fast skeletal muscle myosin. By elevating the level of phosphate in the buffer (15 mM) under varying ATP concentration, we ensured that myosin remained in a phosphate (Pi) bound state. We observed that myosin generated a powerstroke in the presence of phosphate that was similar in size (∼5 nm) and rate (∼5000 s−1 at 3 pN resistive force) to that observed in the absence of phosphate. This suggests that myosin generates its powerstroke prior to the release of phosphate. Further analyses are under way to determine if elevated levels of phosphate induced reversal of the powerstroke later in the binding event. Additional experiments were done over a range of assistive and resistive loads, from −6pN to +6pN, to determine the load-dependence of the powerstroke and the effect on myosin detachment rate from different chemomechanical states. Thus these data provide novel insights into the fundamental nature of force generation by muscle myosin.

Characterization of the Cardiac Myosin Inhibitor CK-3773274: a Potential Therapeutic Approach for Hypertrophic Cardiomyopathy

Hypercontractility of the cardiac sarcomere may be essential for the underling pathological hypertrophy and fibrosis in genetic hypertrophic cardiomyopathies. Here, we characterize the small molecule, CK-3773274, as a novel cardiac myosin inhibitor that decreases contractility in vitro and in vivo. In bovine cardiac myofibrils, CK-3773274 decreased myosin ATPase activity in a concentration-dependent fashion (IC50:1.26 μM). CK-3773274 selectively targets cardiac myosin, as it reduced myosin ATPase activity in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. Importantly, CK-3773274 did not inhibit the ATPase activity of smooth muscle myosin. In transient kinetic studies, CK-3773274 substantially slowed the rate of actin-activated phosphate release, likely stabilizing myosin conformations that bind weakly to actin. Binding of CK-3773274 to cardiac myosin is mutually exclusive with the nonspecific myosin II inhibitor blebbistatin, suggesting they bind to the same or overlapping sites. Consistent with its biochemical phenotype, CK-3773274 (10 μM) reduced fractional shortening (FS) by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium (Ca2+) transient. The effect of CK-3773274 on cardiac contractility in vivo was assessed in healthy male Sprague Dawley (SD) rats using single oral doses ranging from 0.5 to 4 mg/kg. Left ventricular dimensions and FS were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced FS in a dose-related fashion by 20% to 70% relative to vehicle treatment without any changes to heart rate. In conclusion, CK-3773274 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo. Cardiac myosin inhibition may be a viable approach to treat the underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.

Groundwater is important for the geochemical cycling of phosphorus in rapidly urbanized areas: a case study in the Pearl River Delta

The fate of phosphorus in groundwater needs to be understood because phosphorus-rich groundwater is discharged into surface water bodies, which causes eutrophication, especially in urbanized areas. The present study investigated the spatial distributions and driving forces related to the groundwater phosphate levels in various aquifers in the Pearl River Delta (PRD), which has undergone three decades of urbanization, as well as the relationship between groundwater phosphate and arsenic was also discussed. The results showed that most of the high-phosphate (>1.53 mg/L) groundwater occurred in granular aquifers. The proportion of high-phosphate groundwater in granular aquifers was more than four times that in fissured aquifers, whereas high-phosphate groundwater was not observed in karst aquifers in the PRD. High-phosphate groundwater primarily occurred in urbanized areas in the PRD, and the proportion of high-phosphate groundwater had a significant positive correlation with the urbanization level. In granular aquifers, reductive environment and alkalization led to enrichment of the groundwater with phosphate. Anthropogenic sources such as wastewater from township-village enterprises (TVE) and animal wastes were the main sources of high-phosphate groundwater in urbanized areas, and the external input of phosphate enriched the groundwater arsenic levels in urbanized areas. By contrast, geogenic sources such as the release of phosphate from the reduction of Fe/Mn (hydr)oxides and the seawater intrusion accompanied by the release of phosphate from secondary minerals were mainly responsible for the occurrence of high-phosphate groundwater in peri-urban and non-urbanized areas, respectively. The high concentrations of both phosphate and arsenic in groundwater in fissured aquifers were mainly attributed to the infiltration of wastewater from TVEs. In contrast to the granular aquifers, the groundwater Eh and pH conditions were not conductive to the occurrence of high-phosphate groundwater in fissured aquifers.

Microbial fuel cell for simultaneous removal of uranium (VI) and nitrate

Nuclear wastes contain high nitrate concentration along with radionuclides like U (VI). Also, several reports suggest the presence of U (VI) and nitrate as co-contaminants in groundwater. The denitrifying bacteria consortia at MFC cathode produced phosphatase enzyme, which catalyzed the controlled release of phosphate from glycerol 3 phosphate. The inorganic phosphate combined with U (VI) resulting in insoluble uranyl phosphate. Ninety percent of initial U (VI) added in the biocathode could be recovered as Uranyl phosphate. XRD analysis confirmed the presence of uranyl phosphate precipitating at the cathode. Nitrate acted as an electron acceptor at the cathode, enabling completion of the MFC circuit and simultaneous nitrate and U (VI) removal. Since the process was performed in the MFC; the remediation was accompanied by power output. The power density of 2.91 Wm−3 and nitrate removal rate of 0.130 kg NO3−-N m−3 d−1 was achieved. The 16S rDNA based community analysis revealed a high abundance of Pseudomonas species in biocathode. This study demonstrates the applicability of MFC for simultaneous nitrate and U (VI) removal while producing electrical energy.

Biological phosphorus removal in seawater-adapted aerobic granular sludge

Seawater can be introduced or intrude in sewer systems and can thereby negatively influence biological wastewater treatment processes. Here we studied the impact of artificial seawater on the enhanced biological phosphate removal (EBPR) process performance by aerobic granular sludge (AGS) with synthetic wastewater. Process performance, granule stability and characteristics as well as microbial community of a seawater-adapted AGS system were observed. In seawater conditions strong and stable granules formed with an SVI5 of 20 mL/g and a lower abrasion coefficient than freshwater-adapted granules. Complete anaerobic uptake of acetate, anaerobic phosphate release of 59.5 ± 4.0 mg/L PO43--P (0.35 mg P/mg HAc), and an aerobic P-uptake rate of 3.1 ± 0.2 mg P/g VSS/h were achieved. The dominant phosphate accumulating organisms (PAO) were the same as for freshwater-based aerobic granular sludge systems with a very high enrichment of Ca. Accumulibacter phosphatis clade I, and complete absence of glycogen accumulating organisms. The effect of osmotic downshocks was tested by replacing influent seawater-based medium by demineralized water-based medium. A temporary decrease of the salinity in the reactor led to a decreased phosphate removal activity, while it also induced a rapid release of COD by the sludge, up to 45.5 ± 1.7 mg COD/g VSS. This is most likely attributed to the release of osmolytes by the cells. Recovery of activity was immediately after restoring the seawater feeding. This work shows that functioning of aerobic granular sludge in seawater conditions is as stable as in freshwater conditions, while past research has shown a negative effect on operation of AGS processes with NaCl-based wastewater at the same salinity as seawater.