Osteoarthritis (OA) is the most prevalent rheumatic disease in the world. Although its etiology is still unknown, one of the key processes in OA progression and development is oxidative stress. In this context, resveratrol, a well-known anti-oxidant from the stilbene family, could be of particular interest in future OA therapeutic strategies. However, currently, because of its low bioavailability, use of resveratrol in human health is very limited. In this study, we tested two resveratrol self-emulsifying systems previously developed in our laboratory in order to determine if they could improve cellular uptake of resveratrol in a human immortalized chondrocytic cell line (T/C28a2) and enhance protection against oxidative stress. Our results showed that resveratrol self-emulsifying systems were able first to increase cellular tolerance towards resveratrol, and thus decrease resveratrol intrinsic cellular toxicity, allowing the use of higher concentrations, second, to increase resveratrol uptake in membrane and intracellular fractions, and finally, to improve protection against oxidative stress-mediated death in human immortalized chondrocytic cell line T/C28a2. These data suggest that new formulations of resveratrol could be considered as potential beneficial effectors in future OA treatments. Chemical compounds cited in this article: Resveratrol (PubChem CID: 445154); isopropyl myristate (PubChem CID: 8042); Polysorbate 80 (PubChem CID: 5284448); ethanol (PubChem CID: 702); Medium-chain triglycerides (PubChem CID: 93356).Osteoarthritis is a degenerative disease of the joint, characterized by irreversible cartilage degradation. It is the most prevalent rheumatic disease in the world , generalin vitro  production is responsible for chondrocyte dysfunction and apoptosis, leading to cartilage destruction . Chondrooperties ) could bin vivo. Indeed, in humans, the oral consumption of resveratrol (25 mg/kg) leads to only 1.7 to 1.9% of free form of resveratrol found in the peak serum concentrations  of resveratrol, previously developed in our laboratory, which demonstrated increased antioxidant properties in endothelial cells . We carrThe aim of this study was to determine whether the use of resveratrol NE generated from SEDDS dispersion could increase cellular uptake of resveratrol in order to improve its effects on chondrocytes submitted to high oxidative stress, as seen in OA development. These new formulations, designed to be injectable intra-articularly, could overcome resveratrol intestinal and hepatic metabolism observed after oral administration, which is responsible for a decrease in the amount of resveratrol reaching target organs.trans-resveratrol into cis-resveratrol, all the resveratrol formulations were protected from light at each step of preparation and testing.All reagents used for cell culture were from Sigma-Aldrich . Reagents and chemicals used for other experiments are specified in each section. To avoid any isomerization of Trans-resveratrol was purchased from Sigma-Aldrich . Medium-chain triglycerides (Miglyol® 812) were purchased from Sasol , polysorbate 80 (Montanox® 80 VG PHA) from Seppic , and isopropyl myristate and ethanol 96% v/v from Cooper .® 80, and 10% ethanol for SEDDS 1; 20% Miglyol® 812, 70% Montanox® 80, and 10% ethanol for SEDDS 2. The nanoemulsions (NE) resulting from the self-emulsification of the SEDDS in the cell culture medium were characterized by a droplet size of 24 ± 7 and 103 ± 14 nm  and a zeta potential of -15.8 ± 2.6 and -14.7 ± 2.0 mV, for NE 1 and NE 2, respectively. The NE, either free or containing 25 and 50 μM of resveratrol, were evaluated in cytotoxicity and antioxidant assays.Two SEDDS previously formulated to enhance both cellular uptake and antioxidant efficacy of resveratrol were tested . BrieflyImmortalized human T/C28a2 chondrocytes were kindly provided by M. B. Goldring’s laboratory . Briefly, chondrocytes isolated from the human costal cartilage from a 15-year-old female were transfected using the simian virus 40 large T antigen (SV40-TAg). These cells present the classical characteristics of human chondrocytes .2 flasks or in 96-well plates, in DMEM/Ham’s F12 (v/v) medium (5 mM glucose) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (penicillin/streptomycin) at 37°C in a humidified 5% CO2 incubator until they reached 80% confluence.Cells were cultivated in 75-cmCells were used between passages 3 and 10.All treatments were made in DMEM/Ham’s F12 (v/v) supplemented with 1% FBS, 1% antibiotics (penicillin/ streptomycin) and 1% ethanol , as previously published . Besides2 incubator. The plate was emptied by reversal before 100 μL of a neutral red solution  was added to each well. Cells were then incubated for 3 h at 37°C in a humidified 5% CO2 incubator. Afterwards, the plate was emptied and 100 μL of a formol–calcium solution  were added. After 1 min of incubation, the plate was emptied and each well received 100 μL of a solution of ethanol (50% v/v) and pure acetic acid (1% v/v) . After gently mixing the plate for 5 min, absorbance at 540 nm was read on a microplate reader . Results were expressed as mean viability percentage ± SEM (n = 5).In order to evaluate the possible toxicity of the two resveratrol formulations on the chondrocytic cell line, a neutral red assay was performed. A stock solution of 50 mM resveratrol was prepared in 100% ethanol. Cells were incubated with NE 1 and 2 and with resveratrol in ethanolic solution , at both concentrations (25 and 50 μM), in a 96-well plate for 24 h at 37°C in a humidified 5% COCell viability above 95%  was required for further experiments.Table 1.Nomenclature used for each experimental condition is detailed in 2 flasks with the selected resveratrol formulations, i.e., resveratrol 25 μM in 1% ethanol, NE 1 and 2 with resveratrol 25 or 50 μM, for 20, 40, 60, and 180 min. Then, cells were washed with ice-cold PBS and trypsinized in order to collect cell lysates. Pellets were washed twice with ice-cold PBS by centrifugation for 10 min, at 1500 g, at 4°C and then harvested into 0.4 mL of lysis buffer supplemented with 5% proteases inhibitors. Finally, protein concentrations were determined in cell lysates using a Bradford assay kit .Cells were incubated in 75-cmg). Then, 100 μL of methanol  were added to the supernatant containing the intra-cellular fraction, and 200 μL of mobile phase (distilled water/methanol (v/v), with 0.5% acetic acid) were added to the pellet containing the membrane fraction. After centrifugation for 10 min at 2500 g, 100 μL of supernatant of each fraction were injected in the column. Resveratrol concentrations in each cellular compartment were expressed as means ± SEM in nmol/mg of proteins (n = 4).Measurement of resveratrol concentrations in membrane and intracellular compartments were performed by HPLC as previously described . Briefly2O2  or co-incubated with these H2O2 concentrations and resveratrol in the selected formulations, for 24 h at 37°C in a humidified 5% CO2 incubator. Then, a neutral red assay was performed as described above . Results were expressed as mean viability percentage ± SEM (n = 4).The effect of the two resveratrol formulations against oxidative stress-mediated death was evaluated thanks to cell viability assessment. Cells were pre-incubated in a 96-well plate overnight either with the selected resveratrol formulations (NE 1 and NE 2 at 25 and 50 μM resveratrol) in culture medium, or with unloaded NE 1 and NE 2 in culture medium, or with resveratrol 25 μM in 1% ethanol in culture medium, or with the conventional unsupplemented medium. Then, the plate was emptied before cells were further incubated either with different concentrations of H2O2.Control cells were cultured in conventional medium and incubated with neither resveratrol nor Hp < 0.05.Results were expressed as means ± SEM. All statistical analyses were performed with GraphPad Prism 5©, using a Kruskal–Wallis test. Data were considered statistically significant at Figure 1).NE 1 and 2 at 25 and 50 μM resveratrol, and 25 μM resveratrol in ethanolic solution induced no change in cell viability in the chondrocytic cell line after incubation for 24 h. Nevertheless, 50 μM resveratrol in the ethanolic solution induced a cell viability of 91%, thus under the total innocuity threshold defined at 95%. Consequently, this concentration of resveratrol in the ethanolic solution was no longer studied (p < 0.05). When 25 μM or 50 μM of resveratrol was brought by NE 1, a significant increase in resveratrol concentration was observed after 20 min of incubation (p < 0.05 vs. 25 μM resveratrol-1% ethanol), followed by a significant decrease at 60 (25 μM) and 180 min (25 and 50 μM) (p < 0.05 vs. 25 μM resveratrol-1% ethanol) .In the membrane fraction, 25 or 50 μM of resveratrol brought by NE 2 allowed a significant increase in resveratrol concentration at any time of incubation compared with 25 μM of resveratrol as an ethanolic solution (p < 0.05 vs. 25 μM resveratrol-1% ethanol). 25 and 50 μM of resveratrol brought by NE 2 induced a significant increase in resveratrol concentrations after incubation for 20, 60, and 180 min compared with 25 μM resveratrol-1% ethanol (p < 0.05) .In the intracellular fraction, 25 μM of resveratrol in NE 1 induced a significant increase in resveratrol concentration after 180 min  induced a significant decrease in cell viability whatever H2O2 concentration (p < 0.05 vs. control cells) . Of note, resveratrol at 50 μM in 1% ethanol was not tested due to its cytotoxic effect.As expected in the cellular model of oxidative stress, incubation for 24 h with HFigure 3A), 50 μM of resveratrol were able to protect cells against oxidative stress-mediated death at each H2O2 concentration .In the NE 1 panel , 50 μM of resveratrol allowed an increased cell viability after incubation with 500 and 750 μM H2O2 compared with control cells and cells incubated with 25 μM of resveratrol in ethanolic solution (p < 0.05).In the NE 2 panel (2O2 (p < 0.05), but no significant difference was observed after incubation with 200 and 750 μM H2O2 (Table 2).In addition, 50 μM of resveratrol brought by NE 1 was more efficient than brought by NE 2 after incubation with 500 μM Hin vitro studies have shown a beneficial effect of resveratrol in OA. Osteoarthritis is the first rheumatic disease in the world and the main cause of disability in the older population, making it a major public health issue as no curative treatment currently exists to stop its progression and development. Resveratrol, a polyphenol from the stilbene family, is known for its multiple beneficial properties like antioxidant, anti-diabetic and anti-inflammatory activities. Some In order to overcome this bioavailability issue, numerous new formulations of resveratrol are under development . One of Although commonly tested on various cultured cells, resveratrol could present deleterious effects on cell viability, especially when used at elevated concentrations as previously demonstrated by several studies . As an e® 812, making them potentially more or less lipophilic. Miglyol® 812 is composed of medium-chain triglycerides while isopropyl myristate is an ester of isopropanol and tetradecanoic acid. It is known that triglycerides are able to interact with the phospholipidic bilayer of biological membranes , as well as phospholipid-rich surface monolayers of plasma lipoproteins, in a highly specific way .However, despite its main localization in membranes, resveratrol brought by NE 2 was able to protect chondrocytes against cell death mediated by Hchanisms . Resverachanisms , silent chanisms , or NF-κchanisms   is responsible for severe damages on the gastrointestinal tract . When paIn conclusion, our study suggests that nano-emulsions of resveratrol could be considered as beneficial effectors in future OA therapeutic strategies, notably by increasing the concentration of resveratrol reaching the target cells and by limiting the deleterious effect of oxidative stress on chondrocytes.SLC, CC, DB-R, DB, and FR contributed to the conception and design of the study. SLC, TC, and CC carried out the experiments. SLC performed the statistical analysis and wrote the first draft of the manuscript. SLC and CC wrote sections of the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.CC is the guest editor of Frontiers research topic “Future opportunities for resveratrol delivery.” The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lipid droplets (LDs) are essential organelles for cellular energy homeostasis, but how metabolic cues are integrated in their life cycle is unclear. Teixeira et al. find that two protein isoforms, Ldo16 and Ldo45, differentially regulate LD dynamics under nutrient-rich and -deprived conditions, linking energy metabolism and storage. Storage and consumption of neutral lipids in lipid droplets (LDs) are essential for energy homeostasis and tightly coupled to cellular metabolism. However, how metabolic cues are integrated in the life cycle of LDs is unclear. In this study, we characterize the function of Ldo16 and Ldo45, two splicing isoforms of the same protein in budding yeast. We show that Ldo proteins interact with the seipin complex, which regulates contacts between LDs and the endoplasmic reticulum (ER). Moreover, we show that the levels of Ldo16 and Ldo45 depend on the growth stage of cells and that deregulation of their relative abundance alters LD morphology, protein localization, and triglyceride content. Finally, we show that absence of Ldo proteins results in defects in LD morphology and consumption by lipophagy. Our findings support a model in which Ldo proteins modulate the activity of the seipin complex, thereby affecting LD properties. Moreover, we identify ER–LD contacts as regulatory targets coupling energy storage to cellular metabolism. Cells live in fluctuating environments, and the capacity to store energy during nutrient surplus to mobilize upon deprivation is essential for their survival. In eukaryotic cells, energy is accumulated in the form of highly reduced neutral lipids, mainly triglycerides (TAGs) and steryl esters (SEs), in dedicated organelles called lipid droplets LDs; . LD dereLDs consist of neutral lipids in their core surrounded by a phospholipid monolayer and specific proteins essential in regulating their growth and consumption . During YMR147W and YMR148W.To identify putative regulators of the yeast Fld1–Ldb16 seipin complex, we immunoprecipitated endogenous TAP-tagged Fld1 and Ldb16, and copurifying proteins were analyzed by mass spectrometry. Both purifications identified two proteins encoded by adjacent ORFs: YMR147W generating a hybrid transcript with YMR148W . This was confirmed by fluorescence microscopy of representative LD proteins such as Erg6, Pet10, and Tgl1 .Seipin has a central role in controlling the LD proteome . To testescribed . In contldo45Δ ldo16Δ cells . Consistently, in ldo45Δ ldo16Δ mutant, Pdr16-GFP was diffuse throughout the cytoplasm . In agreement with the mass spectrometry data, Pdr16-GFP was detected not only in LD aggregates of fld1Δ and ldb16Δ cells but also in ldo45Δ ldo16Δ fld1Δ and ldo45Δ ldo16Δ ldb16Δ, which lack Ldo proteins . These results indicate that the drop in Ldo45 levels in stationary phase is not caused by changes in splicing, RNA processing, or protein degradation but likely by reduced activity of the YMR147W promoter in stationary phase cells. Thus, Ldo45 levels are tightly coupled to cellular metabolism.Transcriptional analysis suggested that Ldo16 and Ldo45 expression is controlled by independent promoters . To testYMR147W/LDO45 promoter by the strong glyceraldehyde-3-phosphate dehydrogenase (GPD) or galactokinase (GAL1) promoters for constitutive or inducible expression, respectively  cells, LDs were normal during the exponential phase but appeared enlarged during early stationary phase  and disordered domains. These gradually evolve to form a reticulate pattern at late stationary phase . During 6Δ cells . Interes6Δ cells . How LDs6Δ cells , 2017. TIn summary, our study identified Ldo16 and Ldo45 as regulatory subunits of the seipin complex with opposite effects on LDs. Ldo45 appears to modulate TAG storage by reducing Dga1 LD localization and promote LD targeting of some proteins, including Pdr16. Given the roles of seipin in controlling the LD proteome, future studies should dissect the distinct effects of Ldo45 on LD protein targeting. In contrast, Ldo16 does not affect LD proteome and functions primarily upon nutrient depletion, facilitating LD consumption by lipophagy. Remarkably, the relative abundance of these Ldo isoforms is coupled to cellular metabolism. Thus, our data are consistent with a model in which the seipin complex at ER–LD contacts is fine-tuned according to nutrient availability to promote either LD proliferation or consumption.493/503 (Invitrogen) and MDH (Abgent) were used at 1 µg/ml and 0.1 mM, respectively. Anti-HA  antibody was purchased from Roche, anti-Pgk1  was from Thermo Fisher Scientific, anti-GFP  was from ChromoTek, anti-Dpm1  was from Thermo Fisher Scientific, and anti-Kar2  was from Santa Cruz Biotechnology, Inc. Polyclonal anti-Usa1 (rabbit), anti-Fld1 (rabbit), anti-Ldb16 (rabbit), anti-Erg6 (rabbit), anti-Dga1 (rabbit), and anti-Lro1 (rabbit) antibodies were previously described  or to BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), and primers are listed in Tables S1 and S2, respectively. Plasmids expressing the different combinations of Ldo proteins were gifts from J. Vilardell .Protein tagging, promoter replacements, and individual gene deletions were performed by standard PCR-based homologous recombination . Strains600) between 0.5–1.2 or in early stationary phase at 4–6. For expression of galactose-inducible genes, cells were derepressed for 12–20 h at 30°C in media containing 2% raffinose as the sole carbon source and subsequently diluted into the corresponding media containing 2% galactose for 16–22 h. For lipophagy assays, cells were grown overnight in SC media and then diluted to the log phase (OD = 0.6) culture starting from OD = 0.15. The days 1, 4, and 6  are defined as when the same cultures of log phase were grown at 30°C for additional 24, 96, and 144 h, respectively.Cells were grown in yeast extract peptone dextrose (YPD) media , YPGal , synthetic complete (SC) media , SCGal , or synthetic dropout media supplemented with the corresponding amino acids for plasmid selection. Cells were grown at 30°C and assayed in exponential growth phase at an optical density at λ = 600 nm (OD600) were washed and resuspended in 1.4 ml lysis buffer . Cells were lysed with glass beads, and lysates were cleared by low-speed centrifugation at 4°C. Membranes were pelleted at 50,000 rpm for 25 min at 4°C in an Optima Max Tabletop Ultracentrifuge in a TLA 100.3 rotor (Beckman Coulter). The crude membrane fraction was resuspended in 600 µl LB. 700 µl of LB supplemented with 2% of detergent  were added, and membranes were solubilized 2–3 h on a rotating wheel at 4°C. Solubilized membranes were cleared for 15 min at 4°C at full speed in a tabletop centrifuge, and 1.1 ml was used for the immunoprecipitation. TAP-, HA-, and FLAG-tagged proteins were affinity isolated by overnight incubation with calmodulin sepharose 4B beads , anti-HA magnetic beads , and anti-FLAG M2 magnetic beads , respectively. Eluted proteins were analyzed by SDS-PAGE and immunoblotting. In all experiments, the input lane corresponds with 5% of the total extract used for immunoprecipitation.Large-scale purification of TAP-tagged proteins for mass spectrometry analysis was performed as described previously . Small-s493/503, mCherry, and MDH signals were detected using GFP, RFP, and DAPI filters, respectively, with standard settings. Cells were washed twice with PBS, pH 7.4, before visualization. All quantifications were performed from at least three independent experiments, and in each experiment, ≥100 cells/condition were scored. Data distribution was assumed to be normal, but this was not formally tested. Images were cropped, and contrast and intensity were adjusted with Photoshop CC and grouped with Illustrator CC .Fluorescence microscopy was performed at room temperature (∼23°C) in a Cell Observer high speed microscope (ZEISS) equipped with a CMOS camera  controlled by Slidebook 6.0 software (3i). A 100× 1.4 NA Plan Apochromat oil immersion objective was used. GFP, BODIPYFor lipophagy quantification, the vacuole border was determined by the Vph1-tdTomato signal  or the dg for 5 min , washed in milliQ water, preincubated in 0.1 M Tris-HCl, pH 9.5, and 10 mM DTT for 10 min at 30°C, washed, and resuspended in 50 ml spheroplasting buffer . For spheroplast preparation, Zymolyase 20T (Seikagaku Biobusiness) was added (10 µg/OD600 U cells) followed by incubation in a water bath at 30°C until a 10-fold drop in OD600 was observed (45–60 min). Spheroplasts were recovered by centrifugation , washed with spheroplasting buffer, and resuspended in breaking buffer  at a final concentration of 0.3 g of cells (wet weight)/ml, and PMSF (1 mM) and cOmplete were added before homogenization  in a Dounce homogenizer on ice. The homogenate was centrifuged  using rotor JS13.1. The resulting supernatant was transferred into 38-ml Ultra-Clear centrifuge tubes (Beckman Coulter) and adjusted to 19 ml (cell lysate fraction), overlaid with an equal volume of BB, and centrifuged for 45 min at 30,000 rpm in an optima l-100K centrifuge (Beckman Coulter) with an SW-32 swinging bucket rotor. The floating layer was collected from the top of the gradient, and the LDs were further purified. The pellet was collected, resuspended in PBS, pH 7.4, with help of a Dounce homogenizer, and fully dissolved with SDS sample buffer (membranes fraction). The floating layer was gently resuspended in BB (five strokes in a Dounce homogenizer with a loose-fitting pestle), adjusted to 19 ml with BB, transferred to a 38-ml Ultra-Clear tube, and overlaid with 19 ml 10 mM MES-Tris, pH 6.9, 8% (wt/wt) Ficoll 400, and 0.2 mM EDTA. Centrifugation was repeated as before . The floating layer was collected and resuspended in 19 ml 10 mM MES-Tris, pH 6.9, 0.6 M sorbitol, 8% (wt/wt) Ficoll 400, and 0.2 mM EDTA, transferred to 38-ml Ultra-Clear tubes, overlaid with 19 ml 10 mM MES-Tris, pH 6.9, 0.25 M sorbitol, and 0.2 mM EDTA, and then centrifuged once more for 30 min at 30,000 rpm. The recovered high-purity top LD fraction was snap frozen, stored at −80°C, and used subsequently for proteomics.LD purification was performed as previously described . In brieTCA-precipitated proteins were resuspended in 6 M urea and 200 mM ammonium bicarbonate before reduction (10 mM DTT) and alkylation (20 mM iodoacetamide). Samples were diluted to 2 M urea and digested with trypsin (1:10 wt/wt) overnight at 37°C. Tryptic peptide mixtures were desalted using a C18 UltraMicroSpin column using three washes with 0.1% formic acid in water followed by an elution step with 0.1% formic acid in a 1:1 mix of water and acetonitrile .Samples were analyzed in a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) coupled to nano-LC (Proxeon) equipped with a reverse-phase chromatography 12-cm column with an inner diameter of 75 µm, packed with 5-µm C18 particles . Chromatographic gradients were set from 93% buffer A, 7% buffer B to 65% buffer A, or 35% buffer B in 60 min with a flow rate of 300 nl/min. Buffer A contained 0.1% formic acid in water, and buffer B contained 0.1% formic acid in acetonitrile. The instrument was operated in data-dependent acquisition mode and full mass spectrometry scans, and 1-µm scans at a resolution of 60,000 were used over a mass range of m/z 250–2,000 with detection in the Orbitrap. After each survey, scans of the top 20 most intense ions with multiple charged ions above a threshold ion count of 5,000 were selected for fragmentation at a normalized collision energy of 35%. Fragment ion spectra produced via collision-induced dissociation were acquired in the linear ion trap. All data were acquired with Xcalibur software .Acquired data were analyzed using the Proteome Discoverer software suite , and the MASCOT search engine  was used for peptide identification. Data were searched against a database containing all yeast proteins according to the Saccharomyces Genome Database  plus thelmFit function and the empirical Bayes statistics implemented in eBayes and topTable functions of the Bioconductor limma package were used to perform a differential protein abundance analysis  were resuspended in 1 ml of 155 mM ammonium formate and lysed at 4°C with 400 µl of acid-washed glass beads using a cell disruptor (3 × 1 min). Lysates corresponding with 0.4 ODU per 200 µl were spiked with 30 µl of internal standard mixture containing 250 pmol cholesterol ester (10:0), 60 pmol cerimide , 60 pmol diacylglycerol (17:0/17:0 + [2]H5), 75 pmol phosphatidylcholine (16:0/16:0 + [2]H6), 110 pmol internal standard phosphatidylethanolamine (15:0/18:1 + [2]H7), 65 pmol phosphatidylinositol (15:0/18:1 + [2]H7), and 35 pmol TAG (17:0/17:1/17:0 + [2]H5). The samples were subsequently extracted with 990 µl chloroform/methanol  for 2 h  using a ThermoMixer (Eppendorf). The lower organic phase was collected after centrifugation  and vacuum evaporated. Finally, the lipid extracts were reconstituted in 100 µl chloroform/methanol  before their analysis by mass spectrometry.Yeast cell pellets (∼10 ODALL using an Orbitrap Fusion Tribrid (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source, TriVersa NanoMate (Advion Biosciences). In positive ion mode, aliquots of 2:1 lipid extracts were infused in chloroform/methanol/2-propanol  with 7.5 mM ammonium formate using a back pressure of 1.25 psi and an ionization voltage of +0.96 kV. MSALL analysis was performed using high-resolution Fourier transform mass analyzer (FTMS) analysis of the m/z range 500–1,400 and sequential FTMS2 analysis of all precursor ions in the m/z range of 400–1,050 . Freeze substitution of frozen samples was performed in an automatic freeze substitution system  using acetone containing 0.1% of uranyl acetate and 1% water for 3 d at −90°C. On the fourth day, the temperature was slowly increased by 5°C/h to −45°C. At this temperature, samples were rinsed in acetone and subsequently infiltrated and embedded in Lowicryl HM20 for 3 d. 90-nm sections were obtained using a diamond knife (Diatome) on a UC7 ultramicrotome (Leica Microsystems), transferred to formvar-coated 100 mesh Cu grids, and then poststained for 5 min with 2% uranyl acetate and 10 min with Reynold’s lead citrate. Grids were imaged using a Tecnai 12 transmission electron microscope (FEI) operated at 120 kV with a OneView digital camera (Gatan).Fig. S1 shows two Ldo isoforms interacting with the seipin complex and localizing to LDs. Fig. S2 shows how Ldo45 is required for LD recruitment of Pdr16. Fig. S3 shows how Ldo proteins are required for normal LD morphology. Table S1 shows yeast strains used in this study. Table S2 shows primers used in this study.


Leishmania spp. has opened a new era in deciphering Leishmania biology. The method was recently improved using a PCR-based CRISPR Cas9 approach, which eliminated the need for cloning. This new approach, which allows high-throughput gene deletion, was successfully validated in L. mexicana and L. major. In this study, we validated the toolkit in Leishmania donovani targeting the flagellar protein PF16, confirming that the tagged protein localizes to the flagellum and that null mutants lose their motility. We then used the technique to characterise CK1.1, a member of the casein kinase 1 family, which is involved in the regulation of many cellular processes. We showed that CK1.1 is a low-abundance protein present in promastigotes and in amastigotes. We demonstrated that CK1.1 is not essential for promastigote and axenic amastigote survival or for axenic amastigote differentiation, although it may have a role during stationary phase. Altogether, our data validate the use of PCR-based CRISPR Cas9 toolkit in L. donovani, which will be crucial for genetic modification of hamster-derived, disease-relevant parasites.The recent adaptation of CRISPR Cas9 genome editing to Leishmania is the causative agent of leishmaniasis, which has several clinical forms depending on the species, including cutaneous , diffuse cutaneous, mucocutaneous, and fatal visceral leishmaniasis  [ Leishmania goes through several extracellular developmental stages in the insect vector, from nonvirulent procyclic to virulent metacyclic promastigote forms [ Leishmania biology [ Leishmania as more than 50% of the genes encode hypothetical proteins [ Leishmania-specific genetic toolbox that could overcome different parasite-specific limitations such as the absence of RNA-interference machinery in the subgenus Leishmania, a stark contrast to Trypanosoma brucei, where this technique greatly contributed to a better understanding of the biology of this parasite over the past decade [The protozoan parasiteonovani) , 2. Leiste forms , and onete forms , 5. Thes biology . Howeverproteins . One majt decade , 8. Leishmania parasites [ In locus tagging of a gene of interest (GOI) requires multiple steps of cloning to assemble a cassette that could be integrated at the 5′ or the 3′ end of the gene [ Leishmania, at least two rounds of transfection are required [ L. donovani, as several studies have shown its ability to adapt to stressful conditions by copy number variations leading to gene amplification, gene deletion, or aneuploidy [ L. donovani biology.Although possible, genetic engineering has been particularly challenging and time-consuming inarasites . In locuthe gene . Furtherthe gene , 11. Thithe gene : (i) forrequired , and /CRISPR-associated protein 9 (Cas9) has been used, since 2013, as a genome editing tool for a large number of organisms including yeast and mammalian cells, and has subsequently been adapted for several unicellular human pathogens such asma cruzi . The recpulation –19. Howe Leishmania, there are six isoforms, of which only two have been studied: LdCK1.4 (LdBPK_2716800.1) and LdCK1.2 (LdBPK_351030.1). LdCK1.4 is secreted by the promastigotes and may play an important role in virulence and parasite survival [Casein Kinase 1 isoform 1  is a member of the CK1 family, which are signalling serine/threonine protein kinases involved in the regulation of various cellular processes such as the cell cycle or protein trafficking . They cosurvival . LdCK1.2survival . We showsurvival  and reprsurvival . In cont.4 LdBPK_16800.1 asurvival , whereassurvival , 29. Leishmania donovani Bob cell line expressing Cas9 and T7 RNA polymerase. In order to validate the CRISPR Cas9 toolkit in Leishmania donovani, we targeted PF16 gene, which encodes a central pair protein of the axoneme, essential for parasite motility. We successfully deleted the PF16 gene, which resulted in loss of motility, and we obtained the expected flagellar localization of PF16, by C-terminal tagging. We then applied the CRISPR Cas9 toolkit for a first functional genetic analysis of CK1.1. We showed that tagged CK1.1 protein was detected in both life stages but at a very low level. We demonstrated that CK1.1 was not essential for parasite survival as the null mutant parasites could survive as promastigotes and axenic amastigotes but may have a function in stationary phase.In this study, we first generated a L. donovani strain 1S2D (MHOM/SD/62/1S-CL2D) clone LdBob was obtained from Steve Beverley, Washington University School of Medicine, St. Louis, MO, and cultured as described previously [5 logarithmic promastigotes per mL were incubated at 26°C in M199 media (Gibco) supplemented with 10% heat-inactivated FCS, 20 mM HEPES, pH 6.9, 4.1 mM NaHCO3, 2 mM glutamine, 8 μM 6-biopterin, 10 μg/mL folic acid, 100 μM adenine, 30 μM hemin, 1x RPMI 1640 vitamins solutions (Sigma), 100 U/mL of Penicillin/Streptomycin (Pen/Step), and adjusted at pH 7.4. Axenic amastigotes were obtained by incubating 106 logarithmic promastigotes per mL at 37°C and 5% CO2 in RPMI 1640 + GlutaMAX™ -I medium (Gibco) supplemented with 20% of heat-inactivated FCS, 28 mM MES, 2 mM glutamine, 1x RPMI 1640 amino acid mix (Sigma), 1x RPMI 1640 vitamins solutions (Sigma), 10 μg/mL folic acid, 2 mM glutamine, 100 μM adenine, 100 U/mL of Pen/Step, and adjusted at pH 5.5. Relevant selective drugs were added to the medium at the following concentrations: 30 μg/mL hygromycin B (Invitrogen), 30 μg/mL puromycin dihydrochloride (Sigma), and 20 μg/mL blasticidin S hydrochloride (Invitrogen). When appropriate, axenic amastigotes cell aggregates were dispersed by passing cell suspensions five times through a 27-gauge needle before analysis.Axeniceviously –32. Brieμg/mL propidium iodide (Sigma-Aldrich). Cells were analysed with a CytoFLEX flow cytometer  to determine the incorporation of propidium iodide  and to monitor mNG levels in PF16::mNG::3xMyc (PF16-mNG-myc) or CK1.1::mNG::3xMyc (CK1.1-mNG-myc) transgenic parasites . The percentage of cell death, cell growth, and the mean mNeonGreen (mNG) fluorescence intensity were calculated using CytExpert (v2.0.0.153) software . Graphs were generated with GraphPad Prism (v7.03).Cultured parasites were diluted in DPBS (Gibco) and incubated with 2 7 LdBob cells in logarithmic phase were transfected with 15 μg of pTB007, with or without PCR reactions (mock) in 1x Tb-BSF buffer  [2 flasks and left to recover overnight at 26°C before adding or not the appropriate selection drugs. Survival of drug-resistant transfectants became apparent 7–10 days after transfection.Parasite transfections were performed as described previously . 1 × 107 pH 7.3)  using 2 μM each of gene-specific forward and reverse primers, and 1 unit HiFi polymerase (Roche) were mixed in 1x HiFi reaction buffer (Roche), supplemented with 1.875 mM MgCl2 to reach a final concentration of 3.375 mM and 3% (v/v) DMSO. The PCR conditions were as follows: 5 min at 94°C then 40 cycles of 30 s at 94°C, 30 s at 65°C, and 2 min 15 s at 72°C, and lastly a final elongation step of 7 min at 72°C. The presence of the expected product was assessed by running 2 μL of the 40 μL reaction on a 1% agarose gel. The sample was then heat-sterilized at 94°C for 5 min and used for transfection without further purification. Primer sequences are detailed in PCR reactions were performed as described previously . BrieflyμM each of primer G00 (sgRNA scaffold), 2 μM of gene-specific forward primer, and 1 unit HiFi polymerase (Roche) were mixed in 1x HiFi reaction buffer with MgCl2 (Roche). The PCR conditions were 30 s at 98°C followed by 35 cycles of 10 s at 98°C, 30 s at 60°C, and 15 s at 72°C and a final elongation step of 7 min at 72°C. To assess the presence of the expected product, 2 μL of the 20 μL reaction was run on a 1% agarose gel. The sample was heat-sterilized at 94°C for 5 min and transfected without further purification. Primer sequences are detailed in Supplementary Materials in In order to amplify the sgRNA templates, 0.2 mM dNTPs, 2 μM forward primer and reverse primer, 3% (v/v) DMSO, 2.5 units LongAmp Taq DNA polymerase (NEB), and 1x LongAmp Taq Reaction Buffer supplemented with Mg2+ . The PCR conditions were 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, 2 min 30 s at 65°C, and a final elongation step of 10 min at 72°C. Three microliters of reaction was then run on a 1% agarose gel to assess for the presence of the expected product. Primer sequences are detailed in Supplementary Materials in To assess the loss of the target gene in the knockout cell lines, genomic DNA was isolated from parasites collected after 1 passage post-transfection with the DNeasy Blood & Tissue Kit (Qiagen). One hundred nanograms of genomic DNA was mixed with 0.3 mM dNTPs, 0.5 7 and 2 × 108 logarithmic phase parasites (depending on the experiment) were resuspended in RIPA lysis buffer containing 150 mM NaCl, 1% Triton X-100, 20 mM Tris HCl, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, and inhibitor cocktails for proteases  and supplemented with 1 mM sodium orthovanadate and 1 mM PMSF. The cells were sonicated using the Bioruptor® (Diagenode) with the high power mode for 5 min  followed by 5 more minutes  and then centrifuged. Total protein quantity was assessed by the Pierce Coomassie Plus (Bradford) Assay. Twenty micrograms of total proteins was denatured, separated by SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (Pierce). Depending on the experiment, proteins were revealed as described in Supplementary Materials in Between 5 × 10L. donovani promastigotes expressing the fluorescent fusion protein PF16-mNG-myc were imaged by live microscopy. Samples were prepared as previously described [g for 5 min and washed three times in PBS with Hoechst 33342 at 5 μg/mL. The cells were resuspended in 50 μL PBS, and 2 μL was placed on a microscope slide, then a coverslip was applied, and the cells were immediately imaged with a 60x NA 1.42 plan-apochromat oil immersion objective lens (Olympus AMEP4694) on a EVOS FL microscope  with a ICX445 monochrome charge-coupled device (CCD) camera (Sony) at room temperature.escribed . BrieflyLeishmania promastigotes from early stationary phase were filmed for 10 s (200 frames) with a Leica DMI 4000B microscope, using a 40x objective and an Evolv EMCCD camera, with Metaview software. Tracking was performed using the Spot Detector and Spot Tracking tools from Icy software [for 10 s 00 framesMultiple sequence alignments (MSA) were computed using the PSI-Coffee mode of T-Coffee . The res Streptococcus pyogenes Cas9 nuclease gene (hSpCas9) [ L. donovani promastigotes ( L. mexicana promastigotes [We first transfected LdBob promastigotes with the plasmid pTB007 (LdB pTB007) expressing (i) the humanizedhSpCas9)  with a nhSpCas9) . We confstigotes , similarstigotes . We foun L. donovani, we performed C-terminal tagging and the generation of null mutants on the well-studied PF16 gene (LdBPK_201450.1), which encodes a central pair protein of the flagellar axoneme. Previous experiments in L. mexicana showed flagellar localization of PF16, and its deletion abrogates parasite motility [ L. donovani using the same gene editing strategy to validate the toolkit in this parasite species.To assess the efficiency of the CRISPR Cas9 gene editing toolkit inne LdBPK_1450.1, w PF16 gene [ Leishmania spp. using CRISPR Cas9. There are important differences between the genome of LdBPK282A1 reference strains from South-Eastern Nepal [https://www.ncbi.nlm.nih.gov/bioproject/396645) to design the primers required to generate the donor DNA and the corresponding sgRNAs. Sequences were identified using the EuPaGDT CRISPR gRNA Design Tool [ L. donovani. This approach is simple and fast and will greatly improve the way we study Leishmania genes compared to previous methods of gene tagging , since part of their endogenous regulation may be better maintained through the conservation of either the 5′ or 3′UTR .To fuse PF16 with the mNeonGreen-3xmyc (mNG-myc) in LdBob, we produced two PCR fragments: the donor DNA cassette, containing mNG-myc and the puromycin-resistance marker, as well as the sgRNA template to generate a Cas9 cleavage downstream of theF16 gene . It is crn Nepal  and thatrn Nepal ). Thus, ign Tool . LdB pTBign Tool . Fluores PF16 locus to generate null mutants in a single round of transfection. LdB pTB007 promastigotes were transfected with four PCR fragments corresponding to the two sgRNA templates to generate a double-strand break upstream and downstream of the PF16 CDS, and the two repair cassettes containing the resistance marker genes for blasticidin and puromycin [ PF16 has been successfully deleted in the whole population, without the need for subcloning as observed previously with L. mexicana and L. major [ L. donovani [ L. donovani. First, because L. donovani adapts very fast to its environment by copy number variations [ L. donovani strain Ld1S2D, purified from the spleen or the liver of infected hamsters, is particularly sensitive to in vitro culture, as this strain loses virulence after only 5 to 10 passages in culture to become unable to infect hamsters , LdCK1.2, or TbCK1.2, but similar to that of TbCK1.1  and in T. cruzi TcCK1.1 , which are also adjacent to TbCK1.2 and TcCK1.2, respectively. The two isoforms present in T. cruzi have a distinct feature compared to the orthologues in other trypanosomatids; TcCK1.2 is encoded by an array of five copies, while TcCK1.1 is encoded by a single copy  is 324 amino acids long and has a predicted molecular weight of 37.2 kDa. It contains a kinase domain, a N-terminal domain longer than that of CK1 of other eukaryotes including LdCK1.2, but similar to that of TbCK1.1, and a C-terminal domain shorter than that of the human CK1 ( TbCK1.1 . LdCK1.1 TbCK1.1 . Since te strain ). Finallhologues , leadingNext, we applied the CRISPR Cas9 toolkit to gain insight into CK1.1 function in the parasite. We tagged the protein to determine its localization and we deleted it to determine whether this kinase is essential for promastigote or amastigote survival. CK1.1 and a repair cassette containing the puromycin-resistance marker and the mNG-myc tag in frame. We then performed a Western blot analysis to determine whether CK1.1-mNG-myc was expressed in the transfected promastigotes and revealed a band at about 70 kDa corresponding to the expected size of the tagged protein . We did not observe an increase in fragmented DNA or cell debris (data not shown), suggesting that the difference in cell density might not be a consequence of cell death. Interestingly, these data indicate that CK1.2 cannot compensate for the loss of CK1.1 and conversely, the absence of CK1.1 does not influence the regulation of CK1.2 abundance. Indeed, there is no difference in CK1.2 level between the mutant and the control strains in promastigotes  dramatically limit parasite adaptation, by decreasing any compensatory mutation that could mask the phenotype, and (ii) allow the use of hamster-derived parasites for genetic manipulation by preventing the nonspecific loss of virulence occurring during in vitro culture. The use of CRISPR Cas9 in hamster-derived L. donovani opens new possibilities of studying the phenotype of nonessential genes in the context of the relevant mammalian host, thus moving beyond in vitro studies for the medically most relevant Leishmania spp.In this study, we validated the CRISPR Cas9 toolkit for


Physiological analysis indicated that ETH accelerated starch degradation and increased soluble solids content (SSC) and soluble sugars , while CA inhibited starch reduction compared with NA. Using these materials, expression patterns of 24 genes that may contribute to starch degradation (seven previously reported and 17 newly isolated) were analyzed. Among the 24 genes, AdAMY1, AdAGL3 and AdBAM3.1/3L/9 were significantly induced by ETH and positively correlated with starch degradation. Furthermore, these five genes were also inhibited by CA, conforming the likely involvement of these genes in starch degradation. Thus, the present study has identified the genes with potential for involvement in starch degradation in postharvest kiwifruit, which will be useful for understanding the regulation of kiwifruit starch content and metabolism.Starch is one of the most important storage carbohydrates in plants. Kiwifruit typically accumulate large amounts of starch during development. The fruit retain starch until commercial maturity, and its postharvest degradation is essential for consumer acceptance. The activity of genes related to starch degradation has, however, rarely been investigated. Based on the kiwifruit genome sequence and previously reported starch degradation-related genes, 17 novel genes were isolated and the relationship between their expression and starch degradation was examined using two sets of materials: ethylene-treated  vs. control  and controlled atmosphere stored (CA, 5% CO Arabidopsis mutants that synthesize less starch during the daytime and have lower starch degradation capacity at nighttime exhibited reduced growth rates [Starch is widely distributed in plants and accumulates in various organs, such as leaves, seeds and also tubers . Starch th rates ,5. In frth rates  and applth rates . During th rates , mangoesth rates , and banth rates , store ath rates . High stth rates . Thus, eSolanum tuberosum L.) plants [Arabidopsis, where analysis of transgenic or mutants has shown that GWD1, DPE1 and DPE2 are involved in starch degradation in leaves [AMY1, AMY2, AMY3 and PHS1 had limited effects [GWD reduction could inhibit starch synthesis under light [GWD was also involved in starch granule morphology formation [Multiple enzymes have been reported to be involved in starch degradation, such as glucan, water dikinase  2.7.9.4), α-amylase , β-amylase , α-glucosidase , pullulanase , isoamylase , α-glucan phosphorylase  and 4-α-glucanotransferase . However, the key regulators of starch degradation vary between different plants, organs and even developmental stages. For instance, GWD was involved in starch degradation in tubers and leaves of potato  plants ,14, whil) plants . Anothern leaves ,17,18, w effects ,20. Howeer light , and GWDormation .Arabidopsis) and staple crops , starch research in fruit, mainly bananas and kiwifruit, has usually only been conducted at the physiological and biochemical level. During the ripening of bananas, activities of AMY, BAM and AGL increased and showed close correlation with starch degradation [MAmy, Ma-bmy, Maisa [phoI and phoII [Ma-bmy showed increasing expression patterns during postharvest storage [AdAMY1-3, AdBAM1, AdBAM3.1, AdBAM3.2 and AdBAM9) were reported using a kiwifruit expressed sequence tags (EST) database [Unlike model plants  are likely to differ, as starch is degraded to maltose, glucose and glucose 1-phosphate in Arabidopsis leaves, but could not be converted to maltose in kiwifruit [Moreover, the mechanisms of starch degradation in fruit (at least kiwifruit) and model plants  were applied to manipulate kiwifruit postharvest ripening and softening. Using the kiwifruit genome database, genes that encoded AdAMY (AdAMY1-3) and four AdBAM  genes were reported and analyzed for starch metabolism during kiwifruit development [Arabidopsis homologs and the known kiwifruit genes, these seventeen newly isolated genes were designed as three AdAGL , four AdBAM , three AdISA , two AdDPE , AdGWD  and AdPHS , and one AdPU  (Three elopment . In addiX383652) .A. deliciosa “Hayward”) soften after harvest during storage at 20 °C and ethylene treatment (ETH) rapidly accelerates the fruit softening progress [Kiwifruit , while differences in transcript abundance of AdBAM3.1 and AdAGL3 mainly occurred at a later stage (56 days and afterwards)  .Maintenance of texture is important for postharvest fruit storability and transportability. Most fruit undergo softening when detached from the tree, resulting from cell wall degradation and reduction in intracellular adhesion . In someDespite the importance of starch degradation to both softening and flavor of postharvest kiwifruit, starch-related enzymes and genes have only been studied during kiwifruit development and rarely during postharvest softening. Here, 24 genes potentially related to starch degradation were isolated based on the kiwifruit genome, including 17 novel genes and seven previously studied in developing kiwifruit  were invAdAMY1, AdAGL3 and AdBAM3.1/3L/9, were identified as having the greatest potential for involvement in starch degradation in postharvest kiwifruit. As shown in Ma-bmy was reported to be associated with banana fruit starch degradation according to transcript accumulation [GWD and DPE are necessary in potato and Arabidopsis, but their transcript levels were quite low during the major starch degradation process of kiwifruit. On the other hand, AMY showed the potential to regulate both banana and kiwifruit starch degradation, which may suggest some overlap mechanisms between different fruit.Taking the results from ethylene treatment (accelerate fruit softening and starch degradation) and CA storage (inhibit fruit softening and starch degradation) together, five genes, including mulation , and theActinidia deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson var. deliciosa “Hayward”) were chosen as experimental material and were collected and described in our previous report [−1 C2H4, 24 h, 20 °C) vs. control  and controlled atmosphere  vs. NA (0 °C).Mature kiwifruit . SSC measurements were conducted with 12 fruit replicates, with 4 fruit from each biological replicate. After SSC analysis, as well as firmness analysis , the outd-glucose control were read at 510 nm against the reagent blank with UV2550 . Glucose, fructose and sucrose extractions and measurements were conducted according to the protocol of Lisec et al. [g for 10 min, the supernatants were transferred to new tubes and were mixed with 750 µL chloroform and 1400 µL dH2O. Then, the mixtures were centrifuged at 2200× g for 15 min. Aliquots of 100 µL of the supernatants were dried in vacuum  and 20 µL ribitol (0.2 mg·mL−1) was included in each sample as an internal standard. The residue was dissolved in 40 µL of 20 mg/mL pyridine methoxyamine hydrochloride, and incubated for 1.5 h at 37 °C. The sample was then treated with 60 µL Bis (trimethylsilyl) trifluoroacetamide (1% trimethylchlorosilane) for 30 min at 37 °C. The soluble sugars were measured by GC , A volume of 1 µL for each sample was absorbed with a split ratio 10:1 and injected into the gas chromatograph fitted with a fused-silica capillary column . The injector temperature was 250 °C and the helium carrier gas had a flow rate of 1.0 mL/min. The column temperature was held at 100 °C for 1 min, increased to 184 °C with a temperature gradient of 3 °C/min, increased to 190 °C at 0.5 °C/min, held for 1 min, increased to 280 °C at 15 °C/min and then held for 3 min. The significant MS operating parameters were as follows: ionization voltage was 70 eV, ion source temperature was 230 °C and the interface temperature was 280 °C.Starch and soluble sugar  measurements were conducted with frozen materials described above in three biological replicates. Total starch was extracted from 0.1 g frozen samples for each replicate following the procedures of the total starch assay kit , as described by Stevenson et al. . The absc et al. . SolubleTotal RNA extractions were conducted according to the method described by Yin et al. . The genAdAMY1-3) and β-amylase genes  [Starch degradation-related genes mentioned in research on starch metabolism during kiwifruit development, such as α-amylase genes ( AdBAM9) , were se AdBAM9) , additiohttp://tree.bio.ed.ac.uk/software/figtree/). Accession numbers for Arabidopsis genes in The Arabidopsis Information Resource (TAIR) are as follows: AtAMY1-3 ; AtBAM1-9 ; AtISA1-3 ; AtPU1 (Genbank no. At5g04360); AtPHS1-2 ; AtGWD1-3 ; AtDPE1-2 ; and AtAGL1-5 .Alignment was performed using the neighbor-joining (NJ) method in ClustalX  and phylogenetic analysis was performed with online software Figtree  and are listed in AdACT was chosen as the internal control [For real-time PCR, all primers were designed with online software primer3  was calculated with DPS . Figures were prepared with Origin 8.0 .AdAMY1, AdAGL3 and AdBAM3.1/3L/9, were characterized, using various postharvest treatments, as having higher potential to be involved in starch degradation during kiwifruit postharvest ripening. This has provided more specific targets for determining the mechanism and regulation of starch metabolism in kiwifruit. The potential complexity, however, with the involvement of multiple genes, may increase the difficulty for modification by transgenic or classical breeding approaches. Thus, it will be important to find the master regulators  for these five genes in future studies.In conclusion, seventeen genes encoding enzymes related with starch degradation were newly identified and isolated based on the kiwifruit genome, expanded in this catalog in kiwifruit from seven  to 24 ge


Although it is known that left ventricular hypertrophy (LVH) is a predictor of cardiovascular morbidity and mortality, there is no consensus on how LV mass (LVM) should be indexed, particularly in children. The well published difficulties with correctly indexing LVM hamper the confidence in determining the range of normal LVM in growing children. Left ventricular mass to volume ratio (LVMVR) has potential to avoid some of the inherent problems with indexing. Recent literature in adults has also shown a strong association between increasing LVMVR and increased risk of cardiovascular events. We hypothesized that LVMVR remained relatively stable in healthy children and young adults, and could serve as a simple surrogate to identify LVH.Thirty-one patients without cardiac pathology and normal cardiac MRIs (CMR) were identified and designated as the normal LVMVR group (Group 1). We evaluated LVMVR for differences in gender, age, weight, height, BMI, and BSA, by subdividing Group 1 and comparing the mean LVMVR between the two. The demographic data was normally distributed so the median value in each demographic value was used as a cutoff. To determine if LVMVR could discriminate between patients with abnormal LVM, we identified 30 gender, age, weight, height, and BMI-matched patients with a diagnosis of mild or repaired coarctation as a comparison group (Group 2). Patients with a history of coarctation were chosen because they are known to have LVH and increased regional wall thickness secondary to abnormal ventricular-arterial coupling. We also assessed LVM in all patients using CMR-derived LV mass indexed to BSA with normative values obtained from Kawel-Boehm et al. JCMR 2015.Group 1 consisted of 19 males (61%) with a mean age of 15 ± 3.4 years, weight 62.8 ± 19.9 kg, height 163.2 ± 14.4 cm, BMI 23.2 ± 6, and BSA 1.7 ± 0.3. The mean LVMVR of Group 1 was 0.73 g/mL ± 0.11. There were no significant differences in LVMVR by gender (p = 0.88), age (p = 0.55), weight (p = 0.11), height (p = 0.33), BMI (p = 0.15), and BSA (p = 0.22) (Figure The CMR-derived LVMVR remains stable in healthy children and young adults and may serve as a more reliable marker of LVH.


Plants contain a range of aquaporin (AQP) proteins, which act as transporter of water and nutrient molecules through living membranes. AQPs also participate in water uptake through the roots and contribute to water homeostasis in leaves.AQP genes in the B. rapa database and Br135K microarray dataset. Phylogenetic analysis revealed four distinct subfamilies of AQP genes: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs) and small basic intrinsic proteins (SIPs). Microarray analysis showed that the majority of PIP subfamily genes had differential transcript abundance between two B. rapa inbred lines Chiifu and Kenshin that differ in their susceptibility to cold. In addition, all BrPIP genes showed organ-specific expression. Out of 22 genes, 12, 7 and 17 were up-regulated in response to cold, drought and salt stresses, respectively. In addition, 18 BrPIP genes were up-regulated under ABA treatment and 4 BrPIP genes were up-regulated upon F. oxysporum f. sp. conglutinans infection. Moreover, all BrPIP genes showed down-regulation under waterlogging stress, reflecting likely the inactivation of AQPs controlling symplastic water movement.In this study, we identified 59 B. rapa and details the expression of 22 members of the BrPIP subfamily. These results provide insight into stress-related biological functions of each PIP gene of the AQP family, which will promote B. rapa breeding programs.This study provides a comprehensive analysis of AQPs in The online version of this article (doi:10.1186/s12870-017-0979-5) contains supplementary material, which is available to authorized users. Arabidopsis thaliana roots, hypocotyls, leaves, and flower stems [Plants depend on the absorption of water from soil and its subsequent transport to all other plant parts. Water moves inside the plant body through apoplastic, transcellular, and symplastic pathways. The symplastic pathway transports water across membranes  and is ger stems , and riper stems .Arabidopsis encodes 35 different AQPs [Glycine max [Zea mays [Oryza sativa [Populus trichocarpa [Solanum lycopersicum [Glycine max) nodulin-26 (GmNOD26), which is an abundant AQP expressed in the peribacteroid membrane of N2-fixing symbiotic root nodules. It was initially thought that the NIP proteins were found only in the nodules of nitrogen-fixing legumes [Arabidopsis [Arabidopsis encodes 35 different AQPs [Glycine max [Zea mays [Oryza sativa [Populus trichocarpa [Solanum lycopersicum [AQPs are predicted to consist of six membrane-spanning segments with two cytoplasmic termini. AQPs contain Asn-Pro-Ala (NPA) motifs located in two short, fold-back alpha helices following the second  and fifth  trans-membrane helices. Each AQP monomer contains two hemi-pores, which fold together to form the water channel. ent AQPs , whereascine max , 31 in ZZea mays , 33 in Oa sativa , 54 in Pchocarpa  and 47 ipersicum . Based opersicum . The NIP legumes . Howeverbidopsis , and ricbidopsis . The SIPbidopsis . Arabidoent AQPs , 66 AQPscine max , 31 in ZZea mays , 33 in Oa sativa , 54 in Pchocarpa  and 47 ipersicum .Tricticum aestivum TIP2 regulates the responses of plants to abiotic stresses  via an ABA-independent pathway(s) [Arabidopsis, PIP2;5 is up-regulated during cold exposure, and PIP subfamily genes are responsive to drought and salt stresses [NtAQP1 is involved in improving water use efficiency, hydraulic conductivity, and yield production under salt stress in tobacco [AQPs also appear to be involved in responses to abiotic stresses like drought, salt, and cold stresses in various plants. Seven members of the PIP1 subfamily of rice are responsive to cold stresses . Moreovethway(s) . In Arabstresses . In addi tobacco . By contAQP gene family in Brassica rapa to characterize which genes were responsive to biotic and abiotic stresses. Brassica rapa is a species of the genus Brassica, which is economically important worldwide. We performed comprehensive in silico analyses of gene classifications, chromosomal distribution, synonymous and non-synonymous substitution rates, syntenic relationships, evolutionary divergence, subcellular localization, gene duplication, phylogenetic analysis, exon–intron organization, conserved motifs, and predicted functions of AQPs in B. rapa. We further determined the gene expression pattern of PIP subfamily members in B. rapa plants in response to abiotic stresses  and ABA treatment. We also analyzed PIP subfamily expression under biotic stress (infection with Fusarium oxysporum f.sp. conglutinans), and assessed AQP protein similarity to stress response-related proteins from other plants.In this work, we carried out a genome-wide expression profiling of the AQP genes in B. rapa, we searched SWISSPROT of the BRAD (http://brassicadb.org/brad/) [B. rapa (Chiifu & Kenshin), removing any duplicates. A total of 61 gene sequences encoding putative members of the AQP family were identified in B. rapa. Domain searches using SMART confirmed that 59 of the putative AQP genes in B. rapa encoded predicted MIP and trans-membrane domains. In agreement with this result, protein sequence similarity analysis of all 61 sequences using blastp (protein-protein BLAST) showed that all but the two protein sequences lacking functional MIP and trans-membrane domains were most similar to proteins of AQPs. Based on these findings, we concluded that there are 59 functional AQP genes in B. rapa, which we named based on nomenclature used in other plants and guided by sequence similarity and phylogenetic analysis. Tao et al. [AQP genes in B. rapa, and our analysis found these, along with six more AQP genes. Additional file B. rapa AQP genes. These 59 AQP proteins of B. rapa showed a high level of sequence similarity to AQP proteins from different plant species. In silico functional analysis showed that the six newly identified AQP genes are likely involved in water transport in the plant body and leaves and in also root development   and annoo et al.  previousBrNIP1;2a, BrNIP1;2b, BrNIP6;1a and BrNIP6;1b encoded motifs with a variable third residue in which alanine was replaced by glycine and valine. Meanwhile, BrNIP5;1a and BrNIP5;1b encoded dissimilar amino acids in both NPA motifs, where alanine was replaced with serine and valine, respectively. Based on our subcellular localization predictions, all members of the NIP, SIP and PIP subfamilies of B. rapa appear to be present in the cell membrane. However, members of TIP subfamily were predicted to be positioned on vacuoles, with BrTIP 5;1 located in both vacuole and cell membrane  and the fewest were found in chromosome 8 (3.4%)  [B. rapa genome consists of three differentially fractionated sub-genomes, namely the least fractionated (LF), medium fractionated (MF1), and most fractionated (MF2). The 59 BrAQPs were fractionated into three subgenomes , including 26 (44%) in LF, 19 (32%) in MF1, and 14 (24%) in MF2  according to previous reports [BrAQP genes belonged to AK3 (18%), followed by AK1 and AK7 (15%), while only 8% of BrAQP genes were assigned to AK2  Fig. . The phy 10 Fig. . Genome go (MYA) . The B.  reports . The col reports . Most ofAK2 Fig. .Fig. 2DiBrAQP genes in the B. rapa genome implies that some genetic events have affected this gene family during evolution. The distribution of the AQP gene family has likely been influenced by processes such as segmental duplication, tandem duplication, and polyploidization [AQP gene family in B. rapa. We found evidence of at least two tandem duplication events  with total of 43 segmental duplications in the BrAQP gene family  ago and ended at 0.38 mya ago  [The arrangement of dization , 27. In ectively . All BrA~ 9 MYA) .Table 2EAQP genes among B. rapa, B. oleracea and A. thaliana  were found in all PIP subfamily members except BrPIP2;3b and BrPIP2;4c, which were lacked of motif 5, and BrPIP1;2a, which had no motif 4  was found in TIP group members, and motif 6 was found only in subfamily SIP1. The best possible match sequence for each motif is presented in Additional file Conserved motifs among each subfamily were identified using MEME software and compared for providing further support of the grouping of B. rapa AQPs were analyzed using the GSDS program. Most members of the PIP subfamily had three introns, while four members had two introns and two members had four introns. In the TIP subfamily, eight members had two introns and seven members had one intron, but only one gene had no intron. All BrNIP family members had 2 to 4 introns; 7 out of 15 members had 3 introns, another 7 members had 4 introns, and only 1 had 2 introns. BrSIPs formed a small subfamily of BrAQP in which all members had two introns  [BrAQP genes in response to temperature treatments in two inbred lines (chiifu and kenshin) of B. rapa . Most BrPIP genes were present in Cl-1, Cl-2, Cl-4 and Cl-6. The BrPIP genes in Cl-1, Cl-4 and Cl-6 showed higher expression in Chiifu than in Kenshin in response to both cold and freezing temperatures. Five BrPIP genes in Cl-2 showed higher expression in Kenshin than in Chiifu under normal conditions (22 °C). However, Cl-2 and Cl-3 BrAQP genes exhibited higher expression in Kenshin than in Chiifu in response to both cold and freezing temperatures. BrSIP2;1b did not show a significant response in any temperature treatment, whereas BrSIP2;1a did not respond to freezing temperatures. As a whole, we concluded that the majority of BrPIP subfamily genes were highly induced in Chiifu by cold and freezing treatment compared to in Kenshin. These results indicate that BrPIP subfamily genes might play an important role in the cold and freezing tolerance of Chiifu. On the contrary, a few BrPIP and those of other BrAQP subfamilies showed higher expression in Kenshin in response to cold and freezing temperature; those genes might be related to the cold and freezing susceptibility of Kenshin.Expression patterns of the 59 d −4 °C) . The twod −4 °C) . Moreoved −4 °C) . We creaPIP genes in different organs of B. rapa plants  was analyzed by qPCR and semi-quantitative RT-PCR  were expressed in all tested organs but BrPIP1;1b and 2;3a were only slightly expressed in flower buds. Two genes  were abundant in all of the tested organs except flower bud. BrPIP1;1a was highly expressed in roots and leaves and slightly expressed in stem but absent in flower buds. By contrast, BrPIP2;5a, and BrPIP2;5b were highly expressed in roots and flower buds but slightly expressed in stems and leaves. BrPIP1;3a, 1;3b, 1;4, 1;5, 2;4a, 2;5a, 2;5b, 2;6, 2;7a, 2;7b, and 2;7c were highly expressed in flower buds compared to other organs. However, BrPIP1;1a, 1;2b, 2;1, 2;2a, 2;2b, 2;3a, 2;3b and 2;4c were more abundantly expressed in roots compared to other tested parts  different from each other at the mentioned time points  among the genes  but was subsequently down-regulated. The rest of the BrPIP genes were down-regulated soon after drought stress and remained consistent throughout the stress period. These results are in agreement with those for plasma membrane AQPs in response to abiotic stresses in Arabidopsis thaliana [We next used Kenshin for qRT-PCR assays to elucidate the responses of nes Fig. . BrPIP2;ses Fig. . Meanwhithaliana .BrPIP genes were significantly up-regulated during salt-stress (p ≤ 0.01). BrPIP1;3a, 1;3b, 2;4a, 2;4b, 2;7b and 2;7c were up-regulated and showed the highest expression at 24 h and then were down-regulated. BrPIP1;2a, 1;2b, 1;4, 1;5, 2;3b and 2;4c were alternately up- and down-regulated throughout the treatment time course  at 12 h, but again started down-regulation to the end of the time course  exhibited higher expression at 4 h, while BrPIP1;2a and BrPIP2;7c peaked at 1 h and decreased thereafter. BrPIP2;1, BrPIP2;2a, BrPIP2;4b, BrPIP2;6, and BrPIP2;4a genes showed the highest expression at the 24 h time point. By contrast, BrPIP1;2b, BrPIP2;3b and BrPIP2;5a were down-regulated throughout the ABA treatment. BrPIP1;5 exhibited about 8- fold higher expression at 48 h and BrPIP 2;4a showed about 14- fold higher expression at 24 h; the expression change of those genes was statistically significant (p ≤ 0.01) compared to other genes in the same time courses  is an important phytohormone that plays a vital role in plant growth and development as well as in responses to a wide range of stresses. As shown in Fig. ses Fig. .BrPIP genes except BrPIP2;4a exhibited down-regulation compared to control. Some BrPIP genes showed increasing expression from 12 h to the end of treatment, but their relative expression remained below that of the control , respectively. BrPIP2;2a exhibited about 6- fold higher expression at 11 dai compared to mock-treated plants  exhibited similarity to AQPs in crop plants involved in water and solute transport in leaves and fruits during fruit development, pod development, root development, nutrient uptake and arsenic transportation. All of the members of PIP, NIP and SIP subfamily and most of the TIP subfamily members contained the same ar/R selectivity filter and Froger’s positions. In some cases, these were different in TIP subfamily which is consistent with previous research [n tomato . Meanwhiresearch . The ar/BrPIP subfamily showed high similarity to both water flow and abiotic stress-related PIP genes from other plant species, whereas three showed high similarity to proteins involved in water flow between the pollen and stigma papillae, and abiotic stress-related PIP genes from other plant species  in two inbred lines of B. rapa; Chiifu and Kenshin [BrPIP subfamily genes were selected based on their variation in transcript abundance compared to the control, and analyzed for responsiveness to temperature treatments in those two contrasting B. rapa inbred lines  [B. rapa / A. thaliana and B. rapa / B. oleracea, respectively  . Our micely Fig. .BrPIP genes are expressed at different levels in at least one of the tested organs of B. rapa plants. BrPIP1;1a, 1;2a; 2;2a, and 2;3a were more abundantly expressed in roots compared to other tested organs; which is consistent with previous findings [BrPIP1;2b, 1;3a, 1;4, 2;2a, and 2;3a were abundantly expressed in stem while BrPIP1;1a, 1;2b and 2;2a were highly expressed in leaves, like their Arabidopsis counterparts. Previous reports have been suggested that AQP genes are expressed in all plant tissues and are involved in growth and development and responses to environmental stress conditions [BrPIP genes in roots, stem and leaves might be related to different cellular controls of water flow. However, BrPIP1;2a, 1;2b, 1;3a, 1;4, 2;5b 2;6; 2;7a, 2;7b and 2;7c were typically more expressed in flower buds of B. rapa plants . However, the BrPIP1;5, 2;4a, 2;4b, 2;6, and 2;7a exhibited 9-, 16-, 5-, 4- and 4- fold higher expression, respectively, in response to ABA treatment. These results indicate that responsiveness of BrPIP genes to ABA treatment varied greatly. Therefore, it could be deduced that BrPIP gene expression responses are complex, likely due to involvement in both ABA-dependent and ABA-independent signaling pathways.bidopsis , rice [5bidopsis , Brassicca napus , and radca napus . All of ion Fig. . Most ofBrPIP genes were significantly down-regulated. A very few cases showed up-regulation at the end of the time courses, although their expression pattern remained below the control  and could be good sources for breeding targeted abiotic stress-tolerant cultivars. It is interesting to note that all BrPIP genes were significantly down-regulated by water logging stress, while BrPIP1;5 and 2;4a showed the highest expression to ABA treatment. The highly induced BrPIP genes reported here might be involved in maintaining water homeostasis in plant responses to abiotic stresses and ABA, and several of these genes might be functional against multiple stresses. The comprehensive expression analysis under different stress stimuli supplies novel information to assign putative stress-related physiological functions of BrPIP genes and facilitates selection of potential genes for further functional genomics studies in different Brassica crops.In this study, we demonstrated that B. rapa AQP members were identified using the key word “aquaporin” for the SWISSPROT tool of the B. rapa database  and protein sequences of the identified AQPs were processed or deduced using the B. rapa genomic database, after which the AQP protein sequences were further examined to confirm the presence of the characteristic MIP and trans-membrane helical domains using the SMART program  [B. rapa AQPs was carried out using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant/). Additionally, the primary gene structure  was analyzed using ExPasy (http://au.expasy.org/tools/pi_tool.html). Open reading Frame Finder (ORF) was obtained using ORF finder at NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Multiple sequence alignments using the identified protein sequences were made by CLUSTAL Omega . The protein homology study was done using the Basic Local Alignment Search Tool (BLASTp) (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm the identified AQP genes. The exon–intron organization of BrAQP genes was identified by comparing predicted coding sequences (CDS) with the corresponding genomic sequences using the GSDS 2.0 software (http://gsds.cbi.pku.edu.cn). The conserved motifs in the encoded proteins were identified using Multiple Expectation Maximization for Motif Elicitation  with the following parameters: maximum number of motifs 10; width of optimum motif ≥15 and ≤50.dex.php; . We alsoerg.de/;  and TMHM/TMHMM/) . PredictBrAQP genes were downloaded from the B. rapa genomic database (http://brassicadb.org/brad/). Arabidopsis and tomato AQP protein sequences were collected from TAIR (http://www.arabidopsis.org/) and the Sol Genomics network (http://solgenomics.net/), respectively. All sequences were then aligned using Clustal X [http://www.megasoftware.net) [The predicted protein sequences of the 59 lustal X . A phyloare.net) , 66 usinAQP genes along through the ten (10) chromosomes of B. rapa were retrieved from B. rapa database and the locations of the AQP genes were drafted using Map Chart version 2.2 (http://www.wageningenur.nl/en/show/Mapchart.htm). The AQP genes were BLAST searched (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against each other to identify duplicate genes, in which the similarity of the aligned regions covered >80% and the aligned region had identity >80% [Ka), synonymous rate (Ks), and evolutionary constriction (Ka/Ks) between the duplicated AQP gene pairs of B. rapa based on their coding sequence alignments, using the Nei and Gojobori model [Ka/Ks) between duplicated genes was analyzed to identify the mode of selection. Ka/Ks ratio >1, <1 and =1 indicate positive selection, purifying selection and neutral selection, respectively. We calculated the divergence time of duplicated gene pairs using T = Ks/2R Mya (Millions of years), where T refers to divergence time, Ks refers to the synonymous substitutions per site, and R is the rate of divergence of plant’s nuclear genes. For dicotyledonous plants R = 1.5 × 10−8 synonymous substitutions per site per year (38).Sub-genome fractionation, and positional information of all candidate ity >80% . Tandem ri model  as emploAQP genes were collected from the data of Jung et al. (30). For that data, two inbred lines of B. rapa ssp. pekinensis, namely cold-tolerant Chiifu and cold-sensitive Kenshin, were treated with different temperatures viz. 22, 4, 0, −2, and −4 °C for 2 h. A heat map was generated based on transcript abundance value of 59 AQP genes using Cluster 3.0 and tree view software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm#ctv).Temperature-treated microarray data for B. rapa, B. oleracea and A. thaliana were detected using Blast against whole genomes of such crop species. AQP gene positions on chromosomes were collected from databases and the relationship among the three crop species were plotted using Circos software (http://circos.ca/) [The microsyntenic relationship of AQP genes among cos.ca/) .B. rapa ssp. pekinensis) inbred lines cold-tolerant Chiifu and cold-sensitive Kenshin were used for cold-stress experiments, and Kenshin was used for other abiotic stress treatments. Seed sterilization, culture, seedling management were conducted according to the methods described by Ahmed et al. [B. rapa plants were harvested, immediately frozen in liquid nitrogen, and then stored at −80 ° C for RNA extraction. B. rapa (SUN-3061) was used for analysis of organ-specific expression and for biotic stress treatment (with F. oxysporum f.sp. conglutinans). The plants were grown for 3 weeks under culture room conditions with 16 h light and 8 h dark maintaining 25 °C temperature prior to fungus treatment. The fungal spore concentration 1x106 spores per ml solution was used for inoculation using the method described by Ahmed et al. [Chinese cabbage  using the RNeasy mini kit  following the manufacturer’s protocol. The concentration of RNA from each sample was determined by UV spectrophotometry at A260 using a NanoDropND-1000 . DNA contamination was removed using RNase-free DNase  following the manufacturer’s protocol. A 6 μl sample of total RNA was converted to cDNA using the First-Strand cDNA synthesis kit  following the manufacturer’s instructions.2O). The conditions for real-time PCR were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 10 s, and extension at 72 °C for 15 s. The qRT-PCR reactions were normalized using the B. rapa Actin gene as reference for all comparisons [-ΔΔCT method to determine the relative expression. The relative expression data was statistically analyzed (Tukey HSD test) and lettering was done using Minitab 17 software (https://www.minitab. com/products/minitab/).For each treatment, qRT-PCR was performed on three biological replicates. The 10 μl reaction volume consisted of the following: 5 μl 2x Quanti speed SYBR mix, 1 μL (10 pmol) each forward (F) and reverse (R) gene-specific primers, 1 μl template cDNA (50 ng) and 2 μl distilled, deionized water (ddH


By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent Pi transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a−/−) mice compared with their wild-type littermate controls. Rates of VATP normalized in both hypophosphatemic groups after restoring plasma Pi concentrations. Furthermore, VATP was directly related to cellular and mitochondrial Pi uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum Pi content and muscle VATP. After oral Pi repletion and normalization of serum Pi levels, muscle VATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood Pi concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.—Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis.Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using  By udiabetes , 21. In ffspring . HoweverChemicals were obtained from commercial sources and used as received, unless noted otherwise.i, 1.0% Ca, TD 2018S; Harlan Teklad, Madison, WI, USA).All rodent protocols were approved by the Yale University School of Medicine Animal Care and Use Committee. Mice were housed under controlled temperature (22 ± 2°C) and lighting , with free access to water and food  were obtained from The Jackson Laboratory  and bred with C57BL/6J mice. Resulting heterozygotes (HETs) were bred HET by HET, and −/−NaPi2a and wild-type (WT) littermate controls were used for experiments.Mice homozygous for a mutation in the gene of sodium-dependent P−/−NaPi2a mice were evaluated at age 2–3 mo, placed on a low-Pi diet  or a high-phosphate diet , and evaluation was repeated at 2 and 10 wk. Plasma Pi and lactate were measured at baseline, 2 wk, and 10 wk on LPD and HPD. Comprehensive animal metabolic monitoring system  was used to determine activity and respiratory exchange ratio over a period of 72 h of mice that were fed ad libitum with LPD and HPD.For experiments shown in i  or LPD with 0.02% Pi and 0.6% calcium . Mice were matched for age, weight, and food withdrawal time within each experiment, and, if possible, between experiments  between 2 and 3 mo of age were individually housed and maintained on either standard mouse chow diet with 0.6% Periments .31P]MRS by using a 9.4 T superconducting magnet system  interfaced to a Varian console as described earlier (1H coil (25 mm) and an inner [31P] coil (15 mm)]. This stable experimental setup eliminates movement of the region of interest and guarantees acquisition of high signal/noise [31P] spectra. Body temperature was maintained at 37°C by using a heated cradle incorporated into the MR probe, and optimal signals were acquired by using localizers to ensure accurate positioning of the animal in the magnet. To monitor cardiopulmonary status of the animal throughout the experiment and control depth of anesthesia to maintain stable physiologic function, a multiparameter pulse oximeter was used . For shimming, a FastMap sequence over a 2.8- × 2.8- × 2.8-mm volume of the leg was applied. Coil geometries were optimized to match the size of the mouse and to ensure close proximity of the muscle to the coil for a high signal-to-noise ratio while avoiding occlusion of blood flow to the mouse extremity. By using Nuts software  for spectra processing, we are able to calculate VATP. With the help of fully relaxed [31P] spectra, we estimated muscle Pi content. Parameters were calibrated against ATP and Pi measurements that were obtained from muscle tissue after dissection of animals  on the basis of a 72 ml/kg blood volume for a mouse to restore normophosphatemia (∼5.6 mg/dl).−/−NaPi2a mice while inside the MRS instrument. The LPD group received a bolus of 30 μmol NaH2PO4 and the HPD group received 60 μmol NaH2PO4 at pH 7.4. Control group received 60 μmol of iso-osmotic NaCl infusion. Blood samples were obtained at end of study by cardiac puncture for plasma [Pi] measurements.WT mice were divided into 3 groups that each received a phosphate infusion on the basis of the protocol used for VATP was assessed during hyperinsulinemic-euglycemic conditions as shown in 2–cooled aluminum tongs and stored at –80°C for subsequent analysis.VATP and serum Pi before, after 5 mo, and again after a total of 8 mo oral Pi treatment to normalize plasma phosphorous concentrations in a 28-yr-old male individual with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a genetic form of hypophosphatemia as a result of loss-of-function of the proximal tubular sodium-Pi cotransporter NaPi2c, as previously reported by us , carbon dioxide production (VCO2), and respiratory exchange ratio (RER) as well as energy expenditure, food consumption, and activity for body weight and fat-matched animals as previously described . Energy expenditure and RER were calculated from gas exchange data (energy expenditure = [3.815 + 1.232 × RER] × VO2). RER is the ratio of VCO2 to VO2, which changes depending on the substrate that the animal is using. When carbohydrates are the only substrate being oxidized, RER will approximate 1.0, and when only fatty acids are metabolized, 0.7.CLAMS  was used to evaluate oxygen consumption ; Free induction decays were zero-filled and apodized using a 5 Hz Lorentzian function before Fourier transformation. Spectra were manually phased and baseline corrected by fitting to a fifth-order polynomial. Peak areas of [31P] metabolites were determined by integration. Concentrations of [31P] metabolites were determined from fully relaxed spectra assuming a constant 5.5 mmol/kg muscle ATP concentration.ST- . Pi conc−/−NaPi2a and WT mice  and cut into pieces. After washing 3–4 times with 1 ml MiB06, 1 ml of protease solution (2 mg/10 ml MiB06) was added and incubated for 2 min. Sample was then homogenized, transferred to a centrifuge tube, and centrifuged at 800 g for 10 min at 4°C. Supernatant was transferred to a new centrifuge tube after filtering through a cheesecloth and centrifuged at 10,000 g for 10 min at 4°C. Supernatant was discarded and mitochondrial pellet was resuspended in MiB06. After determining mitochondrial protein concentration by Bradford assay, intact mitochondria corresponding to 10 mg of mitochondrial protein were added to the chamber of an a oxygraph 2K  that contained Pi-free mitochondrial respiration buffer MiR05  and oxygen flux (pmol/s/ml) was measured at 37°C. Standardized instrumental calibrations were performed to correct for back-diffusion of oxygen into the chamber from various components, leak from exterior, oxygen consumption by chemical medium, and sensor oxygen consumption. Glutamate (10 mM), malate (2 mM) and ADP (1 mM) was added to the Pi-free medium to favor complex I respiration. Oxygen flux by complex I in dependence of different concentrations of Pi was determined by using DatLab (Oroboros).For isolation of mitochondria from the hind limb of  WT mice A, excise2 in 150 cm2 flasks in 15-ml α-minimal essential medium with 1% penicillin/streptomycin and 2% fetal bovine serum (FBS) after growth to confluency (myocytes). RC13 cells (passage number 5–10) were grown at 37°C under 5% CO2 in 150 cm2 flasks in 15 ml DMEM that was supplemented with 10% FBS and 1% penicillin/streptomycin. For experiments, cells were grown to confluency in 24-well plates. Medium was purchased from Thermo Fisher Scientific Life Sciences . Medium was changed every 3–4 d and cells were passaged (1:10) every 5–7 d after removal from the plate with 1 ml trypsin/EDTA. For experiments, cells were grown to confluency in 12- or 24-well plates in medium that contained 2% FBS and were differentiated into myotubes for 10–20 d.L6 cells (passage number 5–20) were grown at 37°C under 5% COCell lysates were analyzed by PAGE and immunoblotted for phosphorylated Akt (p-Akt) and total Akt as previously described  by using2O for 30 min. Wells were then rinsed with 500 µl buffer A 3 times. Each well received 500 µl of transport buffer B and was incubated for exactly 10 min. Reactions were quenched by washing each well with 500 µl of ice-cold buffer A for Pi transport and buffer A + 5 mM d-glucose for glucose transport. Cells were lysed by treatment with 400 µl of 1 M NaOH and neutralized with 400 µl of 1 M HCl. In both assays, radioactivity was quantified by liquid scintillation counting, normalized to protein content determined by BCA protein assay (Thermo Fisher Scientific Life Sciences), and reported as moles of Pi or glucose transported per minute per mg protein.Cells were starved in serum-free α-minimal essential medium for 14–16 h before the experiment. Cells were then treated with indicated concentrations of inhibitors for 20 min: wortmannin (500 nM) and LY294002 , KP372-1 , and Akt-I-1/2 . ETOH and DMSO were used as vehicle controls. Inhibitors were delivered to each well in 1-µl volumes. After treatment with an inhibitor or vehicle control, cells were treated with 100 nM insulin (Novo Nordisk) or 5 µl Hsem. Multiple comparisons were analyzed by ANOVA—followed, when appropriate, by Turkey post hoc test—by using Prism 6 for Windows software . Single comparisons were performed by 2-tailed unpaired Student’s t test. For statistical analysis of human patient data, means of replicate measurements were compared with reference range. A value of P ≤ 0.05 was considered statistically significant.All results are presented as means ± i in NaPi2a−/− mice was reflected by a 50% reduction in plasma Pi  and decreased further to 2.1 ± 0.3 mg/dl (P < 0.001 vs. baseline) after 2–10 wk on LPD . Plasma Pi levels normalized after 3–10 wk on HPD . Spontaneous physical activity, as assessed over 72 h using CLAMS, was reduced by ∼30% in −/−NaPi2a after 10 wk on LPD compared with HPD  and increased RER  and NaPi2a−/− mice lost 15%   , 29, andNaPi2a−/− mice were hypophosphatemic with a 43% reduction in plasma Pi  and a similar trend for −/−NaPi2a mice .−/−NaPi2a mice with 25 µmol NaH2PO4 (pH 7.4) resulted in normalization of plasma Pi from 3.6 ± 0.1 to 5.6 ± 0.2 mg/dl (P < 0.001) and led to complete normalization of muscle mitochondrial VATP  show that muscle VATP is directly correlated with muscle Pi concentrations . VATP in these mice was reduced by 52% compared with the regular chow-fed group  at 2 µl/min. Basal VATP in hypophosphatemic mice on LPD was 6.9 ± 0.8 µmol/g/min and increased in a dose-dependent manner to 7.6 ± 0.5 µmol/g/min (P < 0.001 vs. preinfusion) after low-dose Pi infusion and 1.9-fold after high-dose Pi infusion to 13.4 ± 0.9 µmol/g/min .To reverse hypophosphatemia and examine the effects on muscle mitochondrial i concentration on oxidative phosphorylation capacity in vitro, we measured the rate of oxygen flux in isolated quadriceps mitochondria from WT and −/−NaPi2a mice in a Pi-free medium in the presence of glutamate, malate, and ADP. Oxygen flux increased upon addition of 1 mM Pi and further increased upon addition of 5 mM Pi to medium . In contrast, insulin stimulation had no effect on VATP in −/−NaPi2a mice (M′/M0), effective MR relaxation time of Pi (T1′) and the rate constant k′ were not changed in response to insulin stimulation but were different between WT and −/−NaPi2a mice .To examine the potential role of plasma P−/− mice B. Plasma−/− mice C, D. Sati concentrations and muscle VATP in healthy, lean, insulin-sensitive control subjects and a severe blunting of this effect in otherwise healthy, young, lean, insulin-resistant offspring of parents with type 2 diabetes (T2D) . Stimulation of Pi uptake was dose dependent and inhibited by wortmannin, a potent and specific PI3K inhibitor. Similar results were obtained by using the PI3K inhibitor LY294002 (50 μmol/min) and the PDK1 inhibitor KP372-1 , whereas Akt-I-1/2, an inhibitor of Akt, had no effect on blocking Pi uptake  when used at a dose of 120 nM, which is sufficient to inhibit glucose uptake by GLUT4 in these cells . These data suggest that insulin-stimulated glucose transport involves activation of PI3K, PDK1, and Akt, whereas insulin-stimulated Pi transport only involves PI3K and PDK1. Similar results were obtained in human RC13 myocyte cells .We have previously observed a strong relationship between insulin stimulation of intramyocellular Pes (T2D) . To furti cotransporter NaPi2c  and increased to 3.3 mg/dl after Pi supplementation. At baseline, muscle VATP was reduced by 50% in the patient with HHRH  and normalized with serum Pi after treatment to 5.4 and, finally, 7.0 µmol/g/min   . We therith VATP  before aVATP is related to intramyocellular Pi in −/−NaPi2a mice, we examined isolated muscle mitochondria from these mice. Oxygen flux, a measure of VATP, is stimulated when Pi is added to the assay buffer; however, no difference was observed between mitochondria isolated from WT and −/−NaPi2a mice, which suggests that intramyocellular Pi regulates VATP and that intramyocellular Pi as a result of hypophosphatemia is likely the cause of reduced VATP in these mice.To further test whether i and VATP in insulin-sensitive and insulin-resistant individuals  that the 50% reduction in plasma Pi was accompanied by a 50% reduction in muscle VATP and also that normalization of plasma Pi levels with oral phosphate repletion resulted in complete normalization of muscle VATP.Finally, we showed in a patient with chronic hypophosphatemia as a result of a loss-of-function mutation in the NaPi2c renal phosphate transporter gene (i uptake is an important determinant of VATP and hypophosphatemic myopathy in mice and men. Future studies in mice in which Pi transporters are selectively deleted in muscle will further clarify the role of Pi uptake for mitochondrial and muscle function.Taken together, our data strongly suggest that cellular Pi transport into myocytes is stimulated by insulin, a hormone markedly induced during refeeding, which may provide a pathophysiologic explanation to the hypophosphatemia observed.Our findings may have broader implications for 2 common clinical conditions: refeeding syndrome and myopathy commonly observed in T2D. Refeeding syndrome is a potentially fatal complication that occurs in patients with starvation, malnutrition or alcohol abuse . Althougi content and decreased insulin-stimulated rates of ATP synthesis (VATP. Furthermore, in RC13 rhabdomyosarcoma cells and L6 myocytes, insulin regulates Pi uptake upstream of Akt, which involves activation of PI3K and PDK1. Similar observations were made by Wang et al (i conditions by the ability of insulin to reduce serum Pi concentrations and promote redistribution of Pi from the circulation into the cell. On the basis of these considerations, decreased muscle ATP synthetic flux may serve as a noninvasive marker to monitor therapy of patients with refeeding syndrome and T2D.Muscle strength and function are also impaired in T2D , 43. Altynthesis , which sng et al , who repVATP may serve as a noninvasive marker for hypophosphatemic myopathy. Our findings further establish insulin as a potential regulator of muscle Pi transport and of VATP. These findings might help to better understand the pathophysiology of refeeding hypophosphatemia and of the myopathy commonly observed in individuals with T2D.Our findings suggest that decreased muscle ATP synthetic flux, in part, may explain muscle weakness in hypophosphatemia, and