Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

Crystal Structure and Enzymology of Solanum tuberosum Inositol Tris/Tetrakisphosphate Kinase 1 (StITPK1).

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates.

Bacterial lipopolysaccharide inhibits colonic carrier-mediated uptake of thiamin pyrophosphate: roles for TLR4 receptor and NF-κB/P38/JNK signaling pathway.

This study investigated the effect of the bacterial endotoxin lipopolysaccharide (LPS) on colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1 that is generated by gut microbiota. We used three complementary models in our study: in vitro (human-derived colonic epithelial NCM460), ex vivo (human differentiated colonoid monolayers), and in vivo (mouse colonic tissue). The results showed that exposure of NCM460 cells to LPS leads to a significant inhibition of carrier-mediated TPP uptake as well as in decreased expression of the colonic TPP transporter (cTPPT) protein, mRNA, and heterologous nuclear RNA (hnRNA) compared with untreated controls. Similarly, exposure of human differentiated colonoid monolayers and mice to LPS caused significant inhibition in colonic carrier-mediated TPP uptake and in cTPPT protein, mRNA, and hnRNA expression. The effect of LPS on colonic TPP uptake and cTTPT expression was also found to be associated with a significant reduction in activity of the SLC44A4 promoter as well as in decreased expression of the nuclear factor Elf-3 (E74-like ETS transcription factor 3), which is needed for promoter activity. Finally, we found that knocking down the Toll-like receptor 4 (TLR4) and blocking the nuclear factor kappa B (NF-κB), JNK, and p38 signaling pathways with the use of pharmacological inhibitors lead to significant abrogation in the degree of LPS-mediated inhibition in TPP uptake and cTPPT expression. These results demonstrated that exposure of colonic epithelia to LPS inhibits colonic TPP uptake via transcriptional mechanism(s) and that the effect is mediated via TLR4 receptor and NF-κB/p38/JNK signaling pathways.NEW & NOTEWORTHY This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1. Three complementary models were used: in vitro (human NCM460 cells), ex vivo (human colonoids), and in vivo (mice). The results showed LPS to significantly suppress TPP uptake and the expression of its transporter, and that these effects are mediated via the membrane TLR4 receptor, and involve the NF-κB/p38/JNK signaling pathways.

Nucleoside Triphosphate Diphosphohydrolase 1 Exhibits Enzymatic Activity toward Tenofovir Diphosphate.

Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice into TFV-diphosphate (TFV-DP) to become pharmacologically active. Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. Of note, TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Thus, it was hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this hypothesis, recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, 6, and 8) were incubated, individually, with TFV-DP to determine their abilities to dephosphorylate TFV-DP in vitro. Following incubations, TFV-DP dephosphorylation was determined using both malachite green phosphate assays and ultrahigh-performance liquid chromatography tandem mass spectrometry. From these, NTPDase 1 exhibited the highest activity toward TFV-DP. Further, enzyme kinetic analysis revealed Michaelis-Menten kinetics for NTPDase 1-mediated TFV-DP dephosphorylation. Next, immunoblot analyses were conducted to confirm the expression of NTPDase 1 protein in human colorectal tissue. Liquid chromatography coupled to mass spectrometry proteomics analysis was used to measure the relative abundance of NTPDases in human colorectal tissue among healthy adult individuals (n = 4). These analyses confirmed the high abundance of NTPDase 1 in human colorectal tissue. Taken together, results suggest that NTPDase 1 may contribute to the regulation of TFV-DP levels. The above data provide important insights into the dephosphorylation of TFV-DP. SIGNIFICANCE STATEMENT: Nucleoside triphosphate diphosphohydrolases (NTPDases) that are involved in enzymatic ATP dephosphorylation may contribute to tenofovir-diphosphate (TFV-DP) dephosphorylation, leading to its inactivation. In this study, the NTPDases responsible for TFV-DP dephosphorylation in vitro and their expression in human colorectal tissue were investigated. Through this work, it was demonstrated that NTPDase 1 has the highest activity toward TFV-DP dephosphorylation, and it was abundant in human colorectal tissue. Importantly, these studies will increase our understanding of TFV-DP disposition.

Reactivity of Fe(III)-containing pyrophosphate salts with phenolics: complexation, oxidation, and surface interaction.

Mixed pyrophosphate salts with the general formula Ca2(1-x)Fe4x(P2O7)(1+2x) potentially possess less iron-phenolic reactivity compared to ferric pyrophosphate (FePP), due to decreased soluble Fe in the food-relevant pH range 3-7. We investigated reactivity (i.e., complexation, oxidation, and surface interaction) of FePP and mixed salts (with x = 0.14, 0.15, 0.18, and 0.35) in presence of structurally diverse phenolics. At pH 5-7, increased soluble iron from all salts was observed in presence of water-soluble phenolics. XPS confirmed that water-soluble phenolics solubilize iron after coordination at the salt surface, resulting in increased discoloration. However, color changes for mixed salts with x ≤ 0.18 remained acceptable for slightly water-soluble and insoluble phenolics. Furthermore, phenolic oxidation in presence of mixed salts was significantly reduced compared to FePP at pH 6. In conclusion, these mixed Ca-Fe(III) pyrophosphate salts with x ≤ 0.18 can potentially be used in designing iron-fortified foods containing slightly water-soluble and/or insoluble phenolics.

A rational design of functionalized black phosphorus cooperates with piperazine pyrophosphate to significantly suppress the fire hazards of polypropylene.

The flammability of polypropylene (PP) not only has negative effects on human health but also causes environmental pollution. Herein, from the molecular polarity point of view, rationally designed hyperbranched charring foaming agents (HCFA) modified black phosphorus nanosheets by in situ polymerization to solve the fire hazards of PP. Based on the UL-94 test V-0 rating, the conventional flame retardant of piperazine pyrophosphate (PAPP) is substituted partly by the BP@PPC. Surprisingly, compared with 27 wt% of PAPP/PP, composites consisting of only 2 wt% of BP@PPC and 20 wt% PAPP/PP also passes the V-0 rating. The results of the cone calorimeter test confirmed that adding BP@PPC decreases the total heat release (THR) and peak heat release (PHRR) by a large amount, which are decreased by 23.4%, 85.8% respectively compared with PP. Moreover, it is uncommon for the fire growth index of BP@PPC composites to be 66.7% lower than that of PAPP/PP composites. In addition, the incorporation of BP@PPC has almost no impact on the mechanical characteristics of PP composites. This study offers a reference for combining established flame retardants with novel compounds to modify the burning behaviors of PP.

Impact of the fortification of a rice beverage with different calcium and iron sources on calcium and iron bioaccessibility.

Rice beverages are commonly fortified with minerals to improve their nutritional value. However, the effect of fortification on mineral bioaccessibility is poorly understood. Thus, the effects of fortification of a rice beverage on mineral concentration and bioaccessibility using calcium carbonate (CaCO3), tricalcium phosphate (Ca3(PO4)2), sodium iron EDTA (NaFeEDTA) and ferric pyrophosphate (Fe4(P2O7)3) individually and in combination were studied. Recovery of the added minerals in the rice beverage ranged from 71.4 % to 92.0 % and 61.0 % to 93.3 % for Ca and Fe, respectively. Mineral bioacessibility was shown to be higher for CaCO3(≤39.0 %) compared to Ca3(PO4)2 (≤14.4 %) and for NaFeEDTA (≤50.7 %) compared to Fe4(P2O7)3 (≤3.9 %). No interaction of the different Ca sources was identified; the addition of iron sources did not have a significant effect on Ca bioaccessibility. The addition of NaFeEDTA to the rice beverage was found to be better than the addition of iron pyrophosphate and the simultaneous addition of this iron sources did not result in an additive effect on Fe bioaccessibility. These results may be used to develop plant-based beverages with an improved mineral bioaccessibility.

Carotenoids in Drug Discovery and Medicine: Pathways and Molecular Targets Implicated in Human Diseases.

Carotenoids are isoprenoid-derived natural products produced in plants, algae, fungi, and photosynthetic bacteria. Most animals cannot synthesize carotenoids because the biosynthetic machinery to create carotenoids de novo is absent in animals, except arthropods. Carotenoids are biosynthesized from two C20 geranylgeranyl pyrophosphate (GGPP) molecules made from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via the methylerythritol 4-phosphate (MEP) route. Carotenoids can be extracted by a variety of methods, including maceration, Soxhlet extraction, supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), accelerated solvent extraction (ASE), ultrasound-assisted extraction (UAE), pulsed electric field (PEF)-assisted extraction, and enzyme-assisted extraction (EAE). Carotenoids have been reported to exert various biochemical actions, including the inhibition of the Akt/mTOR, Bcl-2, SAPK/JNK, JAK/STAT, MAPK, Nrf2/Keap1, and NF-κB signaling pathways and the ability to increase cholesterol efflux to HDL. Carotenoids are absorbed in the intestine. A handful of carotenoids and carotenoid-based compounds are in clinical trials, while some are currently used as medicines. The application of metabolic engineering techniques for carotenoid production, whole-genome sequencing, and the use of plants as cell factories to produce specialty carotenoids presents a promising future for carotenoid research. In this review, we discussed the biosynthesis and extraction of carotenoids, the roles of carotenoids in human health, the metabolism of carotenoids, and carotenoids as a source of drugs and supplements.

The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection.

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis. IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.

Selective adsorption of various phosphorus species coexistence in water-soluble ammonium polyphosphate on goethite: Experimental investigation and molecular dynamics simulation.

The geochemical processes of polyphosphates (poly-Ps) are important for phosphorus (P) management and environmental protection. Water-soluble ammonium polyphosphate (APP) containing various P species has been increasingly used as an alternative P-fertilizer. The various P species coexistence and the chelation of poly-Ps with mental would trigger the P's competitive adsorption and affect the APP's adsorption intensity on goethite, compared to single orthophosphate (P1). P adsorption behaviors of APP1 with two P species and APP2 with seven P species on goethite were investigated via batch experiments in comparison to the traditional P-fertilizer of mono-ammonium phosphate (MAP). Coadsorption of P1 and pyrophosphate (P2) on goethite was investigated by molecular dynamics (MD) simulation. The more Fe3+ dissolved from goethite as a bridge due to the chelation of poly-Ps in APP and contributed to the stronger APP adsorption on goethite compared with MAP. Ion chromatography and spectral analysis showed P1 and P2 in APP were mainly adsorbed by goethite via mainly forming bidentate complexes. The goethite preferentially adsorbed P1 at lower APP concentration but increased the poly-Ps' adsorption at higher APP concentration. MD simulation showed that electrostatic interaction and hydrogen bonds played a key role in water-phosphates-goethite systems. The P1 pre-adsorbed on goethite could be replaced by P2 at high P2 concentration. The results develop new insights regarding the selective adsorption of various P species coexistence in goethite-rich environments.

Effects of dicyandiamide, phosphogypsum and superphosphate on greenhouse gas emissions during pig manure composting.

This study investigated the effects of dicyandiamide, phosphogypsum and superphosphate on greenhouse gas emissions and compost maturity during pig manure composting. The results indicated that the addition of dicyandiamide and phosphorus additives had no negative effect on organic matter degradation, and could improve the compost maturity. Adding dicyandiamide alone reduced the emissions of ammonia (NH3), methane (CH4) and nitrous oxide (N2O) by 9.37 %, 9.60 % and 31.79 %, respectively, which was attributed that dicyandiamide effectively inhibited nitrification to reduce the formation of N2O. Dicyandiamide combined with phosphogypsum or superphosphate could enhance mitigation of the total greenhouse gas (29.55 %-37.46 %) and NH3 emission (18.28 %-21.48 %), which was mainly due to lower pH value and phosphoric acid composition. The combination of dicyandiamide and phosphogypsum exhibited the most pronounced emission reduction effect, simultaneously decreasing the NH3, CH4 and N2O emissions by 18.28 %, 38.58 % and 36.14 %, respectively. The temperature and C/N content of the compost were significantly positively correlated with greenhouse gas emissions.

Azotobacter chroococcum and Pseudomonas putida enhance pyrroloquinazoline alkaloids accumulation in Adhatoda vasica hairy roots by biotization.

Adhatoda vasica is used in the treatment of cold, cough, chronic bronchitis, asthma, diarrhea, and dysentery. The biological activities of this species are attributed with the presence of alkaloids, triterpenoids, and flavonoids. Agrobacterium rhizogenes-mediated transformation of A. vasica, produces pyrroloquinazoline alkaloids, was achieved by infecting leaf discs with strain ATCC15834. The bacterial strain infected 82.7% leaf discs and 5-7 hairy root initials were developed from the cut edges of leaf discs. In this study, seven strains of Azotobacter chroococcum and five strains of Pseudomonas putida were used for the biotization of hairy roots. Plant growth-promoting rhizobacteria (PGPR) develops symbiotic association with roots of plants and increases the growth parameters of plants. PGPR (A. chroococcum and P. putida) increased the profiles of nitrogenase and acid phosphatase enzymes, biomass, dry matter contents, anthranilate synthase activity and accumulation of pyrroloquizoline alkaloids in the biotized hairy roots. Both enzymes (nitrogenase and acid phosphatase) maintain sufficient supply of nitrogen and dissolved phosphorus to the cells of hairy roots therefore, the levels of anthranilate synthase activity and pyrroloquinazoline alkaloids are increased. Total seven pyrroloquinazoline alkaloids (vasicine, vasicinone, vasicine acetate, 2-acetyl benzyl amine, vasicinolone, deoxyvasicine and vasicol) were identified from the biotized hairy roots of A. vasica. In our study, biotization increased the profiles of pyrroloquinazoline alkaloids therefore, this strategy may be used in increasing the production of medicinally important secondary metabolites in other plant species also. Our hypothetical model demonstrates that P. putida cell surface receptors receive root exudates by attaching on hairy roots. After attachment, the bacterial strain penetrates in the biotized hairy roots. This endophytic interaction stimulates acid phosphatase activity in the cells of biotized hairy roots. The P. putida plasmid gene (ppp1) expression led to the synthesis of acid phosphatase in cytosol. The enzyme enhances phosphorus availability as well as induces the formation of phosphoribosyl diphosphate. Later, phosphoribosyl diphosphate metabolizes to tryptophan and finally tryptophan converts to anthranilic acid. The synthesized anthranilic acid used in the synthesis of alkaloids in A. vasica.

Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers.

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling, and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologues display phosphohydrolase activity with a high specificity for the 5-ß-phosphate of inositol pyrophosphates and only minor activity against the ß-phosphates of 4-InsP7 and 6-InsP7. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-ß-phosphate-specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

Iron Bioavailability from Ferrous Ammonium Phosphate, Ferrous Sulfate, and Ferric Pyrophosphate in an Instant Milk Drink-A Stable Isotope Study in Children.

Ferrous ammonium phosphate (FAP) is an iron salt that has been developed for the fortification of food matrices sensitive to color and flavor changes. The objective of the study was to measure iron absorption from FAP in young children and compare it to a previous evaluation of FAP in young women. A double-blind randomized crossover study with two parallel arms was used to evaluate the iron absorption from FAP added to reconstituted milk powder in comparison to that from ferrous sulfate (FeSO4) and ferric pyrophosphate (FePP). Iron absorption was measured in 39 children aged 3- to 6-years-old using erythrocyte incorporation of stable Fe isotopes (57Fe, 58Fe). The geometric mean iron absorption in iron replete children from FAP, FeSO4 and FePP from milk was 8.3%, 7.6% and 2.1%, respectively. Iron absorption from FAP and FeSO4 fortified milk was not significantly different (p = 0.199); however, it was significantly higher than from FePP fortified milk (p < 0.001). Iron bioavailability from FAP and FePP relative to FeSO4 (relative bioavailability (RBV)) was 110% and 33%, respectively. The RBV of FAP (110%) in iron replete children was higher than previously reported RBV (71%) in mainly iron deficient women. The difference in iron status between the children and women in the respective studies may explain the different RBV values and is discussed.

Functional characterization of two different decaprenyl diphosphate synthases in the vetch aphid Megoura viciae.

Long-chain polyprenyl diphosphate synthases play a critical role in the formation of the prenyl side-chain of ubiquinones, but up to date, their functions have scarcely been characterized in insects. Here, we first cloned the complementary DNAs encoding the subunits of decaprenyl diphosphate synthase (DPPS) in the vetch aphid Megoura viciae, an important agricultural pest insect. The results showed that there existed three DPPS subunits, designated as MvDPPS1, MvDPPS2a, and MvDPPS2b, with an open reading frame of 1218, 1275, and 1290 bp, and a theoretical isoelectric point of 7.91, 6.63, and 9.62, respectively. The sequences of MvDPPS1s from different aphid species were nearly identical, while the sequences of MvDPPS2a and MvDPPS2b shared only moderate sequence similarity. Phylogenetic analysis clearly separated MvDPPS2a and MvDPPS2b, indicating a functional differentiation between them. Functional coexpression analysis in Escherichia coli showed that MvDPPS1 plus MvDPPS2a and MvDPPS1 plus MvDPPS2b, respectively, catalyzed the formation of the prenyl side-chain of the ubiquinone coenzyme Q10 (CoQ10). Interestingly, MvDPPS1 plus MvDPPS2b catalyzed the formation of the prenyl side-chain of a ubiquinone other than CoQ10. RNA interference-mediated knockdown of MvDPPS2a imposed no significant effect on MvDPPS2b, and vice versa, suggesting no compensatory action between them. In the end, we detected the product CoQ10 in the aphid, the first identification of CoQ10 in an insect species. Taken together, we characterized two functional DPPSs in M. viciae, one of which might be multifunctional. Our study helps to understand the functional plasticity of the terpenoid backbone biosynthesis pathway in insects.

Liposomal ferric pyrophosphate and ascorbic acid supplementation in pregnant women with iron deficiency anaemia: haematochemical, obstetric, neonatal and psychological outcomes in a prospective observational study.

This study aimed to determine the effects of liposomal iron pyrophosphate/ascorbic acid on clinical and psychological outcomes in pregnant women. Women at the 11th-13th weeks of gestation with iron deficiency anaemia assuming Sideremil™ from April 2018 to May 2019 were recruited. Haematochemical, obstetric, neonatal and psychological outcomes were investigated at the enrolment, 21-23 weeks of gestation, 30-32 weeks of gestation and after 6 weeks from childbirth. Results showed significant positive effects on haemoglobin, ferritin, sideremia and transferrin levels, compared to baseline data. A significant improvement of anxiety and depression levels was also observed. Regarding the quality of life, all the domains significantly improved, especially the Physical Role domain. Our results indicate that Sideremil™ may be a valid treatment for iron deficiency anaemia in pregnant women, since it significantly improves haematological and mental health outcomes. However, further studies are needed to confirm these results.

Oral supplementation of inorganic pyrophosphate in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7  has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.

ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions.

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

THE EFFECT OF DIET ENRICHED WITH PYROPHOSPHATE (E450) ON EXPRESSION OF GENES ENCODING BONE MORPHOGENETIC PROTEIN AND OSTEOCALCIN IN MOUSE EMBRYONIC MANDIBLE TISSUES.

OBJECTIVE: The aim: Of our study was to measure the mRNA expression of the investigated odontogenesis factors in mandible tissue of mouse embryos (17th day of pregnancy) gestated by females, kept on a E450 rich diet since 30 days before fertilization to gestation. PATIENTS AND METHODS: Materials and methods: The effect of food supplements was studied in «Overload phosphates model¼. Experiments were carried out on white nonlinear outbred mice with mass 25-28g (n=40). The females from the control group were fed with standard rodent food, whereas the experimental females were fed with pyrophosphate-enriched food. The materials, used for the molecular genetic study, were the lower jaws of 17-days old mouse embryos (E-17). RESULTS: Results: The investigated BMP2 and osteocalcin genes are expressed at approximately the same level. Pyrophosphate-rich diet does not alter BMP2 gene expression, but it significantly increases the expression of osteocalcin. CONCLUSION: Conclusions: The present study is the first one to describe the impact of the pyrophosphate-rich diet on mRNA expression of key osteogenesis regulators - osteocalcin and BMP2.

High mesoporosity phosphorus-containing biochar fabricated from Camellia oleifera shells: Impressive tetracycline adsorption performance and promotion of pyrophosphate-like surface functional groups (C-O-P bond).

In China, more than 3.5 million tons of Camellia oleifera discarded shells are produced every year. This work first prepared phosphorus-containing biochar (PBC) from C. oleifera shells and was successfully applied to the efficient removal of tetracycline (TC) from solutions. The prepared PBC exhibits superior TC adsorption capacity of 451.5 mg/g, and TC uptake rapidly reached 315.5 mg/g at the first 5 min (C0 = 50 mg/L). Furthermore, PBC also shows excellent applicability to the broad range pH value (1-9) and superior selective removal in the presence of various high concentration coexisting ions (1 mM). Mechanisms underlying TC adsorption were also put forward, and analysis suggested that pyrophosphate-like surface functional groups (C-O-P bond) played a critical role in this process. Notably, treating pharmaceutical wastewater with PBC can efficiently reduce chemical oxygen demand (COD) and total organic carbon (TOC) concentration below the discharge standard of China (GB21904-2008).