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The development of portable, cost-effective, and straightforward DNA biosensors holds immense importance in various fields, including healthcare, environmental monitoring, and food safety. This study contributes to the objective by introducing an innovative approach for synthesizing carbon dots (Cdots) with high quantum yield (QY) and remarkable selectivity for Fe 3+ ions. Utilizing o-phenylenediamine as a precursor, the study achieved a straightforward and environmentally friendly synthesis method, enabling the efficient detachment of metal ions from the Cdot surface upon introducing pyrophosphate (PPi). The presence of surface hydroxyl and amino groups facilitated specific Fe 3+ recognition. Employing D-optimal response surface methodology, the study optimized Cdot synthesis parameters, identifying temperature and heating time as critical factors influencing QY. Statistical analysis confirmed the model's reliability, predicting maximum QY of 48.8 % with minimal deviation from experimental results. Characterization studies revealed the amorphous nature of Cdots through HR-TEM, XRD, and FTIR analysis. Furthermore, the proposed LAMP/PPi biosensing technique demonstrated higher sensitivity, specificity, and repeatability, with negligible interference from common anions and efficacy across varying pH levels. The limit of detection (LOD) of 0.079 (±0.01) μM and the detection range of 0.1 μM-2 mM underscore the biosensor's practical utility. This study highlights a promising direction for developing paper-based LAMP/PPi biosensors with potential diagnostics and environmental monitoring applications. Significantly, the biosensing technique is applicable to any DNA amplification method generating pyrophosphate (PPi) as a by-product., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dinesh Prasad reports financial support was provided by Birla Institute of Technology. DINESH PRASAD reports a relationship with Birla Institute of Technology., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study investigated the effects of sodium pyrophosphate (SPP) and catechin (C) on the in vitro enzymatic digestion of oxidatively damaged myofibrillar protein (MP) gel. The results indicated that SPP increased the β-sheet content and the gastric digestibility of the MP gel, while C hindered the transition from α-helix to β-sheet structure, leading to decreased digestibility. Notably, neither compound significantly affected intestinal digestibility. Furthermore, SPP and C significantly enhanced the antioxidant activity of MP gel digestion products. Notably, their synergistic hydrolysis products, simulating both gastric and gastrointestinal stages, chelated 91.4 % and 89.1 % of Fe 2+ and scavenged 59.4 % and 77.6 % of hydroxyl radicals, respectively. Moreover, the final digestion products of the MP gel treated with SPP and C exhibited the highest content of negatively charged amino acids and absolute Zeta potential values. Overall, this study demonstrated that incorporating SPP and C could positively impact the digestion of oxidatively damaged MP gels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Pseudo-gout is caused by the deposition of highly insoluble calcium pyrophosphate dihydrate (CPPD) crystals in the joints of sufferers. This leads to inflammation and ultimately joint damage. The insolubility of CPPD is driven by the strong attraction of di-cationic calcium ions with tetra-anionic pyrophosphate ions. One of the challenges of dissolving CPPD is that a related mineral, hydroxy apatite (HA) is present in larger amounts in the form of bone and also contains strongly interacting calcium and phosphate ions. Our aim in this work was to selectively dissolve CPPD in preference to HA. To accomplish this, we used a known receptor for pyrophosphate that contains two complexed zinc ions that are ideally spaced to interact with the tetra-anion of pyrophosphate. We hypothesized that such a molecule could act as a preorganized tetra-cation that would be able to outcompete the two calcium ions present in the crystal lattice of CPPD. We demonstrate both visually and through analysis of released phosphorous that this molecule is able to preferentially dissolve CPPD over the closely related HA and thus can form the basis for a possible approach for the treatment of pseudo-gout., (© 2024 Wiley-VCH GmbH.)

Oligoadenylate synthetase 1 (OAS1) catalyzes the dsRNA-dependent polymerization of ATP to form oligoadenylate, a second messenger of the innate immunity system. This paper reports kinetic and mechanistic studies of OAS1-catalyzed dimerization of ATP to form 2'-5'-diadenylate and pyrophosphate (PP i ), the first step in ATP polymerization. Major findings include the following: (1) Reaction progress curves for the production of PP i are biphasic, characterized by a presteady-state lag followed by the linear, steady-state production of PP i . (2) The dependence of steady-state velocity on ATP concentration is sigmoidal and can be described by a rate law derived for a mechanism involving enzyme-catalyzed substrate dimerization. (3) Steady-state velocities were determined as a function of ATP concentration at fixed concentrations of poly(I:C), a synthetic dsRNA activator of OAS1. The data suggest a random mechanism in which either ATP or poly(I:C) can add first to the enzyme. (4) The dependence of k lag on poly(I:C) and ATP concentration requires expansion of this mechanism to include slow conformational isomerization of various poly(I:C)- and ATP-bound complexes of inactive OAS1 to form complexes comprising an active enzyme, to ultimately form the reactive Michaelis complex of active OAS1, poly(I:C), and two molecules of ATP. Finally, within this complex, the two molecules of ATP dimerize to form 2'-5'-diadenylate and pyrophosphate. (5) The pH dependence and solvent deuterium isotope effect for k cat suggests that proton transfer occurs in the rate-limiting transition state, which likely involves proton abstraction from the 2'-hydroxyl of the adenylate acceptor ATP as the oxygen of this hydroxyl attacks the a-phosphate of the adenylate donor ATP in an S N 2 fashion.

Reversible protein phosphorylation is a central signaling mechanism in eukaryotes. Although mass-spectrometry-based phosphoproteomics has become routine, identification of non-canonical phosphorylation has remained a challenge. Here we report a tailored workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing, to our knowledge, the first direct evidence of endogenous protein pyrophosphorylation. We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. Detection was consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). When the biosynthesis of PP-InsPs was perturbed, proteins expressed in this background exhibited no signs of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1 and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation and should be considered in future phosphoproteomic analyses., (© 2024. The Author(s).)

Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 10 5  m -1 ·s -1 , thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 10 4  M -1 ·s -1 . LC-MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The residues of organophosphorus pesticides (OPs) are increasing environmental pollution and public health concerns. Thus, the development of simple, convenient and sensitive method for detection of OPs is crucial. Herein, a multifunctional Fe-based MOF with fluorescence, catalytic and adsorption, is synthesized by a simple one-pot hydrothermal method. The ratiometric fluorescence sensor for detection of OPs is constructed by using only one multifunctional sensing material. The NH 2 -MIL-101(Fe) is able catalyze the o-phenylenediamine (OPD) into 2,3-diaminophenazine (DAP) in the presence of H 2 O 2 . The generated DAP can significantly quench the intrinsic fluorescence of NH 2 -MIL-101(Fe) by the fluorescence resonance energy transfer (FRET) and internal filtration effect (IFE), while producing a new measurable fluorescence. Without immobilization or molecular imprinting, pyrophosphate ion (PPi) can inhibit the peroxidase-like activity of the NH 2 -MIL-101(Fe) by chelating with Fe 3+ /Fe 2+ redox couple. Moreover, PPi can also be hydrolyzed by alkaline phosphatase (ALP), the presence of OPs inhibits the activity of ALP, resulting in the increase of extra PPi preservation and signal changes of ratiometric fluorescence, the interactions of ALP with different OPs are explored by molecular docking, the OPs (e.g., glyphosate) interact with crucial amino acid residues (Asp, Ser, Ala, Lys and Arg) are indicated. The proposed sensor exhibits excellent detection performance for OPs with the detection limit of 18.7 nM, which provides a promising strategy for detection of OPs., Competing Interests: Declaration of competing interest The authors have no conflict of potential competing interest, financial or otherwise., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sensitive and accurate analysis of pyrophosphate (PPi) is of great importance for preventing health hazard in environment. Nevertheless, most of sensors focus on sensitivity and selectivity, but practicality is also a significant quota. How to reconciling sensitivity, selectivity and practicability in one single sensor is desirable but remains challenging. Here, we created a novel metal-carbon nanozyme V 2 O 5 @C with two-dimensional (2D) morphology and high yet exclusive peroxidase (POD)-like activity via a glucose and NH 4 NO 4 -co-directed avenue, and further showed its application in constructing a portable and disposable paper-based analytical chip (PA-chip) for rapid, visual and onsite analysis of PPi. PPi etched V 2 O 5 to prevent the decomposition of H 2 O 2 into ·OH, resulting in weakened POD-like activity. In comparison with PPi deficiency, colorless TMB couldn't be oxidized into oxidized TMB with a dropped absorption at 652 nm. Therefore, obviously shallowed blue color on PA-chip surface was recorded, and demonstrated a negative relationship with PPi dosage, enabling rapid and visual detection of PPi with a limit of detection of 2.6 nM. This study demonstrated the burgeoning applications of nanozymes with POD-like activity in construction of PA-chips for PPi and will quicken the advancement of practical sensors, guaranteeing environmental safety., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This work describes a novel analytical method using capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C 4 D) for simultaneous, simple, and rapid determination of three inorganic phosphates (orthophosphate, pyrophosphate, and tripolyphosphate) widely used as food additives and in pharmaceutical formulations. A background electrolyte composed of 0.5 mol L -1 acetic acid provided fast separation (around 3.0 min) and good separation efficiency and peak resolution. Linearity in the concentration range of 10-500 mg L -1 was confirmed by the coefficients of determination (R 2 ) higher than 0.99. The limits of detection varied from 0.41 to 0.58 mg L -1 . The accuracy of the proposed method was assessed by recovery tests conducted at three concentration levels in tap water samples, food, and personal hygiene products. Recovery values varying from 81% to 118% were achieved, indicating an acceptable accuracy. The proposed CE-C 4 D successfully determined the three inorganic phosphates in the analyzed samples., (© 2024 Wiley‐VCH GmbH.)

The in vitro transcription (IVT) reaction used in the production of messenger RNA vaccines and therapies remains poorly quantitatively understood. Mechanistic modeling of IVT could inform reaction design, scale-up, and control. In this work, we develop a mechanistic model of IVT to include nucleation and growth of magnesium pyrophosphate crystals and subsequent agglomeration of crystals and DNA. To help generalize this model to different constructs, a novel quantitative description is included for the rate of transcription as a function of target sequence length, DNA concentration, and T7 RNA polymerase concentration. The model explains previously unexplained trends in IVT data and quantitatively predicts the effect of adding the pyrophosphatase enzyme to the reaction system. The model is validated on additional literature data showing an ability to predict transcription rates as a function of RNA sequence length., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Cupric ions (Cu 2+ ), pyrophosphate (PPi), and alkaline phosphatase (ALP) are involved in a variety of biochemical processes such as DNA replication, cellular metabolism and play an important role in human growth and development. It is of great significance to establish a method for the sensitive detection of Cu 2+ , PPi and ALP. In this work, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were successfully synthesized by a one-pot method using hydrazine sulfate as reductant, exhibiting a unique strong fluorescence emission in the near-ultraviolet region at ∼339 nm. Since the fluorescence of PEI-AgNCs can be quenched by Cu 2+ through inner filtering effect (IFE), then recovered by competitive binding of pyrophosphate and Cu 2+ , and later weakened again by catalytic hydrolysis of alkaline phosphatase, a sensitive and selective strategy based on the changes of fluorescence "ON" or "OFF" was established to detect Cu 2+ , PPi and ALP. The LODs of these three analytes were 36 nM, 0.2 μM, and 0.14 U L -1 at a S/N ratio of 3, respectively. A series of logic gate circuits for sensing cupric ions, pyrophosphate, and alkaline phosphatase were successfully constructed. The established methods have the potential for biosensing and environmental analysis and the specific UV-A fluorescence property of PEI-AgNCs may be helpful in photonic and optical areas., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In this study, the protein and salts distribution (Ca, P, Na and Mg) in processed cheese (PC) samples prepared with 180 or 360 mEq/kg of the calcium sequestering salts (CSS) disodium phosphate (DSP), disodium pyrophosphate (DSPP), sodium hexametaphosphate (SHMP) and trisodium citrate (TSC) was studied. For this purpose, a water-soluble extract (WSE) of PC samples was prepared. All PC samples contained 45-46% moisture, 26-27% fat and 20-21% protein and had a pH of 5.2 or 5.7. Ultracentrifugation slightly reduced the protein content of the WSE of PC, indicating that most protein in the WSE was non-sedimentable. At equal concentration of CSS, the protein content of the WSE was higher for PC at pH 5.7 compared to PC at pH 5.2. Approximately 55-85% of the Ca and P in the WSE of samples was 10 kDa-permeable for PC prepared with DSPP and SHMP. This suggests that the formation of non-permeable Ca-polyphosphate-casein complexes. For PC prepared with TSC, >90% of Ca in the WSE was 10 kDa-permeable, indicating that micellar disruption arises from sequestration of micellar Ca. These results indicate that the WSE method is an appropriate method to understand how salts present in PC are distributed. However, the WSE and ultracentrifugal supernatant of the WSE can include both soluble and protein-associated salts. Therefore, determining levels of salts in 10 kDa permeate of ultracentrifugal supernatant of the WSE is most appropriate.

Dissolution dynamic nuclear polarization ( d -DNP) has enabled applications such as the real-time monitoring of chemical reactions. Such applications are mainly for 13 C and 15 N spins with long spin-lattice relaxation times in the molecules of interest. However, the only applications for phosphorus using d -DNP are pH imaging and nucleation during crystallization due to the short relaxation times. Here we show that it is possible to observe enzyme reactions using d -DNP with phosphorus. Hyperpolarized 31 P spins in pyrophosphate were obtained using bullet-DNP, which requires less dilution of highly polarized solid samples. Real-time monitoring of the hydrolysis reaction of pyrophosphate by inorganic pyrophosphatase from baker's yeast at physiological pH and was successfully achieved and the reaction rate was determined. This is an important reaction for a wide range of applications related to medicine, agriculture, and quantum life science.

Developing a neurovascular bone repair scaffold with an appropriate mechanical strength remains a challenge. Calcium phosphate (CaP) is similar to human bone, but its scaffolds are inherently brittle and inactive, which require recombination with active ions and polymers for bioactivity and suitable strength. This work discussed the synthesis of amorphous magnesium-calcium pyrophosphate (AMCP) and the subsequent development of a humidity-responsive AMCP/cassava starch (CS) scaffold. The scaffold demonstrated enhanced mechanical properties by strengthening the intermolecular hydrogen bonds and ionic bonds between AMCP and CS during the gelatinization and freeze-thawing processes. The release of active ions was rapid initially and stabilized into a long-term stable release after 3 days, which is well-matched with new bone growth. The release of pyrophosphate ions endowed the scaffold with antibacterial properties. At the cellular level, the released active ions simultaneously promoted the proliferation and mineralization of osteoblasts, the proliferation and migration of endothelial cells, and the proliferation of Schwann cells. At the animal level, the scaffold was demonstrated to promote vascular growth and peripheral nerve regeneration in a rat skull defect experiment, ultimately resulting in the significant and rapid repair of bone defects. The construction of the AMCP/CS scaffold offers practical suggestions and references for neurovascular bone repair.

This work investigated the cryoprotective effect of trehalose (TH) and sodium pyrophosphate (SPP) alone and in combination on myofibrillar protein (MP) oxidation and structural changes in silver carp surimi during 90 days of frozen storage (-20 °C). TH combined with SPP was significantly more effective than single TH or SPP in preventing MP oxidation (P < 0.05), showing a higher SH content (6.05 nmol/mg protein), and a lower carbonyl (4.24 nmol/mg protein) and dityrosine content (1280 A.U.). SDS-PAGE results indicated that TH combined with SPP did not differ significantly from TH and SPP in inhibiting protein degradation but was more effective in inhibiting protein crosslinking. Moreover, all cryoprotectants could stabilise the secondary and tertiary structures and inhibit unfolded and aggregation of MP, with the combination of TH and SPP being the best. It's worth noting that TH combined with SPP had a synergistic effect on inhibiting the decrease in α-helix content and gel-forming ability, and the increase in surface hydrophobicity. Overall, TH combined with SPP could significantly inhibited MP oxidation and structural changes in surimi during frozen storage and improve the gel-forming ability, which was significantly better than single TH or SPP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H + /Na + pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences., Competing Interests: Rodolfo García-Contreras, Héctor Manuel Mora-Montes and Bernardo Franco are Academic Editors at PeerJ. The other authors declare that they have no competing interests., (©2024 García-Contreras et al.)

Elucidation on the emulsifying behaviors of goose liver protein (GLP) from interfacial perspective was scarce when protein charging was altered. This work aimed to elucidate the role of phosphorylation on the interfacial associative interaction and then emulsion stabilizing properties of GLP using three structurally relevant phosphates of sodium trimetaphosphate (STMP), sodium tripolyphosphate (STPP) and sodium pyrophosphate (TSPP). A monotonic increment of protein charging treated from STMP, STPP to TSPP caused progressively increased particle de-aggregation, surface hydrophobicity and structural flexibility of GLP. Compared with STMP and TSPP, STPP phosphorylation rendered the most strengthened interfacial equilibrium pressure (11.98 ± 0.24 mN/m) due to sufficient unfolding but moderated charging character conveyed. Desorption curve and interfacial protein microstructure indicated that STPP phosphorylation caused the highest interfacial connectivity between proteins adsorbed onto the same droplet, as was also verified by interfacial elastic modulus (10.3 ± 0.21 mN/m). STPP treated GLP also yielded lowest droplet size (8.16 ± 0.10 μm), flocculation (8.18%) and Turbiscan stability index (8.78 ± 0.36) of emulsion but most improved microrheological properties. Overall, phosphorylation functioned itself in fortifying the intradroplet protein-protein interaction but restraining the interdroplet aggregation, and STPP phosphorylation endowed the protein with most enhanced interfacial stabilization and emulsifying efficiency., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, K M , was 13 μM, and the turnover number, k cat , was 8 s -1 . The effect of inhibitors was investigated using pyrophosphate (PP i ). PP i caused a dose-dependent decrease in the apparent k cat and increase in the apparent K M under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis., Result: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu 2+ ) ions. The off state of LA@AuAg&#95;Cu 2+ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu 2+ . The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu 2+ ions, and again quenched off state was retrieved due to interaction of free Cu 2+ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples., Significance: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hydrazine substituted thienopyrimidine, a new fluorophore, was used to synthesize a novel Schiff base R1 as a chemosensor via the condensation with p-formyltriphenylamine, and the structure was confirmed using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. When treated with Cu 2+ in dimethylsulfoxide (DMSO)/H 2 O buffer, R1 showed a phenomenon of fluorescence quenching, which was reversible with the action of ethylenediaminetetraacetic acid (EDTA). When treated with Fe 3+ in dimethylformamide (DMF)/H 2 O buffer, R1 exhibited the same phenomenon, but fluorescence was recovered with inorganic pyrophosphate (PPi) quantitatively. The complexation ratios for R1-Cu 2+ and R1-Fe 3+ were both 1:2, which were manifested by MS titrations and corresponding Job's plots. The limits of detection of R1 for Cu 2+ and Fe 3+ were 3.11 × 10 -8 and 1.24 × 10 -7  M, respectively. The sensing mechanism of R1 toward Cu 2+ and Fe 3+ was confirmed using density functional theory calculations and electrostatic potential analysis. Test strips of R1 were fabricated successfully for on-site detection of Cu 2+ and Fe 3+ . In addition, R1 was applied to recognize Cu 2+ and Fe 3+ in actual water samples with satisfactory recovery., (© 2024 John Wiley & Sons Ltd.)

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system ( i.e. , short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white "colony" spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

This paper describes initially the application of the Tyndall effect (TE) of metal-organic framework (MOF) materials as a colorimetric signaling strategy for the sensitive detection of pyrophosphate ion (PPi). The used MOF NH 2 -MIL-101(Fe) was prepared with Fe 3+ ions and fluorescent ligands of 2-amino terephthalic acid (NH 2 -BDC). The fluorescence of NH 2 -BDC in MOF is quenched due to the ligand-to-metal charge transfer effect, while the NH 2 -MIL-101(Fe) suspension shows a strong TE. In the presence of PPi analyte, the MOFs will undergo decomposition because of the competitive binding of Fe 3+ by PPi over NH 2 -BDC, resulting in a significant decrease in the TE signal and fluorescence restoration from the released ligands. The results demonstrate that the new method only requires a laser pointer pen (for TE creation) and a smartphone (for portable quantitative readout) to detect PPi in a linear concentration range of 1.25-800 μM, with a detection limit of ~210 nM (3σ) which is ~38 times lower than that obtained from traditional fluorescence with a spectrophotometer (linear concentration range, 50-800 µM; detection limit, 8.15 µM). Moreover, the acceptable recovery of PPi in several real samples (i.e., pond water, black tea, and human serum and urine) ranges from 97.66 to 119.15%., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

It is a challenging task to remove heavy metal ions efficiently from the wastewater containing high concentrations of complexants. In this work, a novel PEI-modified chitosan/activated carbon composite adsorbent (PCA) was prepared and applied to the removal of Cu(II) from pyrophosphate plating rinsing wastewater. The main species of Cu(II) in the pyrophosphate wastewater was [Cu(HP 2 O 7 ) 2 ] 4- or [Cu(P 2 O 7 ) 2 ] 6- , which were denoted as [Cu(II)-PP] anions. The maximum adsorption capacity of PCA for Cu(II) reached 1.41 mmol g -1 under the condition of pH = 8 and molar ratio of pyrophosphate to Cu(II) = 4:1. The adsorption kinetic behavior of Cu(II) on PCA followed the Elovich model best and PCA attained adsorption equilibrium within 36 h. The thermodynamic studies showed that the adsorption process of Cu(II) by PCA was endothermic and spontaneous. The PCA fixed bed column was used to remove Cu(II) from simulated pyrophosphate plating rinsing wastewater. After three consecutive adsorption-desorption cycles, the adsorption performance, hydraulic conductivity, and mechanical stability of PCA column did not decrease. The FTIR and XPS analysis results indicated that [Cu(II)-PP] anions can be adsorbed on PCA by electrostatic attraction with protonated amine groups or coordination with the amine groups of PCA via ligand substitution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential., (© 2023. The Author(s).)

Selective and sensitive detection of inorganic pyrophosphate (PPi) are of great significance both for clinical applications and fundamental research. In this work, a ratiometric fluorescent probe was developed by decorating a porphyrin-based metal-organic framework (PCN-224) with sulfur nanodots (S-dots). The red-fluorescence of PCN-224 was significantly promoted (more than 6-fold) by S-dots through inhibiting molecular motion of porphyrin ligand, which also provided active sites for the detection of Cu 2+ and PPi. Cu 2+ could selectively quench the red-fluorescence of PCN-224 through coordination with porphyrin ligand, and the competition reaction between PPi and Cu 2+ resulted in the decomposing of coordination and recovery of red-fluorescence. The blue-fluorescence from S-dots was deemed as effective reference, which provided a built-in correction in complex environments. Based on these photophysical properties, a ratiometric fluorescence assay was developed for the quantitative detection of Cu 2+ and PPi, with a limit of detection of 0.11 and 2.66 μM, respectively. The accuracy and practical applications of assays were also demonstrated by detection in tap water and human serum samples., (© 2023 Wiley-VCH GmbH.)

Non-covalent interactions have been extensively used to fabricate nanoscale architectures in supramolecular chemistry. However, the biomimetic self-assembly of diverse nanostructures in aqueous solution with reversibility induced by different important biomolecules remains a challenge. Here, we report the synthesis and aqueous self-assembly of two chiral cationic porphyrins substituted with different types of side chains (branched or linear). Helical H-aggregates are induced by pyrophosphate (PPi) as indicated by circular dichroism (CD) measurement, while J-aggregates are formed with adenosine triphosphate (ATP) for the two porphyrins. By modifying the peripheral side chains from linear to a branched structure, more pronounced H- or J-type aggregation was promoted through the interactions between cationic porphyrins and the biological phosphate ions. Moreover, the phosphate-induced self-assembly of the cationic porphyrins is reversible in the presence of the enzyme alkaline phosphatase (ALP) and repeated addition of phosphates.

Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using 31 P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.

Two terpene cyclases were used as biocatalytic tool, namely, limonene synthase from Cannabis sativa (CLS) and 5-epi-aristolochene synthase (TEAS) from Nicotiana tabacum. They showed significant substrate flexibility towards non-natural prenyl diphosphates to form novel terpenoids, including core oxa- and thia-heterocycles and alkyne-modified terpenoids. We elucidated the structures of five novel monoterpene-analogues and a known sesquiterpene-analogue. These results reflected the terpene synthases' ability and promiscuity to broaden the pool of terpenoids with structurally complex analogues. Docking studies highlight an on-off conversion of the unnatural substrates., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

We report an electrochemical biosensor based on the supramolecular host-guest recognition between cucurbit[7]uril (CB[7]) and L-phenylalanine-Cu(II) complex for pyrophosphate (PPi) and alkaline phosphatase (ALP) analysis. First, L-Phe-Cu(II) complex is simply synthesized by the complexation of Cu(II) (metal node) with L-Phe (bioorganic ligand), which can be immobilized onto CB[7] modified electrode via host-guest interaction of CB[7] and L-Phe. In this process, the signal of the complex-triggered electro-catalytic reduction of H 2 O 2 can be captured. Next, due to the strong chelation between PPi and Cu(II), a biosensing system of the model "PPi and Cu(II) premixing, then adding L-Phe" was designed and the platform was applied to PPi analysis by hampering the formation of L-Phe-Cu(II) complex. Along with ALP introduction, PPi can be hydrolyzed to orthophosphate (Pi), where abundant Cu(II) ions are released to form L-Phe-Cu(II) complex, which gives rise to the catalytic reaction of complex to H 2 O 2 reduction. The quantitative analysis of H 2 O 2 , PPi and ALP activity was achieved successfully and the detection of limits are 0.067 μM, 0.42 μM and 0.09 mU/mL (S/N=3), respectively. With its high sensitivity and selectivity, cost-effectiveness, and simplicity, our analytical system has great potential to for use in diagnosis and treatment of ALP-related diseases., (© 2022 Wiley-VCH GmbH.)

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance 1-3 . Teixobactin 4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan 5 . Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin 4 . The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates., (© 2022. The Author(s).)

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP's overall intracellular function is unknown. Here, using 31 P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP's charge. Our results lead to a novel model for DGPP-protein interaction.

X-ray powder diffraction (XRPD) and thermal analysis (differential scanning calorimetry/derivative of thermogravimetry (DSC/DTG)) are solid-state techniques that can be successfully used to identify and quantify various chemical compounds in polycrystalline mixtures, such as dietary supplements or drugs. In this work, 31 dietary supplements available on the Polish market that contain iron compounds, namely iron gluconate, fumarate, bisglycinate, citrate and pyrophosphate, were evaluated. The aim of the work was to identify iron compounds declared by the manufacturer as food supplements and to try to verify compliance with the manufacturer's claims. Studies performed by X-ray and thermal analysis confirmed that crystalline iron compounds (iron (II) gluconate, iron (II) fumarate), declared by the manufacturers, were present in the investigated dietary supplements. Iron (II) bisglycinate proved to be semi-crystalline. However, depending on the composition of the formulation, it was possible to identify this compound in the tested supplements. For amorphous iron compounds (iron (III) citrate and iron (III) pyrophosphate), the diffraction pattern does not have characteristic diffraction lines. Food supplements containing crystalline iron compounds have a melting point close to the melting point of pure iron compounds. The presence of excipients was found to affect the shapes and positions of the endothermic peaks significantly. Widening of endothermic peaks and changes in their position were observed, as well as exothermic peaks indicating crystallization of amorphous compounds. Weight loss was determined for all dietary supplements tested. Analysis of the DTG curves showed that the thermal decomposition of most food supplements takes place in several steps. The results obtained by a combination of both simple, relatively fast and reliable XRPD and DSC/DTG methods are helpful in determining phase composition, pharmaceutical abnormalities or by detecting the presence of the correct polymorphic form.

Chemodynamic therapy (CDT) is an emerging treatment that usually employs chemical agents to decompose hydrogen peroxide (H 2 O 2 ) into hydroxyl radical (•OH) via Fenton or Fenton-like reactions, inducing cell apoptosis or necrosis by damaging biomacromolecules such as, lipids, proteins, and DNA. Generally, CDT shows high tumor-specificity and minimal-invasiveness in patients, thus it has attracted extensive research interests. However, the catalytic reaction efficiency of CDT is largely limited by the relatively high pH at the tumor sites. Herein, a 808 nm laser-potentiated peroxidase catalytic/mild-photothermal therapy of molybdenum diphosphide nanorods (MoP 2 NRs) is developed to improve CDT performance, and simultaneously achieve effective tumor eradication and anti-infection. In this system, MoP 2 NRs exhibit a favorable cytocompatibility due to their inherent excellent elemental biocompatibility. Upon irradiation with an 808 nm laser, MoP 2 NRs act as photosensitizers to efficiently capture the photo-excited band electrons and valance band holes, exhibiting enhanced peroxidase-like catalytic activity to sustainedly decompose tumor endogenous H 2 O 2 to •OH, which subsequently destroy the cellular biomacromolecules both in tumor cells and bacteria. As demonstrated both in vitro and in vivo, this system exhibits a superior therapeutic efficiency with inappreciable toxicity. Hence, the work may provide a promising therapeutic technique for further clinical applications., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H + and Na + transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a "direct coupling" mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na + transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K + -dependent H + -transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PP i analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PP i hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg 2+ (essential cofactor). The replacement of K + , the second metal cofactor, by Na + increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PP i hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell's direct coupling with conformational coupling to catalyze cation transport across the membrane.

The inhibition of platelet aggregation, and the activity of oxidoreductases and microhemocirculation in a burn wound on the treatment of burns with wound dressings based on bacterial nanocellulose (BC)-zinc oxide nanoparticles (ZnO NPs)-betulin diphosphate (BDP) were studied. The control of the treatment by BC-ZnO NPs-BDP on burned rats by the noninvasive DLF method showed an increase in perfusion and the respiratory component in wavelet spectra, characterizing an improvement in oxygen saturation in the wound. The study on the volunteers' blood found the inhibition of ADP-induced platelet aggregation by 30-90%. Disaggregation depends on the dose under the action of the ionized form of BDP and ZnO NPs-BDP in a phosphate buffer; it was reversible and had two waves. It was shown on rats that the specific activity of LDH reverse and LDH direct (control-intact animals) on day 21 of treatment increased by 11-38% and 23%, respectively. The LDH reverse /LDH direct ratio increased at BC-ZnO NPs-BDP treatment, which characterizes efficient NAD+ regeneration. AlDH activity increased significantly in the first 10 days by 70-170%, reflecting the effectiveness of the enzyme and NAD+ in utilizing toxic aldehydes at this stage of burn disease. The activities of GR and G6PDH using NADP(H) were increased with BC-ZnO NPs-BDP treatment.

The purpose of this exploration was to determine the density and volumetric properties of the aqueous solution of Na 2 H 2 P 2 O 7 with the molality varied from 0.08706 to 0.88402 mol·kg -1 measured at temperature intervals of 5 K from 283.15 to 363.15 K at 101.325 kPa using Anton Paar Digital vibrating-tube densimeter. The thermal expansion coefficient (α), apparent molar volume (V Φ ), expansibility (ϕ E ), and partial molar volume (V B ) of Na 2 H 2 P 2 O 7 (aq) against temperature and molality have been evaluated from density data. On the basis of Pitzer ion-interaction apparent molar volume theory, the Pitzer single-salt parameters (β M,X 0v , β M,X 1v , β M,X 2v and C M,X v , MX = Na 2 H 2 P 2 O 7 ), and their correlation coefficients a i of the temperature-dependence formula f (i, p, T) = a 1  + a 2 ln(T/298.15) + a 3 (T - 298.15) + a 4 /(620 - T) + a 5 /(T - 227) for Na 2 H 2 P 2 O 7 were obtained for the first time. It was revealed that predicted apparent molar volumes agreed well with the experimental values indicating the single salt parameters and the temperature-dependent formula are reliable., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of β,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties.

Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of pyrophosphate to phosphates. PPases play essential roles in growth and development, and are found in all kingdoms of life. Human possess two PPases, PPA1 and PPA2. PPA1 is present in all tissues, acting largely as a housekeeping enzyme. Besides pyrophosphate hydrolysis, PPA1 can also directly dephosphorylate phosphorylated c-Jun N-terminal kinases 1 (JNK1). Upregulated expression of PPA1 has been linked to many human malignant tumors. PPA1 knockdown induces apoptosis and decreases proliferation. PPA1 is emerging as a potential prognostic biomarker and target for anti-cancer drug development. In spite of the biological and physiopathological importance of PPA1, there is no detailed study on the structure and catalytic mechanisms of mammalian origin PPases. Here we report the crystal structure of human PPA1 at a resolution of 2.4 Å. We also carried out modeling studies of PPA1 in complex with JNK1 derived phosphor-peptides. The monomeric protein fold of PPA1 is similar to those found in other family I PPases. PPA1 forms a dimeric structure that should be conserved in animal and fungal PPases. Analysis of the PPA1 structure and comparison with available structures of PPases from lower organisms suggest that PPA1 has a largely pre-organized and relatively rigid active site for pyrophosphate hydrolysis. Results from the modeling study indicate the active site of PPA1 has the potential to accommodate double-phosphorylated peptides from JNK1. In short, results from the study provides new insights into the mechanisms of human PPA1 and basis for structure-based anti-cancer drug developments using PPA1 as the target., (© 2021 Wiley Periodicals LLC.)

Phosphates are commonly included in meat processing, where oxidation is inevitable, to improve water binding. This present study attempted to reveal the interactive roles of protein oxidation and tetrasodium pyrophosphate (TSPP) on the crosslinking pattern of myosin mediated by transglutaminase (TGase). Mild oxidation at 1 mM H 2 O 2 facilitated the TGase-initiated crosslinking, with the dominate crosslinking site shifted from S1 (in nonoxidized myosin) to Rod. The introduction of TSPP alleviated the oxidation stress on proteins, and was conductive to the crosslinking reaction notably at the LMM domain. The crosslinking sites in untreated myosin were identified as Gln-613 (S1) and Gln-1498 (LMM) by amino-acid sequence analysis, while strongly oxidation resulted in the loss of Gln-1498. Contrastively, four new reactive crosslinking sites were generated by TSPP, one (Gln-558/Gln-567) located on S1 and three (Gln-1362, Gln-1374, and Gln-1423/Gln-1426) on LMM. Yet, Gln-1362 was eliminated under strong oxidation at 50 mM H 2 O 2 ., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Most kinases utilize ATP as a phosphate donor and phosphorylate a wide range of phosphate acceptors. An alternative phosphate donor is inorganic pyrophosphate (PPi), which costs only 1/1000 of ATP. To develop a method to engineer PPi-dependent kinases, we herein aimed to alter the product of PPi-dependent myo -inositol kinase from d- myo- inositol 1-phosphate to d- myo- inositol 3-phosphate. For this purpose, we introduced the myo- inositol recognition residues of the ATP-dependent myo- inositol-3-kinase into the PPi-dependent myo -inositol-1-kinase. This replacement was expected to change the 3D arrangements of myo -inositol in the active site and bring the hydroxyl group at the 3C position close to the catalytic residue. LC-MS and NMR analyses proved that the engineered enzyme successfully produced myo- inositol 3-phosphate from PPi and myo- inositol.

Protein O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), an essential post-translational as well as cotranslational modification, is the attachment of β-D-N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic proteins. An aberrant O-GlcNAc profile on certain proteins has been implicated in metabolic diseases such as diabetes and cancer. Inhibitors of O-GlcNAc transferase (OGT) are valuable tools to study the cell biology of protein O-GlcNAc modification. In this study we report novel uridine-peptide conjugate molecules composed of an acceptor peptide covalently linked to a catalytically inactive donor substrate analogue that bears a pyrophosphate bioisostere and explore their inhibitory activities against OGT by a radioactive hOGT assay. Further, we investigate the structural basis of their activities via molecular modelling, explaining their lack of potency towards OGT inhibition., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this study, sodium tripolyphosphate (STP) and tetrasodium pyrophosphate (TSPP) were utilized to modify duck egg white protein (EWP). The phosphorylated EWP was prepared as egg white gel (EWG) by adding sodium hydroxide. The phosphorus content of EWP reached 2.18 mg/g and 2.07 mg/g with the addition of STP and TSPP, respectively, after 2 h phosphorylation. The average particle size, absolute zeta potential value, and surface hydrophobicity of EWP increased significantly during phosphorylation. FTIR results indicate that phosphorylation reduced the random structure and α-helical content while increasing the content of β-sheets and β-turn. The mechanical and rheological properties of EWG decreased obviously after phosphorylation. A three-dimensional porous network microstructure was formed, and the gel with added TSPP had larger pores. Adding STP and TSPP to EWG weakened its salt and solvent sensitivity. The findings provide a direction for the exploration of gel properties after protein modification., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Myofibrillar protein isolated from beef muscles were treated with 3 phosphates (Sodium Hexametaphosphate, sodium tripolyphosphate, sodium pyrophosphate) with different concentrations of 0.3%, 0.6%, 0.9%, 1.2% respectively. Protein solubility, surface hydrophobicity and reactive sulfhydryl group was determined. Atomic force microscopy was used to observe the microscopic protein surface. SDS-PAGE was carried out to determine the proteolysis of myofibrillar protein. The solubility and surface hydrophobic bond of myofibrillar protein was highly increased and the diameter decreased by SHMP, TSPP, STPP. Reactive sulfhydryl groups increased after SHMP addition, but slightly decreased in STPP and TSPP treated MP. TSPP and STPP had the same effect on myofibrillar microstructure and was different from SHMP. Three phosphates all caused MP unfolding. The MP gel complexity was increased, and roughness was decreased after phosphates addition, indicating phosphates helped to construct a more ordered and smoother gel microcosmic surface., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate ( k pol ), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.

Phosphate-based glasses (PBGs) offer significant therapeutic potential due to their bioactivity, controllable compositions, and degradation rates. Several PBGs have already demonstrated their ability to support direct cell growth and in vivo cytocompatibility for bone repair applications. This study investigated development of PBG formulations with pyro- and orthophosphate species within the glass system (40 - x)P 2 O 5 ·(16 + x)CaO·20Na 2 O·24MgO (x = 0, 5, 10 mol%) and their effect on stem cell adhesion properties. Substitution of phosphate for calcium revealed a gradual transition within the glass structure from Q 2 to Q 0 phosphate species. Human mesenchymal stem cells were cultured directly onto discs made from three PBG compositions. Analysis of cells seeded onto the discs revealed that PBG with higher concentration of pyro- and orthophosphate content (61% Q 1 and 39% Q 0 ) supported a 4.3-fold increase in adhered cells compared to glasses with metaphosphate connectivity (49% Q 2 and 51% Q 1 ). This study highlights that tuning the composition of PBGs to possess pyro- and orthophosphate species only, enables the possibility to control cell adhesion performance. PBGs with superior cell adhesion profiles represent ideal candidates for biomedical applications, where cell recruitment and support for tissue ingrowth are of critical importance for orthopaedic interventions., Competing Interests: The authors declare no conflict of interest.

A sensing platform is presented for the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu 2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu 2+ ions. In the presence of ALP, the released Cu 2+ can promote the Cu 2+ -dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10 U L -1 , and the limit of detection (LOD) is about 0.14 U L -1 . The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n = 3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.

Discovering new functional genes, designing perfect crystal structures, and developing high-performance materials are the goals being pursued by scientists. Herein, the first antimony pyrophosphate, K 2 Sb(P 2 O 7 )F, possessing an optimal layered structure, is reported, where the perfect structural arrangement induces excellent optical properties. K 2 Sb(P 2 O 7 )F not only displays a sharply enhanced birefringence (0.157@546 nm) compared to the existing phosphate optical materials, but also exhibits a strong second-harmonic generation response (4.0×KDP). Remarkably, a new bifunctional gene, the square-pyramidal SbO 4 F group, was discovered, and a unique two-dimensional arrangement of Cairo pentagonal tiling units was observed in inorganic compounds for the first time., (© 2020 Wiley-VCH GmbH.)

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nucleoside reverse transcriptase inhibitors (NRTIs) are widely used as antiviral and anticancer agents, although they require intracellular phosphorylation into their antivirally active form, the triphosphorylated nucleoside analogue metabolites. We report on the synthesis and characterization of a new class of nucleoside triphosphate analogues comprising a C-alkyl-phosphonate moiety replacing the γ-phosphate. These compounds were converted into bioreversibly modified lipophilic prodrugs at the γ-phosphonate by the attachment of an acyloxybenzyl (ester) or an alkoxycarbonyloxybenzyl (carbonate) group. Such compounds formed γ-C-(alkyl)-nucleoside triphosphate analogues with high selectivity because of an enzyme-triggered delivery mechanism. The latter compounds were very stable in CD4 + T-lymphocyte (CEM cell) extracts, and they were substrates for HIV-reverse transcriptase without being substrates for DNA-polymerases α, β, and γ. In antiviral assays, the excellent antiviral activity of the prodrugs that was found in CEM/0 cells was completely kept in CEM/TK - cells. The activity was improved by 3 logs as compared to the parent nucleoside d4T.

Inositol pyrophosphates (PP-InsPs) comprise an important group of intracellular, diffusible cellular signals that a wide range of biological processes throughout the yeast, plant, and animal kingdoms. It has been difficult to gain a molecular-level mechanistic understanding of the actions of these molecules, due to their highly phosphorylated nature, their low levels, and their rapid metabolic turnover. More recently, these obstacles to success are being surmounted by the chemical synthesis of a number of insightful PP-InsP analogs. This review will describe these analogs and will indicate the important chemical and biological information gained by using them.