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We report a visualized quantitative detection method for nucleic acid amplification tests based on the coffee-ring effect on colloid-crystal substrates. The solution for loop-mediated isothermal amplification (LAMP) of DNA is drop cast on a colloid-crystal surface. After complete drying, a coffee ring containing the LAMP byproduct (i.e., magnesium pyrophosphate) is formed, and it is found that the width of the coffee ring is linearly correlated to the logarithm of the original DNA concentration before the isothermal amplification. Importantly, compared with other substrates, we found that the colloid-crystal substrate is an appropriate substrate for carrying out the assay of high sensitivity. On the basis of these findings, we develop a coffee-ring-based assay for quantitative readout of trace DNA in a sample. The assay requires 0.50 μL of the sample and is completed in 5 min in a homemade chamber with constant humidity. Semiquantitative detection of trace DNA is performed using naked eyes. With the use of a smartphone, the DNA in a sample can be quantitatively detected with a limit of detection of 20 copies.

Herein, we present a rapid, efficient and regioselective phosphorylation method at the 5'-position of unprotected ribose and ribonucleosides with pyrophosphate in the gas phase, which involves the formation of anionic complexes via electrospray ionization and collisional activation to induce phosphorylation within the complexes.

Single superphosphate (SSP) as an additive could improve phosphorus availability and reduce nitrogen loss for composts, but few studies have explored the influence of SSP on the transformation of carbon fractions in composting. The aim of this work was to assess the effect of different components of SSP, including calcium dihydrogen phosphate (CDP), calcium sulfate (CS) and free acid (FA) on organic matter degradation and maturity during pig manure composting. The results showed that CDP had significantly negative effects on the duration of thermophilic phase and organic matter degradation, but lengthened the curing phase for the transformation of organic matter. FA could intensify the inhibiting effect of CDP and postpone the biodegradation process of composting, but CS could buffer the effect of CDP on the degradation of organic carbon fractions by controlling pH. The study reveals the roles of different components of SSP to the transformation of organic carbon fractions, which lays a foundation for regulating the effects of chemical additives during composting. Regulating the content of CDP in SSP or applying SSP with other chemical additives to control the biotoxicity of excess phosphate on microbial activity should be concerned for complete and efficient composting in further study., (Copyright © 2018. Published by Elsevier B.V.)

The biosynthesis of O antigenic polysaccharides in enteric bacteria from nucleoside diphosphate sugars (donor substrates) is catalyzed by the corresponding glycosyltransferases and proceeds through the intermediate formation of undecaprenyl diphosphate sugars (acceptor substrates). To study this process, a chemical synthesis of the compounds having the natural structure or their modified analogs is necessary. The phosphoroimidazolidate method is a universal method for synthesis of lipid diphosphate disaccharides containing 2-acetamido-2-deoxyglycosyl residue at the reducing end of the disaccharide moiety and 11-phenoxyundecyl residue as lipid fragment of the molecule. We report here protocols to synthesize the disaccharides P 1 -(11-phenoxyundecyl)-P 2 -(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate [D-Rha(α1-3)-D-GlcNAcα-PP-PhU, Compound 1] and P 1 -(11-phenoxyundecyl)-P 2 -(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate [D-Gal(β1-3)-D-GalNAcα-PP-PhU, Compound 6]. We describe the procedures for identification and structure estimation of compounds by TLC, NMR, and MS. We also include the biochemical testing of Compound 6 with α2,3-sialyltransferase WbwA from Escherichia coli O104.

DNA nanotechnology has been developed to construct a variety of functional two- and three-dimensional structures for versatile applications. Rolling circle amplification (RCA) has become prominent in the assembly of DNA-inorganic composites with hierarchical structures and attractive properties. Here, we demonstrate a one-pot method to directly encapsulate horseradish peroxidase (HRP) in DNA flowers (DFs) during RCA. The growing DNA strands and Mg 2 PPi crystals lead to the construction of porous DFs, which provide sufficient interaction sites for spontaneously incorporating HRP molecules into DFs with high loading capacity and good stability. Furthermore, in comparison with free HRP, the DNA flower-encapsulated HRP (termed HRP-DFs) demonstrate enhanced enzymatic activity, which can efficiently biocatalyze the H 2 O 2 -mediated etching of gold nanorods (AuNRs) to generate distinct color changes since the longitudinal localized surface plasmon resonance (LSPR) frequency of AuNRs is highly sensitive to the changes in the AuNR aspect ratio. Through rationally incorporating the complementary thrombin aptamer sequence into the circular template, the synthesized HRP-DF composites are readily used as amplified labels for visual and colorimetric detection of thrombin with ultrahigh sensitivity and excellent selectivity. Therefore, our proposed strategy for direct encapsulation of enzyme molecules into DNA structures shows considerable potential applications in biosensing, biocatalysis, and point-of-care diagnostics.

Iron deficiency is a significant health problem across the world. While many patients benefit from oral iron supplements, some, including those on hemodialysis require intravenous iron therapy to maintain adequate iron levels. Until recently, all iron compounds suitable for parenteral administration were colloidal iron-carbohydrate conjugates that require uptake and processing by macrophages. These compounds are associated with variable risk of anaphylaxis, oxidative stress, and inflammation, depending on their physicochemical characteristics. Ferric pyrophosphate citrate (FPC) is a novel iron compound that was approved for parenteral administration by US Food and Drug Administration in 2015. Here we report the physicochemical characteristics of FPC. FPC is a noncolloidal, highly water soluble, complex iron salt that does not contain a carbohydrate moiety. X-ray absorption spectroscopy data indicate that FPC consists of iron (III) complexed with one pyrophosphate and two citrate molecules in the solid state. This structure is preserved in solution and stable for several months, rendering it suitable for pharmaceutical applications in solid or solution state.

There are several options available for intravenous application of iron supplements, but they all have a similar structure:-an iron core surrounded by a carbohydrate coating. These nanoparticles require processing by the reticuloendothelial system to release iron, which is subsequently picked up by the iron-binding protein transferrin and distributed throughout the body, with most of the iron supplied to the bone marrow. This process risks exposing cells and tissues to free iron, which is potentially toxic due to its high redox activity. A new parenteral iron formation, ferric pyrophosphate citrate (FPC), has a novel structure that differs from conventional intravenous iron formulations, consisting of an iron atom complexed to one pyrophosphate and two citrate anions. In this study, we show that FPC can directly transfer iron to apo-transferrin. Kinetic analyses reveal that FPC donates iron to apo-transferrin with fast binding kinetics. In addition, the crystal structure of transferrin bound to FPC shows that FPC can donate iron to both iron-binding sites found within the transferrin structure. Examination of the iron-binding sites demonstrates that the iron atoms in both sites are fully encapsulated, forming bonds with amino acid side chains in the protein as well as pyrophosphate and carbonate anions. Taken together, these data demonstrate that, unlike intravenous iron formulations, FPC can directly and rapidly donate iron to transferrin in a manner that does not expose cells and tissues to the damaging effects of free, redox-active iron.

Mutation of the gatekeeping residues for prenyl donor selectivity in six dimethylallyl transferases significantly increased their activities toward geranyl diphosphate. Forty-two geranylated derivatives were obtained from 15 cyclic dipeptides by using the engineered enzymes. Taking cyclo-l-Trp-l-Trp as an example, the geranyl moiety can be attached to all seven possible positions of the indole nucleus. This study demonstrates a convenient way to increase the structural diversity of geranylated products by structure-based engineering of the available dimethylallyl transferases.

A multiresponsive microcapsule has been synthesized by incorporating photoswitchable spiropyran units and the thermoresponsive monomer N-isopropylacrylamide into membrane lumens. By using functionalized light or thermoresponsive groups, this multifunctional microcapsule can modulate programmed release and interface reactions between lipase and fluorescein diacetate, alkaline phosphatase and fluorescein diphosphate, and others. Exposing this multifunctional microcapsule in a programmed controlled way allowed us to develop schematics to understand complicated interface interactions on protocells.

Sulfonamide residue in foodstuffs and the environment is a serious global concern for their contribution to the occurrence of antibiotic-resistant bacteria, especially in developing countries. Here, we describe a novel, simple, and low-cost bioassay for sulfonamides, which has high potential versatility for use in low-resource settings. The bioassay method is based on a purpose-built luminescent assay reaction that detects sulfonamide groups. The luminescent assay reaction comprises dihydropteroate synthase, a target enzyme of sulfonamides, and luminescent pyrophosphate detection reagent, which triggers a sequence of biomolecular reactions that convert sulfonamides to emit luminescence. The novel assay detected at least six different sulfonamides with an estimated limit of detection of <25 ng ml -1 in a solution-phase assay using a microplate reader. More importantly, the luminescent assay reaction functioned even after spotting and freeze-drying on a wax pattern-printed paper platform. The paper-embedded luminescent assay reaction showed response signals to sulfadiazine within 30 min at a limit of detection similar to that of the solution-phase assay using a microplate reader. The signal could be recorded using a digital camera in the dark and required no other laboratory infrastructure, freeing the assay from the constraints of a well-fitted laboratory.

Here we report a novel bioluminescence (BL) method for exonuclease I (Exo I) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated pyrophosphate release. An inert hairpin probe with a blocked protruding 3'-terminal biotinylated nucleotide was designed, and a blocked 3'-terminal nucleotide of the probe would be removed only in the presence of Exo I, thus rendering the probe with a free 3'-hydroxyl group. Subsequently, with nucleotide incorporation by TdT, huge amounts of pyrophosphates were generated. After the conversion of pyrophosphate to adenosine triphosphate (ATP) through ATP sulfurylase, BL was emitted by the reaction of d-luciferin and ATP with firefly luciferase. Therefore, Exo I activity was indirectly quantified in the range 1-500 mU with a detection limit of 0.05 mU/μl. Moreover, the developed approach was successfully applied to investigate the inhibitory effect of streptavidin on cleavage of Exo I and also determine Exo I activity in spiked serum samples. Overall, the proposed method has high sensitivity and selectivity, and can be universally extended to the detection of other nucleases using terminal extension as a signal amplification method and BL as a detection signal, having potential application in the diagnosis of nuclease-related diseases or evaluation of nuclease functions in biological systems., (© 2018 John Wiley & Sons, Ltd.)

Isoprenyl chains are found in many important metabolites. These are derived from precursors of the appropriate length produced by isoprenyl diphosphate synthases (IDSs). The human pathogen Mycobacterium tuberculosis makes various isoprenoids/terpenoids, with important roles in their biosynthesis played by two closely related IDSs, encoded by grcC1 (Rv0562) and grcC2 (Rv0989c), with Rv0989c generating the 10-carbon precursor ( E )-geranyl diphosphate (GPP), and Rv0562 the 20-carbon precursor ( E,E,E )-geranylgeranyl diphosphate (GGPP). Intriguingly, while Rv0562 contains the prototypical trans -IDS first and second aspartate-rich (DDxxD) motifs (FARM and SARM, respectively), Rv0989c uniquely contains arginine in place of the second Asp in the FARM and first Asp in the SARM. Here site-directed mutagenesis of the corresponding residues in both Rv0562 and Rv0989c reveals that these play a role in determination of product chain length. Specifically, substitution of Asp for the Arg in the FARM and SARM of Rv0989c leads to increased production of the longer 15-carbon farnesyl diphosphate (FPP), while substitution of Arg for the corresponding Asp in Rv0562 leads to increased release of shorter products, both FPP and GPP. Accordingly, while the primary role of the FARM and SARM is known to be chelation of the divalent magnesium ion co-factors that assist substrate binding and catalysis, the Arg substitutions found in Rv0989c seem to provide a novel means by which product chain length is moderated, at least in these M. tuberculosis IDSs.

The contamination of soil with heavy metals is a severe problem due to adverse impact of heavy metals on environmental safety and human health. It is essential to remediate soil contaminated with heavy metals. This study has evaluated the effects of pine biochar, kaolin, and triple super phosphate (TSP) on multiple heavy metals (Ni, Zn, Cu, and Cd) in contaminated soil and accumulation of heavy metals in plants. The amendments can reduce availability of heavy metals in soil by increasing pH, adsorption, complexation, or co-precipitation. Different amendments have variable effects on accumulation of heavy metals in plants and in soil due to its diverse mechanism of stability. The results showed that application of triple super phosphate (TSP) has significant reduced soil Cd exchangeable (EXC) fraction from 58.59 to 21.30%. Bound to carbonates (CAR) fraction decreased from 9.84 to 5.11%, and bound to Fe-Mn oxides (OX) fraction increased from 29.61 to 69.86%. The triple super phosphate (TSP) has the ability to stabilize Cu and especially Cd. However, triple super phosphate (TSP) has enhanced ecological risk of Zn and Ni. Application of pine biochar has significantly enhanced soil pH. The kaolin has significantly reduced EXC fraction of Cd and increased OX fraction of Cu. The amendments and heavy metals have not caused significant effect on SPAD value of Buxus microphylla Siebold & Zucc (B. microphylla). The triple super phosphate (TSP) has significant decreased biomass of B. microphylla and bamboo-williow (Salix sp.) by 24.91 and 57.43%, respectively. Pine biochar and kaolin have increased the accumulation of Zn and Cd in plants. It is concluded that triple super phosphate (TSP) was effective in remediation of Cd and kaolin was effective in remediation of Cd and Cu. Pine biochar was effective in remediation of Cd, Cu, and Zn.

Detection of EGFR mutations in circulating cell-free DNA (cfDNA) is beneficial to monitor the therapeutic effect, tumor progression, and drug resistance in real time. However, it requires that the mutation detection method has the ability to quantify the mutation abundance accurately. Although the next-generation sequencing (NGS) and digital PCR showed high sensitivity for quantifying mutations in cfDNA, the use of expensive equipment and the high-cost hampered their applications in the clinic. Herein, we propose a highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA (termed as qPCR-Invader). The mutation abundance can be quantified by using the difference of Ct values between mutant and wild-type targets without the need of making a standard curve. The method can quantify a mutation level as lower as 0.1% (10 copies/tube). Thirty-six tissue samples from non-small-cell lung cancer (NSCLC) patients were detected by our method and 14/36 tissues gave EGFR L858R mutation-positive results, whereas ARMS-PCR just identified 12 of L858R mutant samples. The two inconsistent samples were confirmed as L858R mutant by pyrophosphorolysis-activated polymerization method, indicating that qPCR-Invader is more sensitive than ARMS-PCR for mutation detection. The L858R mutation abundances of 19 cfDNA samples detected by qPCR-Invader were close to that from NGS, indicating our method can precisely quantify mutation abundance in cfDNA. The qPCR-Invader just needs a common real-time PCR device to accomplish quantification of EGFR mutations, and the fluorescence probes are universal for any target detection. Therefore, it could be used in most laboratories to analyze mutations in cfDNA. Graphical abstract ᅟ.

Magnetic isolation using magnetic nanoparticles (MNPs) as trapping probes have been widely used in sample pretreatment to shorten analysis time. Nevertheless, to generate MNPs is time-consuming. Furthermore, the generated MNPs have to be further functionalized to gain the capability of recognizing their target species. Thus, an alternative approach that can impose magnetism to nonmagnetic species by simply using magnetic ions as the probes is developed in this study. That is, we employ magnetic ions (Fe 3+ , Co 2+ , and Ni 2+ ) that can interact with nonmagnetic species containing oxygen-containing functional groups as the probes. Pyrophosphate (PPi), bacteria, and mammalian cells were selected as the model samples. Our results show that the as-prepared magnetic ion-PPi conjugates gain sufficient magnetism and can be readily aggregated by applying an external magnetic field. Moreover, the magnetic trapping is reversible. The PPi-containing conjugates can lose their magnetic property simply using ethylenediaminetetraacetic acid or aluminum ions as competing agents to remove or to replace, respectively, the conjugated magnetic ions. In addition, bacteria and mammalian cells that possess abundant oxygen-containing functional groups on their cell surfaces can be selectively probed by magnetic ions and gain sufficient magnetism for magnetic isolation from complex serum samples.

The detection of phosphates and their enzymatic hydrolysis is of great importance because of their essential roles in various biological processes and numerous diseases. Compared with individual sensors for detecting one given phosphate at a time, sensor arrays are able to discriminate multiple phosphates simultaneously. Although nanomaterial-based sensor arrays have shown great promise for the discrimination of phosphates, very few of them have been explored for probing phosphates involved enzymatic hydrolysis. To fill this gap, herein we fabricated two-dimensional-metal-organic-framework (2D-MOF)-nanozyme-based sensor arrays by modulating their peroxidase-mimicking activity with various phosphates, including AMP, ADP, ATP, pyrophosphate (PPi), and phosphate (Pi). The sensor arrays were used to successfully discriminate the five phosphates not only in aqueous solutions but also in biological samples. The practical application of the sensor arrays was then validated with blind samples, where 30 unknown samples containing phosphates were accurately identified. Moreover, the sensor arrays were successfully applied to probing hydrolytic processes involving ATP and PPi that are catalyzed by apyrase and PPase, respectively. This work demonstrates a nanozyme-based sensor array as a convenient and reliable analytical platform for probing phosphates and their related enzymatic processes, which could be applied to other analytes and enzymatic reactions.

As the last resort for intractable Gram-positive bacterial infections, vancomycin is losing efficacy with the emergence of vancomycin-resistant bacteria, especially vancomycin-resistant Enterococci (VRE). To combat this threat, we rationally designed and synthesized 39 novel vancomycin derivatives by respective or combined modifications using metal-chelating, lipophilic, and galactose-attachment strategies for extensive structure-activity relationship (SAR) analysis. In a proposed mechanism, the conjugation of dipicolylamine on the seventh amino acid resorcinol position or C-terminus endowed the vancomycin backbone with binding capacity for the pyrophosphate moiety in lipid II while maintaining the intrinsic binding affinity for the dipeptide terminus of the bacterial cell wall peptidoglycan precursor. The in vitro antibacterial activities were evaluated, and the optimal compounds indicated 16- to 1024-fold higher activity against VRE than that of vancomycin. Compound 11 b (3',5'-bis(dipicolylaminomethyl)tyrosine [1,2,3]triazolylmethoxylethyoxyl ethylaminomethyl-N-decylvancomycin) was found to have particularly potent activity against VRE through synergistic effects brought about by combining two peripheral modifications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: To evaluate the effect of different glass-ceramic surface treatments and aging on the fatigue failure load of a lithium disilicate glass-ceramic adhesively cemented to a dentin analogue material., Methods: One hundred and twenty (120) disc-shaped lithium disilicate specimens (Ø=10mm, thickness=1.5mm) were produced and randomly allocated (n=20) into 6 groups, considering 2 study factors: "surface treatment" in 3 levels (SIL-silane application only; HF5+SIL-5% hydrofluoric acid etching and silane application; ME&P-etching with an one-step ceramic primer), and "storage" in 2 levels (baseline-storage for 7 days; aging-storage for 90 days+12,000 thermal cycles). Ceramic discs were adhesively cemented to discs of a dentin analogue material (Ø=10mm, thickness=2.0mm) following the manufacturers' instructions. The fatigue failure load was determined by the staircase approach (250,000 cycles; 20Hz; initial load=1050N [∼70% of mean load-to-failure]; step size=52.5N [5% of initial load]). Micro-morphologic, fractographic, and atomic force microscope analysis were also performed. Fatigue failure load data were evaluated by one-way ANOVA, Bonferroni and t-tests for independent samples., Results: HF5+SIL presented higher fatigue failure load in both conditions (baseline and aging); ME&P presented intermediary mean values, while the SIL group presented the worst performance. All groups had a statistically significant decrease in the fatigue performance after aging., Significance: Hydrofluoric acid followed by silane application showed the best fatigue performance for an adhesively-cemented lithium disilicate ceramic. Aging negatively influenced the fatigue performance for all tested groups., (Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Thermoluminescence (TL) measurements were carried out on europium (Eu) doped magnesium pyrophosphate (Mg 2 P 2 O 7 ) nanopowders using gamma irradiation in the dose range of 0.1 to 3 kGy. The powder samples were successfully synthesized by chemical co-precipitation synthesis route. The formation and crystallinity of the compound was confirmed by powder X-ray diffraction (PXRD) pattern. The estimated particle size was found to be in nanometer scale by using Debye Scherer's formula. A scanning electron microscopy (SEM) study was carried out for the morphological characteristics of as synthesized Mg 2 P 2 O 7 :Eu phosphor. Photoluminescence (PL) study was carried out to confirm the presence of the rare-earth ion and its valence state. The TL analysis of synthesized samples were performed after the irradiation of Mg 2 P 2 O 7 :Eu with cobalt-60 ( 60 Co) gamma rays. The high and low intensity peaks of TL glow curve appeared at around 400 K, 450 K, 500 K and 596 K respectively. The appreciable shift in peak positions has been observed for different concentrations of Eu ion. The trapping parameters, namely activation energy (E), order of kinetics (b) and frequency factor (s) have been determined using thermal cleaning process, peak shape (Chen's) method and glow curve deconvolution (GCD) functions., (Copyright © 2018 John Wiley & Sons, Ltd.)

Objective: This study aimed at evaluating the effects obtained by administering 30 mg of micronised dispersible ferric pyrophosphate plus 300 mg of alpha-lactalbumin (MDFP-AL) compared to 80 mg of ferrous gluconate (FG) in pregnant women affected by iron-deficiency anemia (IDA)., Patients and Methods: We considered eligible all second-trimester singleton pregnancies in women affected by IDA. We excluded any other disease, twin pregnancies, any other pharmacologic/nutraceutical treatments (besides folic acid) before/during pregnancy. We randomized patients in two groups: one underwent treatment with 1 tablet of MDFP-AL/day, the other one with 1 tablet of FG/day, for 30 days. We evaluated hemoglobin (Hb), ferritin, red blood cells (RBCs), serum iron, hematocrit (Hct), and side effects at baseline (T0), after 15 days (T1) and 30 days (T2)., Results: 50 women met the inclusion/exclusion criteria. We did not observe significant differences between the two groups for mean age, gestational age at the enrollment and parity. In MDFP-AL group, after 15 days (T1) Hb, ferritin, serum iron and Hct and were significantly improved respect to baseline (T0); after 30 days (T2), all the parameters, including RBCs, were significantly improved respect to baseline (T0). Similarly, in FG group the investigated parameters were improved both after 15 (T1) and 30 days (T2) respect to baseline (T0), although less in percentage terms respect to MDFP-AL group. The side effects rate was 24% in FG group, whereas MDFP-AL group did not show any significant side effect., Conclusions: Overall, MDFP-AL is more effective and safe than FG for the treatment of IDA in pregnant women.

Betulin-3,28-diphosphate (BDP) obtained by phosphorylation of betulin using POCl₃ has two main structural forms-BDP-1 and BDP-2-which differ in ethanol solubility, melting point, FTIR spectra, thermoanalytical characteristics and biological activity. Betulin-3,28-diphosphate and its sodium salt (Na-BDP) were characterized using 13 C and 31 P-NMR spectra, powder XRD experiments, as well as differential scanning calorimetry (DSC) and thermogravimetric analysis (TG) methods. The exo-effects at 193 ± 8 °C for ethanol soluble BDP-1 samples (-19.7 ± 0.2 kJ∙mol -1 ) were about three times less than for ethanol insoluble BDP-2 samples f (-70.5 ± 0.7 kJ∙mol -1 ). The DSC curves of Na-BDP-1 and Na-BDP-2 characterized the endo-effects having a maximum at 95⁻112 °C. Water-soluble Na-BDP-1 was obtained as needle-like crystals, unlike poorly crystalline Na-BDP-2, whereas BDP-1 and BDP-2 aged with time and were isolated as amorphous substances. In vitro experiments on rats showed that compared to the control, Na-BDP-1 increased catalase and SOD activity and improved energy metabolism more effectively than Na-BDP-2., Competing Interests: The authors declare no conflict of interest.

A compartmental chemosensor probe HL has been designed and synthesized for the selective recognition of zinc ions over other transition metal ions via fluorescence "ON" strategy. The chemosensing behaviour of HL was demonstrated through fluorescence, absorption and NMR spectroscopic techniques. The molecular structure of the zinc complex derived from HL was determined by X-ray crystallography. A probable mechanism of this selective sensing behavior was described on the basis of spectroscopic results and theoretical studies by density functional theory (DFT). The biological applicability of the chemosensor HL was examined via cell imaging on HeLa cells. The HL-zinc complex served as a secondary fluorescent probe responding to the pyrophosphate anion specifically over other anions. The fluorescence enhancement of HL in association with Zn2+ ions was quenched in the presence of pyrophosphate (PPi). Thus, a dual response was established based on "OFF-ON-OFF" strategy for detection of both cation and anion. This phenomenon was utilized in the construction of a "INHIBIT" logic gate.

Pyrophosphate linkages are important in extant biology and are hypothesized to have played a role in prebiotic chemistry and in the origination of oligonucleotides. Inspired by pyrophosphate as backbones of primordial oligomers, DNA oligomers with varying amounts of pyrophosphate inserts (ppDNA) were synthesized and investigated for their base-pairing properties. As expected, pyrophosphate inserts into the backbone compromised the thermal stability of ppDNA-DNA duplexes. In contrast, the ppDNA-RNA duplex exhibited, remarkably, duplex stability, even with accumulation of pyrophosphate linkages. This seems to be a consequence of an increase in the diameter of the double-helix with eight-bond-repeat units, and higher inclination of the base-pair axis with respect to the backbone in RNA (A-form), compared with that in DNA (B-form). These results suggest that pyrophosphate-linked oligonucleotides could harbor functional capabilities with implications for their roles in the origins of life and chemical biology., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Almost all kinases utilize ATP as their phosphate donor, while a few kinases utilize pyrophosphate (PPi) instead. PPi-dependent kinases are often homologous to their ATP-dependent counterparts, but determinants of their different donor specificities remain unclear. We identify a PPi-dependent member of the ribokinase family, which differs from known PPi-dependent kinases, and elucidate its PPi-binding mode based on the crystal structures. Structural comparison and sequence alignment reveal five important residues: three basic residues specifically recognizing PPi and two large hydrophobic residues occluding a part of the ATP-binding pocket. Two of the three basic residues adapt a conserved motif of the ribokinase family for the PPi binding. Using these five key residues as a signature pattern, we discover additional PPi-specific members of the ribokinase family, and thus conclude that these residues are the determinants of PPi-specific binding. Introduction of these residues may enable transformation of ATP-dependent ribokinase family members into PPi-dependent enzymes.

Reverse transcriptase (RT) is responsible for replicating the HIV-1 genome and is a validated therapeutic target for the treatment of HIV infections. During each cycle of the RT-catalyzed DNA polymerization process, inorganic pyrophosphate is released as the by-product of nucleotide incorporation. Small molecules were identified that act as bioisosteres of pyrophosphate and can selectively freeze the catalytic cycle of HIV-1 RT at the pre-translocated stage of the DNA- or RNA-template-primer-enzyme complex., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Traditionally, genomic DNA detection is relay on a rigorous DNA amplification process, which always accompanied with complicated gel electrophoresis or expensive fluorescence detection methods. In this work, we have translated genomic DNA detection into adenosine triphosphate (ATP) test based on a split aptamer-based electrochemical sandwich assay. The key characteristic of our method are list as follows: first, nucleic acid amplification of the target gene was performed by the use of a loop mediated isothermal amplification (LAMP) process. The pyrophosphate (PPi), which released as the byproduct during the LAMP reaction, were further converted into ATP in the presence of adenosine 5'-phosphosulfate (APS) and ATP sulfurylase. Thereafter, the converted ATP was detected by constructing an electrochemical sandwich aptasensor. With such design, the conversion from the difficult detecting target (genomic DNA) into a convenient measured object (ATP) has been achieved. This proposed strategy was highly sensitive for Nosema bombycis genomic DNA PTP1 detection with a detection limit as low as 0.47 fg/μL and a linear range from 0.001pg/μL to 50ng/μL. And we supposed that this novel target conversion electroanalytical strategy established a universal approach for quantitative analysis of any other kinds of nucleic acid in assistance of nucleic acid polymerization reaction., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nanozymes have been extensively exploited in the construction of colorimetric sensors in the past decade. However, as lack of specific recognition element, these sensors are mostly limited in the detection of hydrogen peroxide and its related agents. In this work, a colorimetric assay for the detection of alkaline phosphatase (ALP) activity was developed by employing copper (II)-based metal-organic frameworks (Cu-MOFs) as peroxidase mimic and pyrophosphate (PPi) as recognition element. Benefiting from its intrinsic porosity, Cu-MOFs built from one-step self-assembly showed a superior catalytic activity over horseradish peroxidase. It was found that, in the presence of PPi, the catalytic activity of Cu-MOFs was greatly inhibited due to the ultrastrong binding ability of Cu 2+ with PPi, and no colorimetric signal was recorded. However, after incubation with ALP, such negative behavior of PPi can be suppressed as the hydrolysis of PPi into orthophosphates (Pi) with low affinity to Cu 2+ , producing a colorimetric signal depending on ALP level. On this basis, a way for quantitative analysis of ALP activity was paved accordingly. Compared with previously reported ALP assays based on nanomaterials, this assay is simple, cost-effective and visualized, which not only allows highly sensitive analysis of ALP activity with a detection limit of 0.19 U/L, but also exhibits a good sensing performance in serum samples. Furthermore, based on the distinct colorimetric signals induced by PPi and ALP, an IMPLICATION logic gate was constructed. We hope that this study could provide a new insight for designing nanozyme-based highly specific colorimetric sensors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions., Results: Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes., Conclusion: A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall that is critical for their survival. The largest structural component of the cell wall, the mycolyl-arabinogalactan-peptidoglycan complex, has at its core a galactan domain composed of d-galactofuranose residues. Mycobacterial galactan biosynthesis has been proposed to involve two glycosyltransferases, GlfT1 and GlfT2, which elongate polyprenol-pyrophosphate linked glycosyl acceptor substrates using UDP-galactofuranose as the donor substrate. We here report the first chemical synthesis of GlfT1 and GlfT2 acceptor substrates containing pyrophosphate and polyprenol moieties (compounds 3, 4, 22 and 23). The approach involves chemical synthesis of an oligosaccharide, subsequent phosphorylation at the reducing end and coupling to a polyprenol phosphate. These compounds were shown to be substrates for either GlfT1 (22 and 23) or GlfT2 (3 and 4) and all were substantially more active than the corresponding alkyl glycoside substrates reported previously. Mass spectrometric analysis of the products formed from the reaction of 3, 4, 22 and 23 with the respective cognate enzyme and UDP-galactofuranose provide additional evidence for the galactan biosynthetic model in which GlfT1 adds the first two galactofuranose residues with the remainder being installed via GlfT2. Overall, these results highlight the importance of the pyrophosphate motif in recognition of acceptor substrates by both enzymes and demonstrate a straightforward route for the preparation of such compounds. The work also provides additional support for the process by which this important glycan is biosynthesized using, for the first time, close structural analogs to the natural substrates.

Crosslinked high-performance polymers have many industrial applications, but are difficult to recycle or rework. A novel class of recyclable crosslinking Cu(II)-metallo-supramolecular coordination polymers are successfully prepared, which possess outstanding thermal stability and mechanical property. More importantly, the Cu 2+ coordination interactions can be further removed via external pyrophosphate to recover the linear polymers, which endow the crosslinking polymers with recyclability., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report a series of Cu(II) and Zn(II) complexes with different ligands containing a dipicolyl unit functionalized with urea groups that may contain or not a phenylboronic acid function. These complexes were designed for the recognition of phosphorylated anions through coordination to the metal ion reinforced by hydrogen bonds involving the anion and NH groups of urea. The complexes were isolated and several adducts with pyrophosphate were characterized using Xray diffraction measurements. Coordination of one of the urea nitrogen atoms to the metal ion promoted the hydrolysis of the ligands containing 1,3-diphenylurea units, while ligands bearing 1-ethyl-3-phenylurea groups did not hydrolyze significantly at room temperature. Spectrophotometric titrations, combined with ¹H and 31 P NMR studies, were used in investigating the binding of phosphate, pyrophosphate (PPi), and nucleoside 5'-polyphosphates (AMP, ADP, ATP, CMP, and UMP). The association constants determined in aqueous solution (pH 7.0, 0.1 M MOPS) point to a stronger association with PPi, ADP, and ATP as compared with the anions containing a single phosphate unit. The [CuL⁴] 2+ complex shows important selectivity for pyrophosphate (PPi) over ADP and ATP., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps., Competing Interests: Conflict of interest statement: C.B. and D.J.K. are collaborating on a forthcoming book., (Copyright © 2018 the Author(s). Published by PNAS.)

The cyclization mechanisms involved in the biosynthesis of sesterterpenes are not fully understood. For example, there are two plausible reaction pathways for sesterfisherol biosynthesis, which differ in the order of ring cyclization: A-D-B/C (Path a) and A-B-C/D (Path b). It is difficult to capture intermediates of terpene cyclization, which is a complex, domino-type reaction, and so here we employed a combination of experimental and computational methods. Density functional theory calculations revealed unexpected intermediates and transition states, and implied that C-H···π interaction between a carbocation intermediate and an aromatic residue of sesterfisherol synthase (NfSS) plays a critical role, serving to accelerate the 1,2-H shift (thereby preventing triquinane carbocation formation) and to protect reactive carbocation intermediates from bases such as pyrophosphate or water in the active site. Site-directed mutagenesis of NfSS guided by docking simulations confirmed that phenylalanine F191 is a critical amino acid residue for sesterfisherol synthase, as the F191A mutant of NfSS produces novel sesterterpenes, but not sesterfisherol. Although both pathways are energetically viable, on the basis of our computational and experimental results, NfSS-mediated sesterfisherol biosynthesis appears to proceed via Path a. These findings may also provide new insight into the cyclization mechanisms in related sesterterpene synthases.

A series of Ba 2 P 2 O 7 :xEu 2+ ,yCe 3+ ,zTb 3+ phosphors was synthesized via a co-precipitation method, then their crystal structure, quantum efficiency and luminescent properties were analyzed by XRD and FL, respectively. The results showed that these phosphors not only presented the excitation characteristics of Ba 2 P 2 O 7 :xEu 2+ ,zTb 3+ , but also exhibited that of the Ba 2 P 2 O 7 :yCe 3+ ,zTb 3+ phosphor. Meanwhile, the tri-doped phosphor showed a stronger absorption around 320 nm in contrast with the Eu 2+ /Ce 3+ :Tb 3+ co-doped phosphor. Not only can energy transfer from Ce 3+ →Eu 2+ be observed; the energy transfer mechanism from Eu 2+ to Tb 3+ is discussed in the tri-doped system. Ce 3+ affects the luminescence properties of Ba 2 P 2 O 7 :xEu 2+ ,yCe 3+ ,zTb 3+ phosphors just as the sensitizer whereas Eu 2+ is considered both as the sensitizer and the activator. The chromaticity coordinates of tri-doped phosphors excited at 320 nm stayed steadily in the bluish-white light region,and the emitted color and color temperature (CCT) of these phosphors could be tuned by adjusting the relative contents of Eu 2+ , Ce 3+ and Tb 3+ . Hence, the single phase Ba 2 P 2 O 7 :xEu 2+ ,yCe 3+ ,zTb 3+ phosphors may be considered as potential candidates for white light-emitting diodes., (Copyright © 2017 John Wiley & Sons, Ltd.)

This paper presents a comparative evaluation of different oral ferric iron formulations for ability to retain Fe 3+ in simulated gastric fluid (SGF), be internalized by cells lining intestinal epithelium, and cross it to reach the bloodstream. In all formulations iron was ferric pyrophosphate, the excipients were different types and fractions of lecithin plus sucrose esters of fatty acids matrix (Sideral® RM; PRT1; PRT2) or lecithin without sucrester (SUN). Dissolution kinetics of formulations in SGF was studied by USP method. The ability of the formulations to promote iron intestinal absorption was evaluated by the Caco-2 cell model, measuring cellular ferritin content, and by the excised rat intestine model, yielding apparent permeability parameters (P app ). All formulations limited iron release in SGF to ≤10%. Sideral® RM was by far the most absorbed by Caco-2, as ferritin content was in the order: Sideral® RM≫PRT2>PRT1>SUN>control. The Fe 3+ crossing the intestinal barrier was in part reduced to Fe 2+ by epithelial enzymes, in part it was carried by formulation rearrangement into nano-structures able to protect it from reduction and apt for internalization by epithelium cells. P app parameters were in the order: Sideral® RM≫PRT1>PRT2>SUN=control. Relevance of transepithelial Fe 2+ carrier, DMT-1, to Fe 3+ transport was ruled out using a DMT-1 inhibitor. In conclusion, Sideral® RM retains iron in SGF, and is the most suitable for Fe 3+ internalization by Caco-2 cells, Fe 3+ protection from enzymatic reduction and promotion of Fe 3+ absorption across intestinal epithelium, non-mediated by DMT-1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Objectives: Nutritional anemia is a significant public health issue with 50-80% prevalence in Indian children. Fortification of food, specifically milk, with iron is a potential approach to increase dietary iron intake. Ferric pyrophosphate [Fe4(P2O7)3] is organoleptically neutral and is less soluble in acid medium and, further, has low bioavailability in milk. However, since ascorbic acid is a potent enhancer of iron absorption, the coadministration of ascorbic acid with Fe4(P2O7)3 might enhance the absorption of iron. We evaluated the effect of ascorbic acid on iron absorption from a Fe4(P2O7)3 and an ascorbic acid fortified milk beverage with respect to milk fortified with Fe4(P2O7)3 alone., Methods and Study Design: A double-blind, two-way crossover, randomized study was conducted in 25 mildly anemic children. The test group received milk fortified with beverage powder containing 7 mg isotopically labeled iron (57Fe/58Fe) as Fe4(P2O7)3, equimolar proportions of ascorbic acid and 200 mg of calcium whereas control group received milk fortified with energy, calcium and iron equivalent beverage powder. Fractional iron absorption was measured by erythrocyte incorporation of stable isotopes of iron (57Fe/58Fe) in both the groups., Results: The fractional iron absorption from the control drink was 0.80% (95% CI: 0.57, 1.12). Fortifying the milk with an equimolar amount of ascorbic acid increased the fractional iron absorption almost 2-fold to 1.58% (95% CI: 1.13, 2.22)., Conclusions: The presence of ascorbic acid in an equimolar ratio with that of iron from Fe4(P2O7)3 salt in milk as a fortificant enhanced iron absorption when compared to milk fortified with only Fe4(P2O7)3.

Gram-positive bacteria endow their peptidoglycan with glycopolymers that are crucial for viability and pathogenesis. However, the cellular machinery that executes this function is not well understood. While decades of genetic and phenotypic work have highlighted the LytR-CpsA-Psr (LCP) family of enzymes as cell-wall glycopolymer transferases, their in vitro characterization has been elusive, largely due to a paucity of tools for functional assays. In this report, we synthesized authentic undecaprenyl diphosphate-linked wall teichoic acid (WTA) intermediates and built an assay system capable of monitoring LCP-mediated glycopolymer transfer. We report that all Bacillus subtilis LCP enzymes anchor WTAs to peptidoglycan in vitro. Furthermore, we probed the catalytic requirements and substrate preferences for these LCP enzymes and elaborated in vitro conditions for facile tests of enzyme function. This work sheds light on the molecular features of glycopolymer transfer and aims to aid drug discovery and development programs exploiting this promising antibacterial target., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Natural product prenyltransferases are known to display relaxed acceptor substrate specificity. Although recent studies with a small set of unnatural alkyl donors have revealed that prenyltransferases are flexible with regard to their alkyl donors, the scope of their alkyl donor specificity remains poorly understood. Towards this goal, we report the synthesis of 20 unnatural alkyl pyrophosphate donors and an assessment of the reactions of these synthetic unnatural alkyl pyrophosphate analogues catalyzed by tyrosine O-prenyltransferase SirD. This study demonstrates that SirD can utilize 16 out of 21 alkyl pyrophosphate analogues (including the natural donor) in catalyzing mostly O-alkylation of l-tyrosine. This study reveals the broad alkyl donor specificity of SirD and opens the door for the interrogation of the alkyl donor specificity of other prenyltransferases for potential utility as biocatalysts for differential alkylation applications., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mode of interaction and thermodynamic stability of complexes formed in binary and ternary Cu(II)/ATP/triamines systems were studied using potentiometric and spectroscopic (NMR, EPR, UV-Vis) methods. It was found that in binary metal-free systems ATP/H x PA species are formed (PA: Spd=spermidine or 3,3-tri=1,7-diamino-4-azaheptane) where the phosphate groups from nucleotides are preferred negative centers and protonated amine groups of amines are positive centers of reaction. In the ternary systems Cu/ATP/H x (PA) as well as Cu/(ATP)(PA) species are formed. The type of the formed Cu(II) complexes depends on pH of the solution. For a low pH value the complexation appears between Cu(II) and ATP molecules via oxygen atoms of phosphate groups. For a very high pH value, where ATP is hydrolyzed, the Cu(II) ions are bound to the nitrogen atoms of polyamine molecules. We did not detect any direct coordination of the N7 nitrogen atom of adenosine to Cu(II) ions. It means that the CuN7 interaction is an indirect type and can be due to noncovalent interplay including water molecule. EPR studies were performed at glassy state (77K) after a fast freezing both for binary and ternary systems. The glassy state EPR spectra do not reflect species identified in titration studies indicating significant effect of rapid temperature decrease on equilibrium of Cu(II) complexes. We propose the molecular structure of all the studied complexes at the glassy state deduced from EPR and optical spectroscopy results., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The exosporium layer of Bacillus anthracis spores is rich in L-rhamnose, a common bacterial cell-wall component, which often contributes to the virulence of pathogens by increasing their adherence and immune evasion. The biosynthetic pathway used to form the activated L-rhamnose donor dTDP-L-rhamnose consists of four enzymes (RfbA, RfbB, RfbC and RfbD) and is an attractive drug target because there are no homologs in mammals. It was found that co-purifying and screening RfbC (dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase) from B. anthracis in the presence of the other three B. anthracis enzymes of the biosynthetic pathway yielded crystals that were suitable for data collection. RfbC crystallized as a dimer and its structure was determined at 1.63 Å resolution. Two different ligands were bound in the protein structure: pyrophosphate in the active site of one monomer and dTDP in the other monomer. A structural comparison with RfbC homologs showed that the key active-site residues are conserved across kingdoms.

In this review, our recent advances in the development of nucleoside di- and nucleoside triphosphate prodrugs is summarized. Previously, we had developed a successful membrane-permeable pronucleotide system for the intracellular delivery of nucleoside monophosphates as well, the so-called cycloSal-approach. In contrast to that work in which the delivery is initiated by a chemically driven hydrolysis reaction, for the di- and triphosphate delivery, an enzymatic trigger mechanism involving (carboxy)esterases had to be used. The other features of the new pronucleotide approaches are: (i) lipophilic modification was restricted to the terminal phosphate group leaving charges at the internal phosphate moieties and (ii) appropriate lipophilicity is introduced by long aliphatic residues within the bipartite prodrug moiety. The conceptional design of the di- and triphosphate prodrug systems will be described and the chemical synthesis, the hydrolysis properties, a structure-activity relationship and antiviral activity data will be discussed as well. The advantage of these new approaches is that all phosphorylation steps from the nucleoside analogue into the bioactive nucleoside triphosphate form can be bypassed in the case of the triphosphate prodrugs. Moreover, enzymatic processes like the deamination of nucleosides or nucleoside monophosphates which lead to catabolic clearance of the potential antivirally active compound can be avoided by the delivery of the higher phosphorylated nucleotides.

One of the promising new antitumor platinum complexes is a large-ring chelate complex [PtCl 2 (cis-1,4-DACH)] (DACH = diaminocyclohexane) (kiteplatin). Recently, new platinum(ii) derivatives of kiteplatin with pyrophosphate as a carrier ligand have been synthesized and tested on a panel of human cancer cell lines. These derivatives of kiteplatin were found to be more effective than clinically used anticancer platinum drugs. The design of kiteplatin pyrophosphate derivatives was based on the concept of pyrophosphate coordinated platinum complexes, phosphaplatins. Phosphaplatins have been shown to function without binding to DNA and hence DNA has been excluded as the target of phosphaplatins in contrast to conventional antitumor platinum drugs. Cytotoxicity, major cellular targets and DNA interactions of the new anticancer platinum drug were characterized by standard biochemical methods and methods of molecular and cellular biology. We demonstrate that, in contrast to what has been reported on closely related phosphaplatins, the derivatives of kiteplatin with the pyrophosphate carrier ligand are activated in the cellular environment. This activation, which yields species capable of platination of DNA, very likely comprises the hydrolytic release of the pyrophosphate ligand that could be enzymatically catalyzed. Collectively, these data provide convincing evidence that unexpectedly DNA is an important target for the biological activity of the kiteplatin pyrophosphate derivatives, although the overall mechanism of action might be different from those of conventional platinum drugs.

Enzymatic reactions usually occur in several steps: a step of substrate binding to the surface of the protein, a step of protein reorganization around the substrate and conduction of a chemical reaction, and a step of product release. The release of inorganic phosphate-PPi-from the matrix of the protein HIV reverse transcriptase is investigated computationally. Atomically detailed simulations with explicit solvent are analyzed to obtain the free energy profile, mean first passage time, and detailed molecular mechanisms of PPi escape. A challenge for the computations is of time scales. The experimental time scale of the process of interest is in milliseconds, and straightforward molecular dynamics simulations are in sub-microseconds. To overcome the time scale gap, we use the algorithm of Milestoning along a reaction coordinate to compute the overall free energy profile and rate. The methods of locally enhanced sampling and steered molecular dynamics determine plausible reaction coordinates. The observed molecular mechanism couples the transfer of the PPi to positively charged lysine side chains that are found on the exit pathway and to an exiting magnesium ion. In accord with experimental findings, the release rate is comparable to the chemical step, allowing for variations in substrate (DNA or RNA template) in which the release becomes rate determining.

Despite the growing knowledge on the mechanisms of fracture healing, bone defects often do not heal in a timely manner. Clinically, tricalcium phosphate (TCP) bone substitutes are used to fill bone defects and promote bone healing. However, the degradation rate of these implants is often too slow for sufficient bone replacement. The use of calcium phosphate material with the crystalline phase Ca 10 [K/Na](PO 4 ) 7 containing different amounts of di- and metaphosphates may overcome this problem, because these materials show an accelerated degradation. Therefore, we generated alkaline substituted Ca-P scaffolds with different amounts of ortho-, di- and metaphosphates. The degradation of these materials was analyzed in TRIS-HCl buffer solution in vitro. Moreover, we measured the compressive strength and porosity of the scaffolds by micro-CT analysis. The biocompatibility of the scaffolds was evaluated in vivo in the mouse dorsal skinfold chamber by means of intravital fluorescence microscopy and histology. We found that higher amounts of incorporated di- and metaphosphates increase the degradation rate and compressive strength of the scaffolds without inducing a stronger leukocytic inflammatory host tissue reaction after implantation. Histological analyses confirmed the good biocompatibility of the scaffolds containing di- and metaphosphates. In summary, this study demonstrates that the compressive strength and degradation rate of Ca-P scaffolds can be improved by incorporation of di- and metaphosphates without affecting their good biocompatibility. Hence, this material modification may be particularly beneficial for the treatment of metaphyseal bone defects in weight bearing locations.

Phosphate oxoanions and phosphorylated biomolecules (such as nucleotides, lipids, and proteins) play key roles in a wide range of biological processes. The ability to selectively detect these ions in the presence of each other has numerous applications in biochemistry and biomedicine. However, receptors and sensors that can discriminate between polyphosphate species with high selectivity and in biologically relevant conditions are rare. In this Account, we show how peptides (both cyclic and linear) can be used to position two zinc(II)dipicolylamine [Zn(II)DPA] binding sites in an appropriate arrangement to provide selective binding of pyrophosphate (PPi) in the presence of other polyphosphate species, including ATP, and in complex media such as cell growth buffer. The use of peptide scaffolds to position the Zn(II)DPA anion binding sites allowed the synthesis of small receptor libraries in which the arrangement of the two binding sites could be subtly altered to evaluate the factors affecting both binding selectivity and affinity for PPi. We altered a number of structural elements including peptide length, cyclic peptide ring size, amino acid composition, the positioning of the binding sites with respect to one another, and the relative stereochemistry of the peptides. Backbone modified cyclic peptides based on the Lissoclinum class of natural products were initially employed to provide an added degree of preorganization to the receptors, although it was subsequently found that short, flexible bis[Zn(II)DPA]-functionalized linear peptides are also effective scaffolds for selective pyrophosphate recognition. The peptidic receptors were successfully employed for the detection of PPi in aqueous media by indicator displacement assays using both colorimetric and fluorescent indicators, with the best compounds able to bind to PPi selectively in both cell growth media and artificial urine and also allow the accurate determination of PPi concentrations in physiologically relevant ranges (micromolar concentrations) in these complex media. Improved pyrophosphate selectivity was observed upon increasing the complexity of the media from HEPES buffer to cell growth media (Krebs saline). Pyrophosphate sensors in which a fluorescent indicator was covalently attached to either a linear or cyclic peptide scaffold through a flexible linker were then constructed. When the Zn(II)DPA binding sites and the indicator were suitably placed with respect to one another on the peptide scaffold, these 'intramolecular indicator displacement assays' showed improved selectivity for PPi over other polyphosphate anions, such as ATP, when compared to the intermolecular indicator displacement assays. This observation provides the basis for the design and application of future PPi sensors in biochemistry and biomedicine.

An efficient near-infrared fluorescence probe has been developed for the sequential detection of Cu 2+ , pyrophosphate (P 2 O 7 4- , PPi), and alkaline phosphatase (ALP), which is based on the "off-on-off" fluorescence switch of branched polyethyleneimine (PEI)-capped NaGdF 4 :Yb/Tm upconversion nanoparticles (UCNPs). The fluorescence is quenched via energy transfer from UCNPs to Cu 2+ for the coordination of PEI with Cu 2+ . The strong affinity between Cu 2+ and PPi leads to the formation of Cu 2+ -PPi complex and results in the detachment of Cu 2+ from the surface of UCNPs, thus the fluorescence is triggered on. ALP-directed hydrolysis of PPi causes the disassembly of Cu 2+ -PPi complex and re-conjugation between Cu 2+ with PEI, which leads to the switch-off fluorescence of UCNPs. The system allows sequential analysis of Cu 2+ , PPi, and ALP by modulating the switch of the fluorescence of UCNPs with detection limits of 57.8nM, 184nM, and 0.019U/mL for Cu 2+ , PPi, and ALP, respectively. By virtue of the NIR feature and excellent biocompatibility, the UCNPs-based probes are suitable for bioimaging. Taking Cu 2+ visualization as a model, the nanoprobes have been successfully applied for intracellular imaging of Cu 2+ in living cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The Fenton process, one of several advanced oxidation processes, describes the reaction of Fe(II) with hydrogen peroxide. Fe(II) is oxidized to Fe(III) that reacts with hydrogen peroxide to Fe(II) and again initiates the Fenton reaction. In the course of the reactions reactive species, e.g. hydroxyl radicals, are formed. Conditions such as pH, ligand concentrations and the hydrogen peroxide/Fe(II) ratio may influence the OH radical yield. It could be shown that at pH < 2.7 and >3.5 the OH radical yield decreases significantly. Two ligands were investigated, pyrophosphate and sulfate. It was found that pyrophosphate forms a complex with Fe(III) that does not react with hydrogen peroxide and thus, the Fenton reaction is terminated and the OH radical yields do not further increase. The influence of sulfate is not as strong as that of pyrophosphate. The OH radical yield is decreased when sulfate is added but even at higher concentrations the Fenton reaction is not terminated., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA-Zn(II) complex) to characterize the enzymatic properties of YhdE and its mutant, establishing a new method for assaying pyrophosphatase catalytic function. Our results reveal for the first time that the new fluorescence sensor confers high sensitivity and specificity and pyrophosphate (PPi) is the direct catalytic product of YhdE. Crystal structures of a mutant in the active-site loop (YhdE&#95;E33A) show conformational flexibility implicated in the catalytic mechanism of YhdE. ITC experiments and computational docking further reveal that Asp70 and substrate dTTP coordinate Mn 2+ . Quantum mechanics calculations indicate that YhdE hydrolysis appears to follow a stepwise pathway in which a water molecule first attacks the α-phosphorus atom in the substrate, followed by the release of PPi from the pentavalent intermediate.

Adenosine diphosphate-ribose (ADP-ribose) and its derivatives play important roles in a series of complex physiological procedures. The design and synthesis of artificial ADP-ribosylated compounds is an efficient way to develop valuable chemical biology tools and discover new drug candidates. However, the synthesis of ADP-ribosylated compounds is currently difficult due to structural complexity, easily broken pyrophosphate bond and high hydrophilicity. In this paper, ADP-ribosyl-N₃ was designed and synthesized for the first time. With ADP-ribosyl-N₃ as the key precursor, a divergent post-modification strategy was developed to prepare structurally diverse ADP-ribosylated compounds including novel nucleotides and peptides bearing ADP-ribosyl moieties., Competing Interests: The authors declare no conflict of interest.

A trivalent rare-earth ion (Sm 3 + )-doped LiNa 3 P 2 O 7 (LNPO) phosphor was synthesized using a conventional high-temperature solid-state reaction route. A predominant orthorhombic phase of LNPO was observed in all X-ray diffraction patterns. The surface states of the LNPO:Sm phosphor were confirmed by X-ray photoelectron spectroscopy. Under 401 nm excitation, the Sm-doped LNPO phosphors showed sharp emission peaks at 563, 600 and 647 nm that are related to the f-f transition of Sm 3 + ions. The optimum concentration of Sm 3 + (9 mol%) produced Commission Internationale de l'Eclairage chromaticity coordinates, color rendering index and correlated color temperature of (0.564, 0.434), 42 and 1843 K, respectively., (Copyright © 2016 John Wiley & Sons, Ltd.)