Pyridoxine challenge reflects pediatric hypophosphatasia severity and thereby examines tissue-nonspecific alkaline phosphatase's role in vitamin B6 metabolism.

Alkaline phosphatase (ALP) is detected in most human tissues. However, ALP activity is routinely assayed using high concentrations of artificial colorimetric substrates in phosphate-free laboratory buffers at lethal pH. Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the ALP isoenzyme expressed in bone, liver, kidney, and elsewhere and is therefore designated "tissue-nonspecific" ALP (TNSALP). Consequently, HPP harbors clues concerning the biological function of this phosphohydrolase that is anchored onto the surface of cells. The biochemical signature of HPP features low serum ALP activity (hypophosphatasemia) together with elevated plasma levels of three natural substrates of TNSALP: i) phosphoethanolamine (PEA), a component of the linkage apparatus that binds ALPs and other proteins to the plasma membrane surface; ii) inorganic pyrophosphate (PPi), an inhibitor of bone and tooth mineralization; and iii) pyridoxal 5'-phosphate (PLP), the principal circulating vitameric form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving several hundred ALPL mutations underlies the remarkably broad-ranging expressivity of HPP featuring tooth loss often with muscle weakness and rickets or osteomalacia. Thus, HPP associates the "bone" isoform of TNSALP with biomineralization, whereas the physiological role of the "liver", "kidney", and other isoforms of TNSALP remains uncertain. Herein, to examine HPP's broad-ranging severity and the function of TNSALP, we administered an oral challenge of pyridoxine (PN) hydrochloride to 116 children with HPP. We assayed both pre- and post-challenge serum ALP activity and plasma levels of PLP, the B6 degradation product pyridoxic acid (PA), and the B6 vitamer pyridoxal (PL) that can enter cells. Responses were validated by PN challenge of 14 healthy adults and 19 children with metabolic bone diseases other than HPP. HPP severity was assessed using our HPP clinical nosology and patient height Z-scores. PN challenge of all study groups did not alter serum ALP activity in our clinical laboratory. In HPP, both the post-challenge PLP level and the PLP increment correlated (Ps < 0.0001) with the clinical nosology and height Z-scores (Rs = +0.6009 and + 0.4886, and Rs = -0.4846 and - 0.5002, respectively). In contrast, the plasma levels and increments of PA and PL from the PN challenge became less pronounced with HPP severity. We discuss how our findings suggest extraskeletal TNSALP primarily conditioned the PN challenge responses, and explain why they caution against overzealous B6 supplementation of HPP.

Failure of Micractinium simplicissimum Phosphate Resilience upon Abrupt Re-Feeding of Its Phosphorus-Starved Cultures.

Microalgae are naturally adapted to the fluctuating availability of phosphorus (P) to opportunistically uptake large amounts of inorganic phosphate (Pi) and safely store it in the cell as polyphosphate. Hence, many microalgal species are remarkably resilient to high concentrations of external Pi. Here, we report on an exception from this pattern comprised by a failure of the high Pi-resilience in strain Micractinium simplicissimum IPPAS C-2056 normally coping with very high Pi concentrations. This phenomenon occurred after the abrupt re-supplementation of Pi to the M. simplicissimum culture pre-starved of P. This was the case even if Pi was re-supplemented in a concentration far below the level toxic to the P-sufficient culture. We hypothesize that this effect can be mediated by a rapid formation of the potentially toxic short-chain polyphosphate following the mass influx of Pi into the P-starved cell. A possible reason for this is that the preceding P starvation impairs the capacity of the cell to convert the newly absorbed Pi into a "safe" storage form of long-chain polyphosphate. We believe that the findings of this study can help to avoid sudden culture crashes, and they are also of potential significance for the development of algae-based technologies for the efficient bioremoval of P from P-rich waste streams.

Effects of limestone solubility on the efficacy of a novel consensus bacterial 6-phytase variant to improve mineral digestibility, retention, and bone ash in young broilers fed low-calcium diets containing no added inorganic phosphate.

This study evaluated the effect of limestone solubility on the capacity of a novel consensus bacterial 6-phytase variant (PhyG) to improve phosphorus (P) and calcium (Ca) digestibility, retention, and utilization in low-Ca broiler diets containing no added inorganic phosphate (Pi). Male Ross 308 broilers (n = 1,152) were fed one of 16 experimental diets from 11 to 21 d of age in a randomized complete design (12 birds/cage, 6 cages/treatment). Diets comprised three positive controls (PC3, PC2, and PC1) containing 1.8, 1.2, or 0.6 g/kg MCP-P and 7.7, 7.0, or 6.2 g/kg Ca, respectively, and a negative control (NC) containing no added Pi (4.4 g/kg P; 2.8 g/kg phytate-P) and 5.5 g/kg Ca from either low or high solubility limestone (LSL or HSL, respectively, [with 42% and 97% solubility after 5 min at pH 3.0]), supplemented with 0, 250, 500, 1,000, or 2,000 FTU/kg of PhyG. Fecal samples collected on days 18 to 20 and ileal digesta collected on day 21 were analyzed for titanium dioxide, Ca, P, and phytate (IP6, inositol hexakisphosphate). Tibias (day 21) were analyzed for ash content. Data were analyzed by factorial analysis (2 limestone solubilities × 4 MCP-P levels and 2 limestone solubilities × 5 phytase dose levels) and exponential regression. Increasing dose levels of PhyG resulted in an exponential increase (P < 0.01) in the apparent ileal digestibility (AID) of P, ileal digestible P content of the diet, ileal IP6 content, and IP6 disappearance in birds fed either HSL or LSL diets, but AID Ca and ileal digestible Ca were exponentially increased by the phytase only in HSL diets (P < 0.01). Relative to HSL, the LSL increased AID P, ileal digestible P, and IP6 disappearance (P < 0.05) but reduced AID Ca, ileal digestible Ca, and retainable Ca (P < 0.05), resulting in reduced retainable P and tibia ash. Phytase exponentially increased the apparent total tract digestibility of P, retainable P, and tibia ash in HSL and LSL diets, but at or above 500 FTU/kg values were higher in HSL than LSL (interaction P < 0.05). The findings highlight that phytase dose-response effects on mineral digestibility and utilization are different for high- and low-solubility limestones, and it is therefore recommended to use digestible rather than total Ca content during diet formulation to ensure an optimal balance of Ca and P, especially in low-Ca diets. In diets containing HSL, higher phytase dose levels may be needed to compensate for the low digestible P content of the basal diet.

In broilers, an excess of dietary calcium (Ca) or imbalance with phosphorus (P) can impair mineral digestion and utilization. As a result, diets are being formulated with less Ca, but the quality of the added Ca (that is mainly from limestone) is also important. This study investigated effects of limestone solubility (high [HSL] vs. low [LSL]) on the capacity of a novel consensus bacterial 6-phytase variant, PhyG, to improve P and Ca digestion and utilization in low-Ca diets containing no added inorganic phosphate. Increasing the phytase dose increased ileal P and phytate digestibility and the digestible P content of the diet at 21 d of age regardless of limestone solubility and reduced the negative effects of HSL (relative to LSL). Total tract digestibility of P and Ca, retainable P and Ca, and tibia ash were also increased by phytase, but responses were reduced with LSL relative to HSL. The findings highlight that phytase dose-responses differ in diets containing different limestones and it is therefore recommended to formulate diets based on the content of digestible rather than total Ca to ensure that Ca requirements are met but not exceeded, with optimal phytase efficacy. In diets containing HSL, a higher PhyG dose level is needed to meet the requirement for P.

Phosphate (Pi) stress-responsive transcription factors PdeWRKY6 and PdeWRKY65 regulate the expression of PdePHT1;9 to modulate tissue Pi concentration in poplar.

Phosphorus (P) is an important nutrient for plants. Here, we identify a WRKY transcription factor (TF) in poplar (Populus deltoides × Populus euramericana) (PdeWRKY65) that modulates tissue phosphate (Pi) concentrations in poplar. PdeWRKY65 overexpression (OE) transgenic lines showed reduced shoot Pi concentrations under both low and normal Pi availabilities, while PdeWRKY65 reduced expression (RE) lines showed the opposite phenotype. A gene encoding a Pi transporter (PHT), PdePHT1;9, was identified as the direct downstream target of PdeWRKY65 by RNA sequencing (RNA-Seq). The negative regulation of PdePHT1;9 expression by PdeWRKY65 was confirmed by DNA-protein interaction assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), co-expression of the promoters of PdePHT1;9 and PdeWRKY65 in tobacco (Nicotiana benthamiana) leaves, and chromatin immunoprecipitation-quantitative PCR. A second WRKY TF, PdeWRKY6, was subsequently identified and confirmed to positively regulate the expression of PdePHT1;9 by DNA-protein interaction assays. PdePHT1;9 and PdeWRKY6 OE and RE poplar transgenic lines were used to confirm their positive regulation of shoot Pi concentrations, under both normal and low Pi availabilities. No interaction between PdeWRKY6 and PdeWRKY65 was observed at the DNA or protein levels. Collectively, these data suggest that the low Pi-responsive TFs PdeWRKY6 and PdeWRKY65 independently regulate the expression of PHT1;9 to modulate tissue Pi concentrations in poplar.

Bioremediation of lead-contaminated soil by inorganic phosphate-solubilizing bacteria immobilized on biochar.

In this study, a bio-composite (IBWS700) was prepared using inorganic phosphate-solubilizing bacteria (iPSB), which were immobilized on biochar produced from wheat straw (WS700). Further, the bio-remediation effects of the composite for lead (Pb) in soil were also investigated. The presence of different Pb species, physicochemical properties, enzyme activities, and immobilization mechanisms of Pb in soil were also evaluated. Compared to free iPSB and biochar, IBWS700 significantly decreased the lead bio-availability whereas increased the residual fraction, also affected available phosphorus (AP), cation exchange capacity (CEC), organic matter (OM) and activity of urease, alkaline phosphatase, sucrase and catalase. Interestingly, the changes in the enzyme activity, AP and OM performed twice increases with increasing Pb concentration, which was rarely reported. The reason might be attributed to the reconstruction of bacteria communities with high Pb load. Further, the immobilization mechanisms mainly included bio-adsorption and bio-precipitation. SEM revealed that the surface of IBWS700 covered with a large number of heterogeneous colonization of iPSB and white stack after Pb2+ adsorption. FTIR spectra showed that O-H, C-O-P, CO, and C =C could play important roles in bio-adsorption. Moreover, XRD analysis indicated that bio-precipitates were mainly Pb5(PO4)3Cl. In general, the use of IBWS700 could effectively immobilize Pb2+ and improve soil quality.

Role of inorganic phosphate concentrations in in vitro activity of fosfomycin.

OBJECTIVES: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). METHODS: The wild-type BW25113 strain, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT, and ΔphoB) and six clinical isolates of Escherichia coli with different fosfomycin susceptibilities were used. EUCAST breakpoints were used. Susceptibility was evaluated by agar dilution using standard Mueller-Hinton agar (Pi concentration of 1 mM similar to human plasma concentration) and supplemented with Pi (13 and 42 mM, minimum and maximum urinary Pi concentrations) and/or glucose-6-phosphate (25 mg/L). Fosfomycin transporter promoter activity was assayed using PglpT::gfpmut2 or PuhpT::gfpmut2 promoter fusions in standard Mueller-Hinton Broth (MHB), supplemented with Pi (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC50 under different Pi concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time-kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (Cmax)), 1053 and 4415 mg/L (urine Cmax range), using MHB with 28 mM Pi (mean urine Pi concentration) + 25 mg/L glucose-6-phosphate. RESULTS: All the strains showed decreased susceptibility to fosfomycin linked to increased Pi concentrations: 1-4 log2 dilution differences from 1 to 13 mM, and 1-8 log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing Pi concentrations higher fosfomycin EC50 bacterial viability was observed, except against ΔglpT-uhpT. The increase in Pi reduced the bactericidal effect of fosfomycin. DISCUSSION: Pi variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated Pi concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.

A novel consensus bacterial 6-phytase variant completely replaced inorganic phosphate in broiler diets, maintaining growth performance and bone quality: data from two independent trials.

Total replacement of dietary inorganic phosphate (Pi) by a novel consensus bacterial 6-phytase variant (PhyG) in phytate-rich diets (>0.3% phytate-P) was investigated in 2 trials using growth performance and bone quality as outcome measures. Both trials utilized a completely randomized design with 5 dietary treatments across 4 phases: starter (0-10 d), grower (10-21 d), finisher 1 (21-35 d), and finisher 2 (35-42 d). Treatments comprised a nutritionally adequate positive control (PC) diet containing monocalcium phosphate and 4 experimental diets (IPF1, IPF2, IPF3, and IPF4), all containing no added Pi and reduced in Ca by 0.2 to 0.3% units vs. PC. IPF1contained PhyG at 1,000 FTU/kg (all phases); IPF2 contained PhyG at 1,000 FTU/kg (all phases) and was additionally reduced in digestible AA, ME, and sodium (-0.2 to -0.4% points, -74 kcal/kg, -0.04% points, respectively, vs. PC); IPF3 contained PhyG at 3,000 FTU/kg in starter, 2,000 FTU/kg in grower, and 1,000 FTU/kg in finisher phases; and IPF4 contained xylanase (2,000 U/kg) and PhyG (2,000 FTU/kg in starter, 1,500 FTU/kg in grower, and 1,000 FTU/kg in finisher phases) and was additionally reduced in ME (-71 kcal/kg vs. PC). Ross 308 broilers were used (trial 1: n = 1,200 mixed sex; 24 birds per pen × 10 replicates; trial 2: n = 1,300 males; 26 birds × 10 replicates). During all phases in both trials, all IPF treatments maintained or improved BW, ADG, ADFI, FCR and BW-corrected FCRc and bone quality parameters vs. PC. vs. PC, treatment IPF3 increased ADG during starter phase (+10.8%) and reduced overall FCRc (-12 points, P < 0.05) in Trial 1, and increased overall ADG (+4.4%), day 35 and day 42 BW (+3.5%, +4.9%), and reduced overall FCRc (-11 points) in Trial 2 (P < 0.05). IPF4 produced equivalent performance to IPF3 (both trials). These are the first data to demonstrate total replacement of Pi by microbial phytase during an entire growth cycle in broiler diets.

Capability of Penicillium oxalicum y2 to release phosphate from different insoluble phosphorus sources and soil.

Due to insufficient amount of soluble phosphate and poor persistence of traditional chemical phosphate fertilizers in agricultural soils, the eco-friendly and sustainable phosphorus sources for crops are urgently required. The efficient phosphate-releasing fungal strain designated y2 was isolated and identified by the internal transcribed spacer of rDNA as Penicillium oxalicum y2. When lecithin, Ca3(PO4)2, or ground phosphate rock were separately used as sole phosphorus source, different phosphate-releasing modes were observed. The strain y2 was able to release as high as 2090 mg/L soluble phosphate within 12 days of incubation with Ca3(PO4)2 as sole phosphorus source. In the culture solution, high concentration of oxalic, citric, and malic acids and high phosphatase activity were detected. The organic acids contributed to solubilizing inorganic phosphate sources, while phosphatase was in charge of the mineralization of organic phosphorus lecithin. Afterwards, the fungus culture was applied to the soil with rape growing. During 50 days of incubation, the soil's available phosphate concentration increased by three times compared with the control, the dry weight of rape increased by 78.73%, and the root length increased by 38.79%. The results illustrated that P. oxalicum y2 possessed both abilities of solubilizing inorganic phosphorus and mineralizing organic phosphorus, which have great potential application in providing biofertilizer for modern agriculture.

Sustained release of arsenic trioxide benefits interventional therapy on rabbit VX2 liver tumor.

The benefit of chemotherapy as a constituent of transcatheter arterial chemoembolization (TACE) is still in debate. Recently we have developed arsenic trioxide nanoparticle prodrug (ATONP) as a new anticancer drug, but its systemic toxicity is a big issue. In this preclinical TACE study, ATONP emulsified in lipiodol behaved as drug-eluting bead manner. Sustained release of arsenic from ATONP within occluded tumor caused very low arsenic level in plasma, avoiding the "rushing out" effect as ATO did. Correspondingly, intratumoral arsenic accumulation and inorganic phosphate deprivation were simultaneously observed, and arsenic concentration was much higher as ATONP was transarterially administered than ATO, or intravenously injected. Tumor necrosis and apoptosis were remarkably more severe in ATONP group than ATO, but no significant hepatic and renal toxicity was perceived. In brief, ATONP alleviated arsenic toxicity and boosted the therapeutic effect of TACE via Pi-activated drug sustainable release.

Phosphorus fractions and oxygen isotope composition of inorganic phosphate in typical agricultural soils.

Phosphorus (P), despite being an essential nutrient element for plants growth in agricultural ecosystem, the low utilization rate of soil P and the environmental problems caused by soil P losses are serious. Therefore, scoping knowledge of the possible sources and utilization extent of soil P by microorganisms is very helpful for better understanding of promoting P utilization for sustainable agriculture. Oxygen isotope of phosphate technology is an effective tool to trace the sources of P. In this study, P contents and oxygen isotope composition of inorganic phosphate (δ18OP) of different pools (H2O-P, NaHCO3-P, NaOH-P, and HCl-P) in typical agricultural soil from Northeast China and Central China were analyzed and quantified. The results showed that fertilizer and land use were important factors influencing the contents of H2O-Pt and NaHCO3-Pt and the soil TP contents from different types of soils were greatly affected by soil weathering degree. The δ18OP of different P pools indicated that the difference in utilization extent of different P fractions by microorganisms and the δ18OP values of different P fractions could be due to accumulation of multiple factors. The results will provide effective information for further study on sources and effective utilization of different P fractions in soil.

Phosphorus Transport in Mycorrhiza: How Far Are We?

Mycorrhizal fungi considerably improve plant nutrition and help them to cope with changing environments. Particularly, these fungi express proteins to transfer inorganic phosphate (Pi) from the soil to colonized roots through symbiotic interfaces. The mechanisms involved in Pi transfer from fungal to plant cells are still largely unknown. Here, we discuss the recent progress made on the description of these mechanisms and we propose the most promising hypotheses and alternative mechanisms for this process. Specifically, we present a phylogenetic survey of candidate Pi transporters of mycorrhizal fungi that might ensure Pi unload into the symbiotic interfaces. Gathering additional knowledge on mycorrhizal Pi transport will improve the Pi-useefficiency in agroecological systems and will guide towards addressing future research challenges.

Metabolic Changes of Amino Acids and Flavonoids in Tea Plants in Response to Inorganic Phosphate Limitation.

The qualities of tea (Camellia sinensis) are not clearly understood in terms of integrated leading molecular regulatory network mechanisms behind inorganic phosphate (Pi) limitation. Thus, the present work aims to elucidate transcription factor-dependent responses of quality-related metabolites and the expression of genes to phosphate (P) starvation. The tea plant organs were subjected to metabolomics analysis by GC×GC-TOF/MS and UPLC-Q-TOF/MS along with transcription factors and 13 metabolic genes by qRT-PCR. We found P starvation upregulated SPX2 and the change response of Pi is highly dependent on young shoots. This led to increased change in abundance of carbohydrates (fructose and glucose), amino acids in leaves (threonine and methionine), and root (phenylalanine, alanine, tryptophan, and tyrosine). Flavonoids and their glycosides accumulated in leaves and root exposed to P limitation was consistent with the upregulated expression of anthocyanidin reductase (EC 1.3.1.77), leucoanthocyanidin dioxygenase (EC 1.4.11.19) and glycosyltransferases (UGT78D1, UGT78D2 and UGT57L12). Despite the similar kinetics and high correlation response of Pi and SPX2 in young shoots, predominating theanine and other amino acids (serine, threonine, glutamate, valine, methionine, phenylalanine) and catechin (EGC, EGCG and CG) content displayed opposite changes in response to Pi limitation between Fengqing and Longjing-43 tea cultivars.

Energy restriction in renal protection.

Energy restriction (ER) has been widely studied as a novel intervention, and its ability to prolong life has been fully demonstrated. For example, ER can significantly extend the lifespans of model flies, worms, rodents and other mammals. The role of ER in renal protection has also been elucidated. In preclinical studies, adjusting total energy intake or consumption of specific nutrients has prophylactic or therapeutic effects on ageing-related kidney disease and acute and chronic kidney injury. Amino acid restriction has gradually attracted attention. ER mimetics have also been studied in depth. The protective mechanisms of ER and ER mimetics for renal injury include increasing AMP-activated protein kinase and sirtuin type 1 (Sirt1) levels and autophagy and reducing mammalian target of rapamycin, inflammation and oxidative stress. However, the renal protective effect of ER has mostly been investigated in rodent models, and the role of ER in patients cannot be determined due to the lack of large randomised controlled trials. To protect the kidney, the mechanism of ER must be thoroughly researched, and more accurate diet or drug interventions need to be identified.

Phosphocreatine Levels in the Left Thalamus Decline during Wakefulness and Increase after a Nap.

Scientists have hypothesized that the availability of phosphocreatine (PCr) and its ratio to inorganic phosphate (Pi) in cerebral tissue form a substrate of wakefulness. It follows then, according to this hypothesis, that the exhaustion of PCr and the decline in the ratio of PCr to Pi form a substrate of fatigue. We used 31P-magnetic resonance spectroscopy (31P-MRS) to investigate quantitative levels of PCr, the γ-signal of ATP, and Pi in 30 healthy humans (18 female) in the morning, in the afternoon, and while napping (n = 15) versus awake controls (n = 10). Levels of PCr (2.40 mM at 9 A.M.) decreased by 7.0 ± 0.8% (p = 7.1 × 10-6, t = -5.5) in the left thalamus between 9 A.M. and 5 P.M. Inversely, Pi (0.74 mM at 9 A.M.) increased by 17.1 ± 5% (p = 0.005, t = 3.1) and pH levels dropped by 0.14 ± 0.07 (p = 0.002; t = 3.6). Following a 20 min nap after 5 P.M., local PCr, Pi, and pH were restored to morning levels. We did not find respective significant changes in the contralateral thalamus or in other investigated brain regions. Left hemispheric PCr was signficantly lower than right hemispheric PCr only at 5 P.M. in the thalamus and at all conditions in the temporal region. Thus, cerebral daytime-related and sleep-related molecular changes are accessible in vivo Prominent changes were identified in the thalamus. This region is heavily relied on for a series of energy-consuming tasks, such as the relay of sensory information to the cortex. Furthermore, our data confirm that lateralization of brain function is regionally dynamic and includes PCr.SIGNIFICANCE STATEMENT The metabolites phosphocreatine (PCr) and inorganic phosphate (Pi) are assumed to inversely reflect the cellular energy load. This study detected a diurnal decrease of intracellular PCr and a nap-associated reincrease in the left thalamus. Pi behaved inversely. This outcome corroborates the role of the thalamus as a region of high energy consumption in agreement with its function as a gateway that relays and modulates information flow. Conversely to the dynamic lateralization of thalamic PCr, a constantly significant lateralization was observed in other regions. Increasing fatigue over the course of the day may also be a matter of cerebral energy supply. Comparatively fast restoration of that supply may be part of the biological basis for the recreational value of "power napping."

HcPT1.2 participates in Pi acquisition in Hebeloma cylindrosporum external hyphae of ectomycorrhizas under high and low phosphate conditions.

Ectomycorrhizal fungi improve tree phosphorus nutrition through transporters specifically localized at soil-hyphae and symbiotic interfaces. In the model symbiosis between the fungus Hebeloma cylindrosporum and the maritime pine (Pinus pinaster), several transporters possibly involved in phosphate fluxes were identified, including three H+:Pi transporters. Among these three, we recently unraveled the function of one of them, named HcPT2, in both pure culture and symbiotic interaction with P. pinaster. Here we investigated the transporter named HcPT1.2, by analyzing inorganic phosphate transport ability in a yeast complementation assay, assessing its expression in the fungus associated or not with the plant, and immunolocalizing the proteins in ectomycorrhizas. We also evaluated the effect of external Pi concentration on expression and localization of HcPT1.2. Our results revealed that HcPT1.2 is involved in Pi acquisition by H. cylindrosporum mycelium, irrespective of the external Pi concentrations.

31 P T2 s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the human brain at 7T.

PURPOSE: To determine the phosphorus-31 T2 s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the healthy human brain at 7T. METHODS: A 3D chemical shift imaging multi-echo sequence with composite block pulses for refocusing was used to measure one free induction decay (FID) and seven full echoes with an echo spacing of 45 ms on the brain of nine healthy volunteers (age range 22-45 years; average age 27 ± 8 years). Spectral fitting was used to determine the change in metabolic signal amplitude with echo time. RESULTS: The average apparent T2 s with their standard deviation were 202 ± 6 ms, 129 ± 6 ms, 86 ± 2 ms, 214 ± 10 ms, and 213 ± 11 ms for phosphoethanolamine, phosphocholine, inorganic phosphate, glycerophosphoethanolamine, and glycerophosphocholine, respectively. CONCLUSION: The determined apparent T2 for phosphoethanolamine, glycerophosphocholine, and glycerophosphoethanolamine is approximately 200 ms. The lower apparent T2 value for phosphocholine is attributed to the overlap of this resonance with the 3-phosphorous resonance of 2,3-diphosphoglycerate from blood, with an apparent shorter T2 . Omitting the FID signal and the first echo of phosphocholine leads to a T2 of 182 ± 7 ms, whereas a biexponential analysis leads to 203 ± 4 ms. These values are more in line with phosphoethanolamine and the phosphodiesters. The short T2 of inorganic phosphate is subscribed to the fast reversible exchange with γ-adenosine triphosphate, which is mediated by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase within the glycolytic pathway. Magn Reson Med 80:29-35, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

1 H NMRS of carnosine combined with 31 P NMRS to better characterize skeletal muscle pH dysregulation in Duchenne muscular dystrophy.

In recent years, quantitative nuclear magnetic resonance imaging and spectroscopy (NMRI and NMRS) have been used more systematically as outcome measures in natural history and clinical trial studies for Duchenne muscular dystrophy (DMD). Whereas most of these studies have emphasized the evaluation of the fat fraction as an assessment for disease severity, less focus has been placed on metabolic indices measured by NMRS. 31 P NMRS in DMD reveals an alkaline inorganic phosphate (Pi ) pool, originating from either leaky dystrophic myocytes or an increased interstitial space. 1 H NMRS, exploiting the pH-sensitive proton resonances of carnosine, an intracellular dipeptide, was used to distinguish between these two hypotheses. NMR data were obtained in 23 patients with DMD and 14 healthy subjects on a 3-T clinical NMR system. Both 31 P and 1 H NMRS data were acquired at the level of the gastrocnemius medialis muscle. A multi-slice multi-echo imaging acquisition was performed for the determination of water T2 and fat fraction in the same region of interest. Whereas nearly all patients with DMD showed an elevated pH compared with healthy controls when using 31 P NMRS, 1 H NMRS-determined pH was not systematically increased. As expected, the carnosine-based intracellular pH was never found to be alkaline in the absence of a concurrent Pi -based pH elevation. In addition, abnormal intracellular pH, based on carnosine, was never associated with normal water T2 values. We conclude that, in one group of patients, both 1 H and 31 P NMRS showed an alkaline pH, originating from the intracellular compartment and reflecting ionic dysregulation in dystrophic myocytes. In the other patients with DMD, intracellular pH was normal, but an alkaline Pi pool was still present, suggesting an extracellular origin, probably revealing an expanded interstitial volume fraction, often associated with fibrotic changes. The data demonstrate that 1 H NMRS could serve as a biomarker to assess the normalization of intramyocytic pH and sarcolemmal permeability following therapy inducing dystrophin expression in patients with DMD.

Standardized total tract digestibility of phosphorus in various inorganic phosphates fed to growing pigs.

An experiment was conducted to determine the standardized total tract digestibility (STTD) of phosphorus (P) in five sources of inorganic phosphate fed to growing pigs, including dicalcium phosphate (DCP), monodicalcium phosphate (MDCP), monocalcium phosphate (MCP), tricalcium phosphate (TCP) and monosodium phosphate (MSP, reagent grade). Six barrows (42.4 ± 1.1 kg) individually housed in metabolism crates were allotted to a 6 × 6 Latin square design with six dietary treatments and six periods. Each experimental period consisted of a 4 day adaptation period and a 5 day collection period. The five experimental diets contained 0.24 to 0.34% of P from each inorganic phosphate as a sole source of P. A P-free diet was also prepared to estimate the basal endogenous loss of P. The STTD of P in MSP (94.9%) was not different from the STTD of P in MCP (93.0%), but was greater (P < 0.05) than that in DCP, MDCP and TCP (87.0, 86.5 and 71.3%, respectively). In conclusion, digestibility of P in reagent-grade MSP was greater than that in feed-grade inorganic phosphates such as DCP, MDCP and TCP, and digestibility of P in DCP and MDCP was greater than that in the TCP.

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions.

Inorganic phosphate (Pi ) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to Pi limitation have been reported in E. coli. Given the essential role of Pi , adaptation to Pi limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting Pi conditions for 75 days. Not surprisingly, adapted cells showed better survival under Pi limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U-13 C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to Pi limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749-758, 2017.

Characterization of six PHT1 members in Lycium barbarum and their response to arbuscular mycorrhiza and water stress.

Phosphorus (P) is vitally important for most plant processes. However, the P available to plants is present in the soil in the form of inorganic phosphate (Pi), and is often present in only limited amounts. Water stress further reduces Pi availability. Previous studies have highlighted the important roles of members of the PHOSPHATE TRANSPORTER 1 (PHT1) family and arbuscular mycorrhizal (AM) associations for Pi acquisition by plants growing in various environments. In order to understand the Pi uptake of Lycium barbarumL., a drought-tolerant ligneous species belonging to the Solanaceae family, we cloned and characterized six L. barbarum genes encoding transporter proteins belonging to the PHT1 family, and investigated their transcriptional response to AM associations and water stress. The six cloned PHT1 genes of L. barbarum had a similar evolutionary history to that of PHT1 genes found in other Solanaceae species. Three of these genes (LbPT3, LbPT4 and LbPT5) were AM-induced; the other three genes (LbPT1, LbPT2 and LbPT7) played distinct roles in Pi acquisition, translocation and remobilization in roots and leaves. AM-induced PHT1 genes maintained their function under water stress, while moderate and severe water stress upregulated non-AM-induced PHT1 genes in roots and leaves, respectively. Moreover, although LbPT1 was upregulated in AM roots under water stress, LbPT2 and LbPT7 were inhibited in AM roots, which suggested that an AM association satisfied the demand for Pi in roots under water stress and that LbPT1 may play a role in translocating Pi from roots to shoots in this situation.