Micractinium, Chlorella, and Scenedesmus, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m2/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m2/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona. Three strains, from the genera  Furthermore, many algae are able to use saline or brackish water resources which should reduce costs and avoid further demands on limited freshwater resources. Lastly, algae can be grown throughout the calendar year in many locations. However, despite the apparent promise of algal biomass, algae have yet to be fully utilized at industrial scales, due in part to the poor correlation of productivities measured in the laboratory to outdoor performance and ultimately lack of cost competitiveness with traditional petroleum-based fuel resources at current prices , connected to a programmable water chiller/heater circulating pump . White LED (light emitting diode) lights  illuminated both of the longer sides of the water bath, and were connected to a programmable dimmer , this provided uniform lighting to all culture tubes. Carbon dioxide was mixed with air to 2% COTo initiate the screening process, 300 strains from diverse environments were first evaluated for their ability to grow in seawater-based media (defined below). Of the 300, 107 strains were found to be halotolerant (growth in seawater-based media) and were further screened in duplicate under simulated winter conditions. In each screening run, approximately 35 unique strains were each grown in single chambers, from a starting optical density (750 nm) of 0.2. To compare the most promising isolates, the top performing strains from each winter run were run for a 3rd time to produce triplicate data. This represented 33 promising winter isolates chosen by the down-selection criteria that included: doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. During all runs, optical density (750 nm) was measured over a time course (measurements every other day), from which doubling time could be calculated, followed by endpoint compositional analysis, which included analysis of moisture, ash, lipid content as converted fatty-acid-methyl-esters (FAME), and carbohydrates. The reported doubling time, calculated during linear growth phase, includes time in which the lights were off simulating night time (15 h a day), and thus is a representation of a real-world winter doubling time including light and temperature variation rather than the shortest doubling time possible under ideal conditions. The cell size reported was calculated by averaging 20 linear phase cells measured with a Nikon Eclipse Ci microscope, utilizing NID Elements v. 4.40.00 software and calibrated with an improved Neubauer hemocytometer; average and the respective standard deviation is reported in the results.4Cl (5.0 × 10−3 M), NaH2PO4⋅H2O (0.313 × 10−3 M), Na2SiO3⋅9H2O (1.06 × 10−4 M), FeCl3⋅6H2O (1.17 × 10−5 M), Na2EDTA⋅2H2O (1.17 × 10−5 M), CuSO4⋅5H2O (3.93 × 10−8 M), Na2MoO4⋅2H2O (2.60 × 10−8 M), ZnSO4⋅7H2O (7.65 × 10−8 M), CoCl2⋅6H2O (4.20 × 10−8 M), MnCl2⋅4H2O, (9.10 × 10−7 M), thiamine HCl (2.96 × 10−7 M), biotin (2.05 × 10−9 M), cyanocobalamin (3.69 × 10−10 M), Tris base (24.76 × 10−3 M).The media used in screening was a modified f/2 formulation . To seaw2O. PCR was carried out with 2 μL of the cell lysate, 25 μL of Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs), 18 μL of H2O, and 2.5 μL each of the forward and reverse primers (10 μM) (TM Yeast DNA Purification kit (Lucigen) from day 0 seed cultures, lyophilized biomass samples, ultimately confirming correct speciation via 18s sequences. 18s sequences were deposited into the Sequence Read Archive, submission ID: SRP136934.Phylogenetic sequencing was conducted using universal eukaryotic primers to amplify the 18S rRNA gene. Reverse primer, 1391RE 5-GGGCGGTGTGTACAARGRG-3 and forward primer 360FE 5-CGGAGARGGMGCMTGAGA-3 were used for all amplifications . Colony  (10 μM) . The the (10 μM)  used as Micractinium reisseri), 4-C12 (Chlorella vulgaris), and 46B-D3 (Scenedesmus rubescens) were deployed outdoors utilizing the AzCATI testbed site located at Arizona State University in Mesa Arizona during February of 2016. Cultures were scaled up indoors using bubble columns and 15 L flat panel reactors as described in 2. Media consisted of 35 g/L salinity achieved using Crystal Sea , 2.14 mM ammonium chloride and 0.134 mM phosphate (NaH2PO4⋅H2O). Trace metals were added to the following final concentrations (standard f/2 media), FeCl3⋅6H2O (1.17 × 10−5 M), Na2EDTA⋅2H2O (1.17 × 10−5 M), CuSO4⋅5H2O (3.93 × 10−8 M), Na2MoO4⋅2H2O (2.60 × 10−8 M), ZnSO4⋅7H2O (7.65 × 10−8 M), CoCl2⋅6H2O (4.20 × 10−8 M), MnCl2⋅4H2O, (9.10 × 10−7 M). Inoculation density was targeted at 0.05 g/L of algal biomass, and sodium carbonate was added as necessary to control the pH drop due to consumption of NH3 from the added NH4Cl. Pond pH was adjusted to 7.9 via a pH controller connected to a solenoid delivering CO2 gas, and PAR was measured continuously with a LiCor LI-190R quantum pyranometer , 4-C12 (Chlorella vulgaris), 46B-D3 (Scenedesmus rubescens); along with their respective cell sizes, 4.2 ± 0.5, 5.7 ± 0.7, and 11.7 ± 1.6 μm.Initial down-selection involved screening for the ability of 300 strains to grow in saline media, as the algae were collected from diverse water sources with varied salinity. Of these 300, 107 showed growth in saline media, and were thus subjected to more detailed growth analysis as described in the methods. All data generated from this screening is available in the Nannochloropsis salina (CCMP 1776) as a baseline control in all down-selection trials. Nannochloropsis sp. are widely regarded as industrially relevant for outdoor growth due to their relatively high productivities, high lipid content, and stability during outdoor cultivation (Nannochloropsis oceanica (KA32) productivity  approach, and thus add additional value to algal biomass . It is cChlorella isolates rapidly drop in productivity, ultimately requiring fresh inoculum in outdoor trials due to problems associated with predation and contaminating organisms, such as Vampirovibrio chlorellavorus . As discussed above, storage carbon accumulation and chlorophyll degradation correlate with nutrient depletion, however, carbon fixation continued, as reflected by AFDW increase. This suggests the cells are initiating a nutrient deprivation response once the nutrients in the media are depleted, despite the fact there are sequestered nutrients in the cells. This is supported by The data presented here validated the indoor screening and selection methodology, with the down-selected strains showing growth and composition suitable for biofuel and bioproducts production when considering the cold temperatures of the ponds Figures –3. Theset = 0 in Figure 4-C12 and 14-F2 show a clear increase in the percentages of specific fatty acid chains lengths, likely due to nitrogen limitation  of seed cultures represents an area of future research optimization as cells that experience a reduced initial transfer shock as the algae are transitioned from a photobioreactor to a pond, will likely have increased productivities.The winter productivity data reported here are unique in that metrics for biomass, along with the associated lipid and carbohydrate composition are reported under real world deployment conditions. Furthermore, algal ponds were used for growth, which is significant as techno-economic analysis has suggested that for algal biofuels to be cost competitive with traditional petroleum sources, algal ponds should be employed due to the low capital expenditure relative to photobioreactors . AdditioOur data demonstrate that algal strain screening under simulated outdoor environmental conditions enables identification of strains with robust biomass productivity and biofuel precursor composition. This can be especially useful in future efforts as algae could be cultivated in a variety of geographic locations representing unique temperature and light regimes, along with other environmental variables. Importantly, we have identified a series of halotolerant strains with the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds, with biomass compositions useful for biofuels and bioproducts. These strains represent promising candidates for further development and deployment. Future studies will focus on optimization of culture conditions, down-selection of superior summer strains, and generation of facile genetic toolkits for these promising halotolerant algae.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.LD, MP, and MG conducted experimental design and wrote the manuscript. LD performed strain screening, characterization, and down-selection. SVW directed FAME and carbohydrate analysis. HG and JM directed the outdoor deployment. All authors reviewed and revised the manuscript.The 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.


Cordycepsmilitaris, mutants were generated from mixed spores of two C. militaris strains with varying cordycepin-producing capacities. Fifteen stable mutants producing from 0.001 to 2.363 mg/mL cordycepin were finally selected. The relative fluorescence intensities of the 15 mutants, two C. militaris strains and an Aspergillus nidulans strain at different concentrations of lyophilized mycelium powder were then investigated using the Nile red method. The mutant CM1-1-1 with the highest relative fluorescence intensity among the eighteen strains was selected for optimizing the Nile red method. Relative fluorescence intensity was linearly correlated with cordycepin concentration in liquid broth (R2 = 0.9514) and in lyophilized mycelium powder (R2 = 0.9378) for the 18 cordycepin-producing strains under identical culture conditions and with cordycepin concentration in liquid broth (R2 = 0.9727) and in lyophilized mycelium powder (R2 = 0.9613) for CM1-1-1 under eight different sets of conditions. In addition, the cordycepin content in lyophilized mycelium powder measured by the Nile red method was linearly correlated with that determined by an HPLC method (R2 = 0.9627). In conclusion, neutral lipids in lipid droplets are required during cordycepin accumulation; these neutral lipids are potential biomarkers of cordycepin biosynthesis.To clarify the relationship between neutral lipid content and cordycepin accumulation in  Cordyceps militaris, has attracted considerable attention due to its diverse therapeutic potentials associated with anticancer, antitumor, anti-inflammatory, antimicrobial and antiviral effects [C. militaris.Cordycepin (3′-deoxyadenosine), a cytotoxic nucleoside analogue found in  effects ,2,3. CorC. militaris and Aspergillus nidulans), the Cns1 and Cns2 proteins, responsible for cordycepin biosynthesis, were determined to be colocalized on a type of organelle in Cordyceps militaris using GFP. Then, the Nile red method was employed to demonstrate that these organelles were lipid droplets in the mycelium of C. militaris [The biosynthetic mechanism of cordycepin in purine biosynthesis has attracted widespread interest in the past seven decades since the discovery of cordycepin in 1950 . Notablyilitaris , suggestLipid droplets, spherical cellular organelles consisting of a neutral lipid core enclosed by a phospholipid monolayer membrane covered with many multifunctional binding proteins, are involved in multiple intracellular processes, such as protein storage and degradation and nucleic acid processing ,7. The nThe relative fluorescence intensity of Nile red-stained smooth muscle cells was first used as a reliable indicator to quantify neutral lipid content , and theC. militaris starting strains after LiCl+60Coγ compound mutagenesis. Lyophilized mycelium powder (LMP) was used as the original material to optimize the Nile red method to investigate the quantitative relationship between the cordycepin concentration in both liquid broth and LMP and the corresponding relative fluorescence intensity (RFI) from Nile red-stained LMP in cordycepin-producing fungi.In this study, fifteen mutant strains producing different levels of cordycepin were obtained from two After exposure to different concentrations of LiCl, mutants were obtained from 12 different groups. The LiCl concentration was screened by lethality and mutation frequency A. Accord60Coγ irradiation, ten groups of mutants were obtained from the mutant mixed spore suspensions (MSSs) (60Coγ irradiation) were preliminarily screened according to the inhibition diameter of each mutant in antimicrobial tests  B. The leal tests B. FifteeUnder a multiscan spectrum with an excitation wavelength of 490 nm and emission wavelength of 550–650 nm, RFI from Nile red-stained LMP of the mutant CM1-1-1 was markedly distinguished from that of unstained LMP and that of Nile red, showing a similar emission peak at 598 nm. The fluorescence intensities from Nile red and unstained LMP were rather low. Therefore, a 490 nm excitation wavelength and a 598 nm emission wavelength were applied in the following experiments.−5 mg/mL, and rapidly decreased when the final LMP concentration was greater than 8 × 10−5 mg/mL, indicating that a final concentration of at most 8 × 10−5 mg/mL LMP should be used when the final concentration of Nile red reached 3.2 µg/mL (2 = 0.9514) (2 = 0.9378) , cordycepin concentration in liquid broth (2363.278 µg/mL) and cordycepin content in LMP (24.14 mg/g) among the 18 strains, was selected for subsequent experiments.The effects of different LMP concentrations of the 18 selected strains on the RFI were investigated A. The RF.2 µg/mL A. Notabl 0.9514) B and cor 0.9378) C when th−5 mg/mL.The effect of different final concentrations of Nile red  on the RFI from Nile red-stained LMP of mutant CM1-1-1 was investigated. −5, 2 × 10−5, 3 × 10−5, 4 × 10−5, 5 × 10−5, 6 × 10−5, 7 × 10−5 and 8 × 10−5 mg/mL) on the RFI from Nile red-stained LMP of mutant CM1-1-1 were investigated by mixing 120 µL of LMP suspension at different concentrations with 30 µL of Nile red solution to a final concentration of 3.2 µg/mL. −5~8 × 10−5 mg/mL, which was quantitatively expressed as y = 41.905x − 8.571 .The effects of different final concentrations of LMP (1 × 10v/v) on the RFI from Nile red-stained LMP of mutant CM1-1-1 was investigated   was individually pretreated with DMSO at different volume fractions  before fluorescence intensities were determined. The RFI slowly increased with increasing DMSO volume fraction, peaked when the DMSO volume fraction was 10% and slightly decreased when the DMSO volume fraction was greater than 10% (The effect of different volume fractions of dimethyl sulfoxide (DMSO)  (stigated C. To maithan 10% C. Theref−4 mg/mL), mixed with 10% (v:v) DMSO to 1 × 10−4 mg/mL, and reacted in darkness at 30 °C for 10 min. Then, 120 μL of LMP suspension was mixed with 30 µL of Nile red solution at a final concentration of 3.2 µg/mL and thoroughly vortexed for 5 min at 30 °C prior to the determination of fluorescence intensities. 2 = 0.9727). 2 = 0.9613).Eight samples of LMP harvested under different culture conditions (#1–#8) were individually diluted in PBS (1.11 × 102 = 0.9627).Single colonies of strain #1, strain #2, CM1-1-1, CM1-2-5 and CM1-3-3, prepared using the method described in 60Coγ compound mutagenesis. The RFI values from the Nile red-stained LMP of these cordycepin-producing strains were linearly correlated with the corresponding cordycepin concentration in liquid broth (R2 = 0.9332) when the LMP final concentration was 8 × 10−5 mg/mL. The mutant CM1-1-1, with the highest RFI from Nile red-stained LMP among the eighteen strains, was selected for optimizing the Nile red method, and the optimal method for RFI determination can be described as follows: 120 μL of LMP suspension, which was obtained by mixing LMP suspension prepared in PBS (1.11 × 10−4 mg/mL) with 10% DMSO (v/v) prior to a reaction in darkness at 30 °C for 10 min, was mixed with 30 µL of Nile red solution at final concentration of 3.2 µg/mL and thoroughly vortexed for 5 min at 30 °C prior to the determination of fluorescence intensities. Using this optimal method, the cordycepin concentration in liquid broth (R2 = 0.9727) and cordycepin content in LMP (R2 = 0.9613) were demonstrated to be linearly correlated with the corresponding RFI from Nile red-stained LMP of eight samples of mutant CM1-1-1 harvested under different culture conditions. In addition, the cordycepin content in LMP measured by the optimized Nile red method was linearly correlated to that determined by an HPLC method using five strains cultured for different times (R2 = 0.9627). In conclusion, in 18 cordycepin-producing fungi, neutral lipids in lipid droplets are required during cordycepin accumulation and considered potential biomarkers of cordycepin biosynthesis.In our study, an optimized methylcellulose-based medium, a type of semisolid medium, was first used for isolating mutant spores of fungi. In brief, a fungal spore suspension was piped into optimized semisolid methylcellulose medium, and the medium thoroughly shaken to homogeneously disperse these spores. Then, the relative positions of these spores in optimized semisolid methylcellulose medium were fixed during static cultivation, and these spores formed single colonies and were separately transferred into downstream medium in 96-deep-well plates using a pipette with a 1000 μL tip. Notably, compared to traditional methods for isolating single fungal spores, such as the spread plate method, this novel method is simple, convenient and efficient for several reasons: (a) optimized semisolid methylcellulose medium is liquid at 0~70 °C, which is suitable for the growth of fungi; (b) pipette operation is simple, convenient and efficient; and (c) single spores can form single colonies inside the medium and on the surface of the medium. In addition, LMP of fungi was first used as the original material to measure neutral lipid content in lipid droplets using the Nile red method, and the quantitative relationships between the cordycepin concentration in liquid broth, cordycepin content in LMP, and corresponding RFI from Nile red-stained LMP were investigated. Eighteen stable mutants (CM1-1-1~CM1-3-5), individually producing from 0.001 (CM1-3-3) to 2.363 (CM1-1-1) mg/mL cordycepin, were finally obtained after LiCl+Cordycepin is a self-toxic component in cordycepin-producing fungi. These fungi can develop the capacity to resist and detoxify this toxic compound. 3′AMP is potentially formed and transferred into lipid droplets, and then cordycepin is biosynthesized by the two enzymes Cns1 and Cns2, which are colocalized on lipid droplets . Our datC. militaris mutant G81-3 produced abundant cordycepin in the mycelial stage by liquid surface culture. The over saturation of cordycepin resulted in crystallization when the cordycepin concentration in liquid broth exceeded approximately 7.4 g/L at 25 °C. The maximum capacity of cordycepin production reached 14.3 g/L through a decrease in the beginning substrate level and a water-supplementing strategy [C. militaris; during this time, signal transduction is markedly more active than it is in mycelia [C. militaris cells.In addition, the multiple functions of lipid droplet-associated proteins may illuminate the positive effect of lipid droplets on cordycepin accumulation. Since lipid droplet-associated proteins vary substantially in different fungi, lipid droplet-resident proteins are involved in different signals, such as those involving signal peptides, posttranslation modification and nucleic acids, which may help these proteins localize lipid droplets . Notablystrategy . Then, s mycelia . As a reC. militaris CGMCC 3.4655 (strain#2), Bacillus subtilis CGMCC 1.1849 and A. nidulans CGMCC 3.15737 (strain#3) were obtained from the China General Microbiological Culture Collection Center . C. militaris CICC 14014 (strain #1) was obtained from the China Center of Industrial Culture Collection . C. militaris and A. nidulans were maintained on PDA and YPD slants, respectively. Bacillus subtilis was cultured on nutrient agar (NA) medium. Basic liquid medium (g/L): glucose, 34; peptone, 17; KH2PO4, 1; and MgSO4·7H2O, 1. Optimized semisolid medium (g/L): basic liquid medium; methylcellulose, 23. Medium #A (g/L): glucose, 16; peptone, 7; KH2PO4, 1; and MgSO4·7H2O, 1. Medium #B (g/L): glucose, 6; peptone, 2; KH2PO4, 1; and MgSO4·7H2O, 1.Nile red, DMSO and methylcellulose (viscosity: 4000 MPa·s) were obtained from Shanghai Sangon Biotech Limited Corporation . Nile red solution: 0.1 mg per 1 mL of acetone, was diluted with acetone to the appropriate concentration just prior to each assay. Cordycepin was obtained from the National Institutes for Food and Drug Control . LiCl was obtained from Sinopharm Chemical Reagent Limited Corporation . Methylene blue solution (0.1%) was obtained from Beijing Solarbio Science and Technology Co., Ltd. .6 living conidia/mL and examined by methylene blue staining, optical microscopy  and hemocytometry. MSSs of strain #1 and strain #2 were prepared by mixing the same volume of each suspension.Spore suspensions of strain #1 and strain #2, which had markedly different cordycepin-producing capabilities, were individually diluted with sterile PBS (pH 7.2) to 1 × 10Mutant MSSs were appropriately diluted with sterile PBS, transferred into 20 mL of optimized semisolid medium in a 50 mL Erlenmeyer flask, fully shaken to homogeneously blend the spores, and incubated statically in darkness at 25 °C for 72 h. The relative positions of mutant MSSs in optimized semisolid medium were fixed during static cultivation. Variable isolates were transferred into downstream medium using a pipette with a 1000 µL tip. Lethality was calculated with the equation below by observing different isolates in optimized semisolid medium.60Coγ compound mutagenesis and screening of stable mutants). Antimicrobial tests were performed with B. subtilis [Eight random isolates under each mutation condition were separately transferred into 1.8 mL of basic liquid medium per well in 96-deep-well plates and incubated statically at 25 °C in darkness for 10 d (LiCl mutation) or 30 d (LiCl+subtilis . Eight sUsing Equations (1)–(4) the mutants which showed an inhibition diameter over 20% larger than that of strain #1 were defined as positive mutants, and the mutants with a 20% or smaller inhibition diameter than strain #2 were considered negative mutants. NB is the number of single colonies before mutagenesis, NA is the number of single colonies after mutagenesis, PMF is the positive mutant frequency, NEC is the number of positive mutant colonies, NVC is the number of viable colonies, NMF is the negative mutant frequency, IMF is the intermediate mutant frequency and NDC is the number of negative mutant colonies.w/v) prepared in sterile PBS and cultured in darkness at 25 °C for 12 h. Lethality assays and antimicrobial tests were performed, and the optimal concentration of LiCl was selected according to lethality and mutant frequency.According to the modified mutation method using LiCl  that sti60Coγ ray [60Coγ ray  at different doses  for 15 min. Positive, intermediate and negative mutants under each dose of 60Coγ irradiation were preliminarily screened according to their inhibition diameters being obviously different from those of their initial strains in antimicrobial tests. Throughout six d of cultivation on PDA slants for six generations, 15 stable mutants were individually cultured at 25 °C for 30 d, selected by comparing the inhibition diameters of liquid broth obtained from mutants of the first generation with those of the corresponding mutants of the sixth generation using antimicrobial test method as described in According to the modified mutation method under 0Coγ ray , which m0Coγ ray , after tv/v), flow rate: 1 mL/min, 35°C, injection volume: 20 µL, and detection wavelength: 260 nm. The mycelia of these strains were used for determining fluorescence intensities.A single colony of each selected strain , prepared using the method described in The mycelia of the stable mutants, strain #1, strain #2 and strain #3 were filtered with three-layer gauze, washed with pure water three times, frozen in a cryogenic refrigerator  at −80 °C for 12 h, dried in a vacuum freeze dryer  for 28 h, ground into LMP in a mortar, and then filtered through a 90 mesh sieve. The mortar and sieve were washed with 95% ethanol three times prior to the next LMP being treated. According to a previously described method , water eLMP (10 ± 0.1 mg) was weighed with an electronic balance  and diluted with 10 mL of PBS prior to each assay. Then, 120 µL of LMP suspension was mixed with 30 μL of Nile red solution at a final concentration of 3.2 μg/mL. Treated LMP was agitated on a 96-well plate oscillator vigorously for 5 min before determining the fluorescence intensities on a microplate reader  with a 490 nm excitation wavelength and a 598 nm emission wavelength. The RFI was calculated after subtracting the self-fluorescence of Nile red and the fluorescence intensity of the LMP suspension at 598 nm.−1, 1 × 10−2, 1 × 10−3, 1 × 10−4, 1 × 10−5 and 1 × 10−6 mg/mL) were mixed with 30 µL of Nile red solution at a final concentration of 3.2 µg/mL and vortexed vigorously in darkness at 30 °C for 5 min [For optimization, 120 µL of LMP suspensions at seven different concentrations  at different final concentrations  and vortexed vigorously at 30°C in darkness for 5 min prior to determining the fluorescence intensity.−4 mg/mL) were individually mixed with 30 µL of Nile red solution at the optimal final concentration and vortexed vigorously at 30 °C in darkness for 5 min before the determination of the fluorescence intensity.Then, 120 µL of LMP suspensions of the selected strain at eight different concentrations  and reacted in darkness at 30 °C for 10 min. For this purpose, 120 µL of pretreated LMP suspension was mixed with 30 µL of Nile red solution at the optimal final concentration and vortexed vigorously in darkness at 30 °C for 5 min before determining fluorescence intensity.To maintain the optimal final concentration of LMP, LMP suspensions were individually mixed with a volume ratio of DMSO  . Statistical differences at the 0.05 level using the least significant difference (LSD) method were considered significant.


MSCs were isolated from dental tissues  following digestion method and were analyzed for the expression of pluripotent markers and cell surface markers. All three types of MSCs were evaluated for their potential to differentiate into mesenchymal lineages. Further, the MSCs were differentiated into pancreatic β cell-like cells using multistep protocol and characterized for the expression of pancreatic lineage specific markers. Functional properties of differentiated pancreatic β cell-like cells were assessed by dithizone staining and glucose challenge test. All three types of MSCs showed fibroblast-like morphology upon culture and expressed pluripotent, and mesenchymal cell surface markers. These MSCs were successfully differentiated into mesenchymal lineages and transdifferentiated into pancreatic β cell-like cells. Among them, dental follicle derived MSCs exhibits higher transdifferentiation potency toward pancreatic lineage as evaluated by the expression of pancreatic lineage specific markers both at mRNA and protein level, and secreted higher insulin upon glucose challenge. Additionally, follicle-derived MSCs showed higher dithizone staining upon differentiation. All three types of MSCs from a single donor possess similar cellular properties and can differentiate into pancreatic lineage. However, dental follicle derived MSCs showed higher potency toward pancreatic lineage than pulp and papilla derived MSCs, suggesting their potential application in future stem cell based therapy for the treatment of diabetes.The present study was carried out to investigate and compare the  Diabetes mellitus (DM) is one of the devastating disease characterized either by absolute insulin deficiency due to the destruction of β cells Type 1) or by relative insulin deficiency due to the reduced insulin sensitivity in peripheral cells Type 2)  assay. In brief, cells were seeded at a density of 9 × 10MSCs derived from human dental pulp, papilla, and follicle tissues were analyzed for cell surface marker’’s  expression using flow cytometer  as previously described . In brie6 cells per ml were fixed in 70% ethanol at 4°C for 4 h. Cells were then washed twice with DPBS and stained with 10 μg/ml propidium iodide (PI) solution for 15 min. The DNA content was measured and categorized as G0/G1, S, or G2/M phase of the cell cycle.For cell cycle analysis, a total of 1 × 10Human dental pulp, papilla, and follicle tissues derived MSCs were evaluated for their differentiation potency toward mesenchymal lineages by following previously published protocol . In brieMSCs derived from human dental pulp, papilla, and follicle tissues were differentiated into insulin producing pancreatic β cell-like cells as described previously with minor modifications . In brieStep I: After reaching 70% confluence, cells were cultured in DMEM containing 10% FBS and 25 mM glucose supplemented with 1 μM retinoic acid for 24 h.Step II: After induction, cells were cultured for another 2 days in DMEM containing 10% FBS and 25 mM glucose, then dissociated with 0.25% trypsin-EDTA solution.Step III: Dissociated cells were plated on to a six-well plate precoated with Geltrex LDEV-free membrane matrix and cultured in DMEM containing 10% FBS and 5.6 mM glucose, supplemented with 10 mM nicotinamide, 10 ng/ml epidermal growth factor (EGF) and 300 nM indolactam V for 9 days. The media were changed for every 3 days interval during differentiation.Step IV: The media were changed to DMEM containing 10% FBS and 5.6 mM glucose, supplemented with 10 mM Exendin-4 and 50 ng/ml Activin A for 7 days. The media were changed for every 3 days interval during differentiation.Control cells were maintained in parallel using DMEM containing 10% FBS and 25 mM glucose.Upon pancreatic differentiation of MSCs, both control and differentiated cells were analyzed for their ability to secrete insulin upon glucose challenge following previous protocol with minor modifications ,29. In bUpon pancreatic differentiation of MSCs, both control and differentiated cells were analyzed for the presence of intracellular zinc by staining with dithizone. In brief, a stock solution of dithizone was prepared by dissolving 100 mg of dithizone in 5 ml of DMSO. Both control and differentiated cells were washed twice with DPBS and stained with 10 μl dithizone stock in 1 ml DPBS solution at 37°C for 15 min. The stained cells were examined under a phase-contrast microscope (Nikon) for crimson-red staining and images were taken.The expression of pluripotent marker genes was analyzed by PCR, and the fold change in the expression of mesenchymal lineage specific genes and pancreatic lineage specific genes was evaluated by RT-qPCR in triplicates from three independent experiments. Total RNA was isolated using easy-spin™ [DNA free] total RNA extraction kit (iNtRON Biotechnology). A total of 2 μg RNA was used to synthesize complementary DNA (cDNA) using HiSenScript™ RH[-] RT PreMix Kit (iNtRON Biotechnology) following reaction in three steps; step I: 42°C for 50 min, step II: 85°C for 10 min, step III: End. The PCR reaction containing 2 μl cDNA, 1 μl each of forward and reverse primers of 10 μM concentration and 16 μl of RNase free water was carried out using Maxime PCR Premix (iNtRON Biotechnology). The PCR reaction was carried out in following conditions: initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55–60°C for 20 s, extension at 72°C for 30 s and final extension at 72°C for 10 min, using Mastercycler® pro . The PCR products were analyzed by agarose gel electrophoresis.−∆∆CT method. The specificity of primers was analyzed by their product length on agarose gel. The primers used in the present study are listed in The Real-time qPCR was carried out in Rotor gene Q (Qiagen) using RealMOD™ Green AP 5× qPCR mix (iNtRON Biotechnology). Each reaction mix with a final volume of 25 μl consisted of a total of 50 ng cDNA, 5 μl qPCR mix, 13 μl RNase free water, and 1 μl each of forward and reverse primers at 400 nM final concentration. The reaction was carried out with an initial denaturation at 95°C for 10 min, followed by 40 PCR cycles of 95°C for 10 s, annealing (Primer specific temperature) for 6 s, and 72°C for 4 s, followed by a melting curve from 60°C to 95°C at 1°C/s and then cooling at 40°C for 30 s, according to manufacturer’s instruction. The threshold cycle (CT) values and melting curves of each sample were analyzed using Rotor-Gene Q series software (Qiagen). YWHAZ  was used as housekeeping gene for normalization of the data. The relative mRNA abundance was calculated according to the 2The expression of pancreatic lineage specific marker proteins was evaluated by immunocytochemistry. In brief, the differentiated pancreatic β cell-like cells and INS-1 rat insulinoma cells were fixed with 3.7% formaldehyde for 30 min and permeabilized with 0.25% Triton X-100 for 10 min at room temperature. After thorough washing with DPBS, cells were blocked with 1% BSA for 1 h and incubated with primary antibodies such as rabbit anti-PDX1 , rabbit anti-NGN3 , mouse anti-Arx , rabbit anti-PAX4 , mouse anti-MafA , rabbit anti-Glut2 , mouse anti-C peptide  or mouse anti rat C peptide , mouse anti-Glucagon , goat anti-Somatostatin , and mouse anti-Insulin  or mouse anti-Insulin  for overnight at 4°C. Cells were then washed twice with DPBS and incubated with CruzFluor 488 conjugated goat anti-rabbit IgG , or CruzFluor 488 conjugated donkey anti-goat IgG , or CruzFluor 594 conjugated donkey anti-rabbit IgG , or CruzFluor 594 conjugated donkey anti-mouse IgG , or FITC conjugated donkey anti-mouse IgG  for 1 h at 37°C. The nuclei were stained with DAPI at a concentration of 1 μg/ml for 5 min and the images were captured using fluorescence microscope .The expression of pluripotent marker proteins in MSCs derived from dental pulp, papilla, and follicle tissues was evaluated by Western blotting. In brief, the lysates of protein were prepared from cultured MSCs using RIPA buffer  containing protease inhibitors. The concentration of protein was measured using Microplate BCA Protein Assay kit  and a total of 25 μg protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The separated proteins were then transferred onto polyvinylidene difluoride membranes  and membranes were incubated with primary antibodies such as rabbit anti-OCT4 , rabbit anti-SOX2 , rabbit anti-NANOG , and mouse anti-GAPDH  for overnight at 4°C. Then membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG , and goat anti-mouse IgG  secondary antibodies for 2 h at room temperature. The immunoreactivity was detected in dark condition by adding enhanced chemiluminescence  and exposed to X-ray films.P<0.05 is considered as significant difference.The statistical differences between experiments groups were analyzed by one-way ANOVA using IBM SPSS 21.0. Tukey’s test was performed for multiple comparisons and data were presented as a mean ± standard error of the estimate of mean value (S.E.M) for each sample analyzed in triplicates obtained from three independent experiments. The value The MSCs isolated from human dental pulp, papilla, and follicle tissues exhibited adherent fibroblast like spindle morphology that become homogeneous at passage 3 upon sub culturing . The pulRUNX2), osteonectin ON, and bone morphogenetic protein 2 (BMP2) showed significant (P<0.05) increase when compared with undifferentiated controls (γ (PPARγ), fatty acid binding protein 4 (FABP4), and lipoprotein lipase (LPL) was found to be significantly (P<0.05) increased when compared with undifferentiated controls (SOX9), aggrecan (AGCAN), and collagen II (COL II) was found to be significantly (P<0.05) increased when compared with undifferentiated controls , NK6 homeobox 1 (NKX6.1), neurogenin 3 (NGN3), aristaless-related homeobox (ARX), paired box 4 (PAX4), insulin (INS), solute carrier family 2 member 2 (GLUT2), MAF bZIP transcription factor A (MAFA), glucagon (GCG), and somatostatin (SST) were found to be significantly (P<0.05) increased after differentiation when compared with control undifferentiated cells, and the relative fold change in expression levels was 3.75 ± 1.6, 4.96 ± 1.2, 6.53 ± 2.0 and 1.69 ± 1.6, 1.9 ± 2.4, 1.86 ± 2.6 and 7.0 ± 2.8, 7.68 ± 1.4, 7.05 ± 2.2 and 9.66 ± 2.0, 10.21 ± 2.4, 11.91 ± 2.0 and 10.4 ± 2.2, 11.71 ± 2.6, 11.08 ± 1.2 and 10.65 ± 1.6, 10.83 ± 1.8, 12.59 ± 2.0 and 14.4 ± 1.8, 14.23 ± 2.8, 14.15 ± 2.0 and 10.97 ± 3.0, 10.91 ± 2.8, 11.18 ± 1.6 and 13.63 ± 2.4, 14.36 ± 2.0, 15.92 ± 3.4 and 17.41 ± 1.8, 17.5 ± 2.0, 14.37 ± 2.6, respectively, in pulp, papilla, and follicle tissues derived differentiated MSCs  treatment (P<0.05) higher insulin upon glucose challenge when compared with pulp, and papilla-derived MSCs. Nevertheless, differentiated cells were positively stained with dithizone cytochemical solution , cells from passage 3 were induced to pancreatic lineage. After 19 days of induction, change in morphology from fibroblastic to spherical was observed and cells tended to form clumps when compared with control undifferentiated counterparts , though ted MSCs A,B. Furtted MSCs A. The exreatment B. Follicsolution C, demonsin vitro differentiation into mesenchymal lineages and pancreatic lineage.The present study was conducted to compare the multipotency of three types of dental MSCs isolated from a single donor tooth. MSCs isolated form dental pulp, papilla, and follicle tissues were compared on the basis of their morphology, proliferation, expression of cell surface markers and pluripotent transcription factors, As dental tissue is considered a biomedical waste in routine dental procedures, it is gaining more attention in the field of regenerative medicine as a potential source of MSCs without imposing any ethical issues. Many dental-derived MSCs have been identified and characterized, including MSCs from human exfoliated deciduous teeth (SHED) , dental All three types of MSCs showed plastic adherent fibroblast-like morphology when cultured under identical conditions. MSCs of dental tissue possess more clonogenic as well as proliferative potentials than BMSCs ,33. Our Present study also demonstrated that MSCs from dental pulp, papilla, and follicle tissues were successfully differentiated into mesenchymal lineages such as osteocytes, adipocytes, and chondrocytes under appropriate culture conditions. Differentiated cells also expressed lineage specific markers and positively stained for lineage specific staining. Pulp and follicle tissue derived MSCs exhibited higher differentiation potential toward mesenchymal lineages when compared with papilla-derived MSCs and these findings are consistent with previous report . The preWe further assessed the transdifferentiation potential of dental pulp, papilla, and follicle tissues derived MSCs toward pancreatic β cell-like cells. Previous studies have demonstrated the ability of various human stem cells including dental MSCs to differentiate into pancreatic lineages with the use of different cytokines, growth factors, and extracellular matrix components ,12–14. HMesenchymal stem cells isolated from dental pulp, papilla, and follicle tissues exhibited largely similar features related to morphology, proliferation, expression of various cell surface, intracellular and pluripotent markers, and differentiation toward mesenchymal lineages such as osteocytes, adipocytes, and chondrocytes. Further, all three types of MSCs displayed greater ability to transdifferentiate into pancreatic β cell-like cells. Nevertheless, differentiated cells were positively stained for dithizone and secreted insulin upon glucose challenge. However, the expression of pancreatic lineage specific markers and insulin secretion assay revealed that follicle-derived MSCs are prominent sources for cell-based diabetic therapy. When compared with other tissue sources of MSCs, the findings of the present study are particularly very interesting when we considered an extracted tooth samples sourced from routine medical waste. To our knowledge, this is the first kind of study in which we compare the pancreatic lineage differentiation potential of MSCs from three different regions of dental tissue from a single donor and identify a potent MSCs source for the generation of pancreatic β cell-like cells.


Background and Objective: In Wilson’s disease, copper metabolism is impaired due to defective copper transporting protein ATP7B, resulting in copper accumulation in liver and brain and causing damage to liver and brain tissues. Published data suggest that one of the possible treatments for Wilson’s disease is curcumin—a compound found in the root of Curcuma longa. In this study, we tested whether curcumin affects copper transport and excretion in HepG2 hepatocytes carrying wildtype ATP7B. Materials and Methods: We examined the impact of 5 µM and 25 µM curcumin on the transport of copper in HepG2 cells incubated with 20 µM and 100 µM CuCl2, as well as copper excretion from cells. First, immunofluorescent staining and co-localization analysis were carried out in HepG2 cells using confocal laser scanning microscope and Nikon NIS Elements software. Second, a concentration of copper extracted into cell culture medium was determined using atomic absorption spectrometry. Results: The analysis of the co-localization between Golgi complex and ATP7B revealed that both 5 µM and 25 µM doses of curcumin improve the ability of liver cells to transport copper to plasma membrane at 20 µM CuCl2, but not at 100 µM CuCl2 concentration. However, atomic absorption spectrometry showed that curcumin rather promotes copper absorption into liver cell line HepG2 than excretion of it. Conclusions: Curcumin accelerates the transport of copper within liver cells, but does not promote copper excretion from HepG2 cells. Copper is an important microelement necessary for the functioning of several cellular processes ,2,3,4. HHeavy metal transport protein ATP7B ,12 playsATP7B gene cause Wilson’s disease [Mutations in  disease ,18, a ge disease . Several disease ,20,21. I disease ,6,7. The disease ,22.trans part of Golgi complex [trans Golgi complex where copper binds with ceruloplasmin; and (2) it is involved in copper excretion through gall [ATP7B is an essential protein for the maintenance of copper homeostasis and excretion of copper excess from a body. In a cell, ATP7B protein is located in the  complex ,23. The ugh gall ,10. To pugh gall .trans Golgi complex to vesicles where copper is stored temporarily. When copper levels exceed 20 µM concentration, the excretion of vesicles containing copper and ATP7B takes place at the apical surface of hepatocytes, and copper is excreted through gall [Upon the elevation of copper levels in a cell, ATP7B transports it from ugh gall .Curcuma longa plant [Curcumin is a hydrophobic polyphenol compound extracted from the roots of ga plant ,27. Curcga plant ,29; howega plant ,31.Zhang et al.  have fouThe aim of this study was to verify whether the experimental settings described by Zhang et al.  would all-Lysine-coated  glass coverslips in MEM cell culture medium supplemented with 10% FCS, glutamine (2 mM), penicillin and streptomycin  until they reached 90% confluence. After that, they were pre-treated with 25 µM or 5 µM curcumin  for 24 h and then incubated with 20 µM or 100 µM CuCl2  for 4 h before immunostaining .2 flasks  until 90% confluence, then treated with 25 µM or 5 µM curcumin for 24 h and then incubated with 100 µM or 20 µM CuCl2 for additional 24 h, respectively. After that, cells were washed with PBS and fresh MEM medium added. Samples for atomic absorption spectrometry measurements were collected after 1.5, 3, 4.5, 6, 7.5 and 9 h and cells counted using Bürker chamber and standard Trypan Blue  staining for evaluation of dead cell proportion at the end of the experiment. Before temporary freezing, medium samples were centrifuged to remove cell debris.For copper measurements, cells were cultured in 25 cmCells were fixed with 4% formaldehyde , immunostained with primary antibodies  at 1:1000 dilution and rabbit α ATP7B  at 1:500 dilution), and incubated overnight at +4 °C. Secondary polyclonal antibodies included Goat α Mouse IgG (H + L), Alexa Fluor 488  and Donkey α Rabbit IgG (H + L), Dylight 550 , diluted at 1:500, and were incubated for 1 h at room temperature. Fluoroshield mounting medium with DAPI  was used.The samples were analyzed with Nikon A1R+  confocal microscope using lasers with 405 nm, 488 nm and 561 nm wavelengths. A 100× oil immersion objective with numerical aperture 1.45  was used.n = 3 was considered the minimal number of replication for data analysis.NIS Elements  software was used for co-localization analysis. The co-localization analysis between Golgi complex marker p230 ant ATP7B was carried on following method described by Manders . ResultsThe analysis was performed using atomic absorption spectrometer Varian Spectra AA 200 Z  with graphite furnace and electrothermal atomizers as better described in ,35. Zeemp-Value less than 0.05 (p < 0.05) was considered as statistically significant.All the graphs, calculations, and statistical analyses were performed using GraphPad Prism software version 7.0 for Mac . To test whether the collected numerical data are normally distributed, the D’Agostino and Pearson and Shapiro-Wilk normality tests were applied. The comparison of means between different groups of numerical variables was performed using one-way or two-way ANOVA. Homogeneity of variances was tested using Brown-Forsythe and Bartlett’s tests. If data were not normally distributed, the comparison of medians between different groups was switched to non-parametric one-way ANOVA on ranks or Kruskal-Wallis test followed by post-hoc analysis. Changes of measured copper and differences among the groups were assessed with RM two-way ANOVA (considering two main sources of variation—time and treatment) followed by two-stage step-up method of Benjamini, Krieger and Yekutieli as post-hoc test. Influence of curcumin was assessed by Multiple t tests with correction for multiple comparisons using Holm-Sidak method. Results are expressed as median and interquartile range (IQR) as dispersion characteristic. n = 14) detected in not exposed cells to 0.11 (0.06–0.17) (n = 25) and 0.06 (0.04–0.12) (n = 20) at 20 μM and 100 μM CuCl2, respectively .We performed a co-localization study between ATP7B and p230 to test the translocation of ATP7B away from Golgi complex in HepG2 cells upon copper treatment. First, we tested the basic effect of increasing copper concentrations on translocation of ATP7B . The co-2-treated HepG2 cells. We registered significantly different (0.05 (0.04–0.09) co-localization, n = 20, vs. 0.11 (0.06–0.17), n = 25, p = 0.013, 2 concentration of 20 μM (0.09 (0.05–0.11), n = 8, vs. 0.06 (0.04–0.12) (n = 20), p = 0.97, We then tested the effect of 25 μM curcumin treatment on the 20 μM and 100 μM CuCl2 when pre-incubated to 5 μM curcumin (0.23 (0.21–0.29), n = 10, vs. 0.45 (0.31–0.64), n = 9, p = 0.0187, After that, we decided to test the effect of a lower curcumin concentration (5 μM) on 20 μM copper-treated HepG2 cells since it had been shown as a relevant concentration at which curcumin could start to affect ATP7B translocation. In this case, we also observed significant decrease in ATP7B co-locallization with Golgi complex in cells exposed to 20 μM CuCl2 with or without 5 µM curcumin pre-incubation (2, we observed rapid excretion of copper as measured 1.5 h after cell washing and medium change, after which the concentration of copper gradually decreased in the medium (To evaluate the potential curcumin effect on copper excretion from hepatocytes, we performed atomic absorption spectrometry measurements in samples collected after incubation of cells exposed to 100 or 20 µM CuClcubation . When cee medium . At all e medium .2-treated samples, a smaller increase in copper levels was observed right after the medium change which dropped until 6 h but then started to increase again (In the 20 µM CuClse again . Moreovese again .Our observation of ATP7B translocation away from Golgi complex to cytoplasmic vesicular compartment upon elevated copper levels  is consiAt first, we used relatively high curcumin dose, 25 μM . The res2-treated cells at a concentration of 5 μM. We demonstrated that, even at 5 μM dose, curcumin can improve ATP7B-mediated copper transport in cells making it suitable for testing in vivo, followed by clinical trials in humans, since the dose is not cytotoxic.However, there are published data that curcumin concentration over 20 μM can cause cytotoxic effect ,43,44, t2 released copper into cell culture medium, which might be explained by medium change (2, copper re-absorption rate was similar in curcumin-treated and non-treated cells confirming that curcumin has no effect on cells treated with toxic copper levels (2-treated cells had stopped at 7.5 h, cells started to excrete copper again (To estimate the curcumin effect on copper excretion from liver cells, we performed repeated, time-controlled atomic absorption spectrometry measurements of copper in cell culture medium. We observed that HepG2 cells treated with both 100 and 20 μM CuClm change . In the r levels . In contr levels  as indicer again . SignifiDespite the lack of research on curcumin potential to influence copper transport in hepatocytes, a plethora of data has been collected on different biological properties of curcumin. A variety of curcumin properties has been listed in relation to different diseases ,47,48,49We suggest that, although curcumin has shown positive effect on stimulation of copper excretion from induced pluripotent and embryonic stem cell-derived liver-like cells , there iCurcumin at both 25 and 5 μM doses accelerates intracellular copper transport from Golgi complex to cytoplasm in HepG2 hepatocytes. This effect is observed at 20 μM, but not at the relatively toxic 100 μM copper level. In HepG2 cells, rather copper absorption than excretion takes place at 20 μM and 100 μM copper concentrations. Moreover, curcumin accelerates absorption of copper into HepG2 cells unless the cells have already absorbed toxic levels of copper.


To gain further insight into the mechanisms involved in the NPP1 downregulation induced starch overaccumulation, in this study we conducted photosynthesis, leaf proteomic, and chloroplast phosphoproteomic analyses of wild-type (WT) and npp1 plants cultured under HT + ECO2. Photosynthesis in npp1 leaves was significantly higher than in WT. Additionally, npp1 leaves accumulated higher levels of sucrose than WT. The proteomic analyses revealed upregulation of proteins related to carbohydrate metabolism and the protein synthesis system in npp1 plants. Further, our data indicate the induction of 14-3-3 proteins in npp1 plants. Our finding demonstrates a higher level of protein phosphorylation in npp1 chloroplasts, which may play an important role in carbohydrate accumulation. Together, these results offer novel targets and provide additional insights into carbohydrate metabolism regulation under ambient and adverse conditions.Rice nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides including ADP-glucose and ATP. Under high temperature and elevated CO Gas exchange of WT and npp1 leaves was recorded in the central segment of top leaves attached to the main culm between 3 and 8 h after the start of the photoperiod. Leaf cuvette conditions were set as follows: block temperature was set at ambient  and high (33 °C) temperatures; [CO2] was set at 400 to 2000 µmol·mol−1; relative humidity was maintained equal to that in the growth chamber; and Photosynthetically Active Radiation (PAR) was set at 1200 µmol·m−2·s−1, resulting in light-saturated photosynthesis and no decline as a result of photorespiration.Photosynthetic rate (v/v) acetone was assayed by the method described by Porra et al. [Sucrose content was measured according to the methods described previously ,68. Chloa et al. . Proteinnpp1 grown for 7 days under normal  and elevated temperature and CO2  conditions were ground in liquid nitrogen and suspended in 7 M urea, 2 M thiourea, 3% (w/v) CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate), 1% (v/v) Triton X-100, and 10 mM dithiothreitol. After centrifugation at 10,000× g at 4 °C for 5 min, the supernatants were mixed with 1/10 volume of 100% (w/v) TCA, incubated on ice for 15 min, and then centrifuged at 10,000× g at 4 °C for 15 min. The resulting precipitates were washed three times with ice-cold acetone and resuspended in 8 M urea. Two hundred milligrams of leaves of WT and http://www.uniprot.org/) O. sativa subsp. japonica database  with the following parameters: enzyme, trypsin; maximum missed cleavages site, 2; peptide charge, 2+ or 3+; MS tolerance, 5 ppm; MS/MS tolerance, ±0.5 Da; dynamic modification, carboxymethylation (C), oxidation , iTRAQ 4-plex . False discovery rates were <1%. The procedure of quantitative shotgun proteomic analysis was the same as previously described . The prohttps://repository.jpostdb.org/).The mass spectrometry proteomics data (JPST000338) have been deposited in the jPOST repository (2) and elevated temperature and CO2  conditions. Thirty grams of leaves were homogenized with an equal volume of solution A mixture consisting of 50 mM HEPES-KOH pH 7.5, 0.33 M sorbitol, 5 mM MgCl2, 5 mM MnCl2 and 5 mM EDTA (ethylenediaminetetraacetic acid), 50 mM sodium ascorbate, and then the homogenates were passed through four layers of gauze and four layers of Miracloth . The filtrate was layered onto an 80% (v/v) Percoll  cushion containing solution A, and centrifuged at 2000× g at 4 °C for 4 min. The crude chloroplasts on the Percoll surface were diluted with more than twice the volume of solution A, then layered onto a discontinuous density gradient consisting of 40% and 80% Percoll solutions. The gradient was centrifuged at 4000× g at 4 °C for 10 min. Intact chloroplasts enriched around the 40%/80% Percoll interface were collected and subjected again to the Percoll gradient centrifugation. Intact chloroplasts were diluted with five times the volume of solution A, and centrifuged at 2000× g at 4 °C for 4 min, followed by chlorophyll and protein extractions. The procedure of chloroplast isolation was essentially identical to the method described earlier ,71. Ricew/v) CHAPS, 1% (v/v) Triton X-100, and 10 mM dithiothreitol. After centrifugation at 10,000× g at 4 °C for 5 min, the supernatants were mixed with 1/10 volume of 100% (w/v) TCA, incubated on ice for 15 min, and then centrifuged at 10,000× g at 4 °C for 15 min. The resulting precipitates were washed three times with ice-cold acetone and resuspended in 8 M urea. The protein preparations (100 µg) were digested with 2% (w/w) endoproteinase Lys-C and trypsin in 25 mM NH4HCO3 and 0.8 M urea at 37 °C for 12 h. The reaction mixtures were dried on a Centrifugal Concentrator  and then dissolved in buffer A consisting of 60% (v/v) acetonitrile (ACN), 5% (v/v) glycerol, 0.1% (v/v) Trifluoroacetic acid (TFA). A MonoSpin TiO column  was pre-equilibrated with buffer B consisting of 80% (v/v) ACN, 0.1% (v/v) TFA, followed by buffer A, and the samples were centrifuged at 3000× g for 1 min each. The obtained peptide samples were applied to the tip column and centrifuged at 3000× g for 5 min. The flow-through fraction was applied to the tip column again. Subsequently, the tip column was washed three times with buffer A and centrifuged at 3000× g for 1 min. The binding phosphopeptides were eluted with 100 μL of 5% NH4OH with centrifugation at 1000× g for 5 min. After elution with 5% NH4OH, the tightly binding phosphopeptides were eluted with 100 µL of 1 M (w/v) bis-Tris propane with centrifugation at 1000× g for 1 min. The phosphopeptides eluted with 5% NH4OH solution were dried on a Centrifugal Concentrator and then dissolved in 5% (v/v) formic acid (FA). The phosphopeptides eluted with 1 M bis-Tris propane were acidified with 900 μL of 5% FA and then desalted using a MonoSpin C18 column .Analyses of phosphoproteins were carried out according to the method described by Fukuda et al. . Intact v/v) FA and 2% (v/v) ACN using a DiNa-A system . A linear gradient from 0% to 33% buffer D consisting of 0.1% FA and 80% ACN for 600 min, 33% to 100% D for 10 min and back to 0% D in 15 min was applied, and peptides eluted from the HiQ sil C-18 W-3 column were directly loaded on a MonoCap C18 High Resolution 2000 separation column. The separated peptides were introduced into a LTQ-Orbitrap XL mass spectrometer  with a flow rate of 300 nL·min−1 and an ionization voltage of 1.7–2.5 kV. The mass range selected for MS scan was set to 350–1600 m/z and the top five peaks were subjected to MS/MS analysis. The full MS scan was detected in the Orbitrap, and the MS/MS scans were detected in the linear ion trap. The normalized collision energy for MS/MS was set to 35 eV for collision-induced dissociation (CID).Each phospho-peptide fraction was loaded on a HiQ sil C-18 W-3 trap column with buffer C consisting of 0.1% (Operation of protein identification with software and database was carried out as described above . The phohttps://repository.jpostdb.org/).The chloroplast phosphoproteome data (JPST000462) have been deposited in the jPOST repository  cleaning enzyme. The molecular physiological phenotype of the npp1 mutant was further analyzed in the present study. Lower temperatures and changes in the transpiration rates of npp1 leaves were observed, indicating that the disruption of the NPP1 gene caused the stomatal opening of rice leaves. Furthermore, the analysis of the nA/Ci curve indicated that the enhancement of photosynthesis in npp1 resulted from multiple causes in addition to the stomatal conductance. The proteome of carbohydrate metabolism and protein synthesizing system in npp1 leaves was strongly upregulated by HT and ECO2. Furthermore, the protein phosphorylation status in npp1 chloroplasts was significantly higher than in WT chloroplasts. An increase in ATP in chloroplasts might be a key stimulus, because NPP1 preferentially hydrolyzes ATP. Judging from the overall results, we consider that the remarkable enhancement of plant growth and carbohydrate accumulation in npp1 mutant plants under HT and ECO2 conditions was a consequence of the non-controlled activation of photosynthesis and protein synthesis by loss of function of a fine-tuning enzyme NPP1.Previous investigations have revealed that


Upon addition of potassium pyroantimonate (PA) ligand, it was adsorbed on the GONRs surface to inhibit the catalysis to cause the RRS peak decreasing. When the analyte of Na+ was added, the coordination reaction between PA and Na+ took place to form the stable complexes of [Na2(PA)] to release free GONRs catalyst that resulted in the RRS peak increasing linearly. Accordingly, a new and sensitive RRS method for Na+ was established, with a linear range of 0.69-25.8 nmol/L and a detection limit of 0.35 nmol/L Na+.The gold nanoparticle reaction of HAuCl Zhu et al.  on the system can be excluded. The CH3CH2OH-Na+-PA-GO-H2O2-HAuCl4-VB4r system generated the characteristic nanogold peaks at 370 nm and 545 nm , the recovery was 96.5% to 98.2%, and relative standard deviation is 3.3% to 6.7% , a model of 3K-15 high- speed refrigerated centrifuge , a model of 79-1 magnetic stirrer with heating , a model of HH-S2 electric hot water bath , a model of Zetasizer Nano nanometer particle size and zeta potential analyzer , S-4800 field emission scanning electron microscope , and a model of SYZ-550 quartz subboiling distilled water  were used.A model of Hitachi F-7000 fluorescence spectrophotometer  by synchronous scanning technique with a volt of 400 v, excited slit and emission slit of 5 nm, emission filter of 1% T attenuator and 4 , 10 μmol/L VB4r, 10 mol/L H2O2, KMnO4(s), multiwall carbon nanotube , 50% CH3CH2OH, 50 mmol/L KOH, 129 mmol/L NaCl standard solution, 39.33 μmol/L sodium pyroantimonate solution (118 μmol/L potassium pyroantimonate solution and 129 mmol/L NaCl solution were mixed in a volume ratio of 1:2 according to the desired volume) were prepared. A 1.0 mg grapheme oxide (GO) was dissolved in 100 mL water with ultrasonic treatment to obtain 10 μg/mL GO solution. It was treating for 15 min by ultrasonic process before use. All reagents were of analytical grade and the water was doubly distilled.A 11.8 mmol/L potassium pyroantimonate (PA) solution: 1.5 g of potassium pyroantimonate  was added to a conical flask before 150 mL distilled water and 0.15 g KOH was added and boiled to dissolve. After cooling to room temperature, the solution was transferred into a 250 mL volumetric flask and added water to the mark. It was diluted 100 times for use. A 2.9 mmol/L HAuCl2SO4 before reacting for 1 h. Then 300 mg of KMnO4 was added to the solution, mixed well, and heated at 60°C for 2 h. Next, the product was poured into 200 mL of ice water containing 5 mL of 30% H2O2. Then the solution was ultrasonicated for 10 min before centrifuging at 7000 rpm for 10 min. The supernatant was taken and the manganese dioxide generated in the reaction was removed. The final concentration of GONRs solution was 238 μg/mL. It was neutralized with 50 mmol/L NaOH and then diluted to the desired concentration before use.  .GONRs were prepared by the dissociating of MWCNTs. 50 mg MWCNT powder was added to a 50 mL round bottom flask containing 10 mL of concentrated HμL 50% CH3CH2OH, 50 μL 118 μmol/L PA and a certain amount of NaCl standard solutions were added to a 5 mL marked test tube, mixed well, and reacted for 10 min at room temperature. Then a 100 μL 238 ng/mL GONRs, 100 μL 2.9 mmol/L HAuCl4, and 50 μL 0.1 mmoL/L H2O2 solutions were added before diluting to 1.5 mL with water. The mixed solution was placed in a 60°C water bath for reacting 20 min and the reaction was terminated by tap water. The mixture was transferred into a quartz cell, and its RRS spectra were recorded. The RRS intensity I at 550 nm and the blank I0 without Na+ were recorded, and ΔI = I–I0 was calculated.A 100 + with CAP to control the GONR catalysis of the reduction of HAuCl4 with H2O2 to produce gold nanoparticles with strong RRS effect, a new RRS method for the determination of Na+ was established. It has the advantages of simple operation, high sensitivity, and good selectivity.Based on the selective reaction of Na


Lactose synthesis rate is an important factor in milk production and quality in mammals. Understanding the lactose synthesis mechanism is crucial for the improvement of milk quantity and quality. However, research on the temporal gene changes regarding lactose synthesis during the whole lactation is still limited. The objective of this study was to determine gene expression profiles related to lactose synthesis in sows during lactation, and further identify the critical steps or key factors in the lactose synthesis pathway.To determine the temporal change of factors related to lactose synthesis in sows, milk from eight multiparous Yorkshire sows (parity 3 to 6) was collected at 0 h, 2 h, 6 h, 12 h, 24 h, day 2, 3, 4, 7, 14, and 21 after birth of the first piglet. Lactose content, prolactin and progesterone concentration, and gene or protein expression related to lactose synthesis were measured.P < 0.05). A similar trend was observed in IGF-1 and insulin concentrations in milk, both of which were greatest at D3 with a subsequent decrease during middle to late lactation. Conversely, milk prolactin and progesterone concentrations moderately decreased with the progression of lactation. The mRNA or protein expressions related to glucose transportation (GLUT1), glucose-galactose interconversion (HK1 and UGP2), UDP-galactose transportation (SLC35A2), and lactose synthetase (LALBA and B4GALT1) in the lactose synthesis pathway were significantly upregulated during early to middle lactation and plateaued by late lactation (P < 0.05).The lactose yield increased gradually from D2 to D21 and reached a maximum at D14 (3-fold from D2) during lactation  and lactose synthetase (LALBA and B4GALT1) might be the critical steps in the lactose synthesis pathway of sows during lactation. Milk yield and composition of sows is crucial for growth and performance of their piglets . In pastSow milk contains approximately 5.5% lactose, 4.5% protein, and 6% fat –9. As thLactose synthesis is a complicated process that requires the coordination of many genes encoding for enzymes involved in glucose uptake –18, glucTherefore, the objective of this study was to determine gene expression profiles related to lactose synthesis in sows during lactation, and further identify the critical genes or key factors in the lactose synthesis pathway.A total of 8 multiparous sows  were selected from the Changjiang Swine Breeding Center in Guangdong Province (China) and were managed by standard procedures. The diets were corn-soybean meal based and were formulated to meet or exceed nutrient requirements for lactating sows recommended by the National Research Council (NRC) . The dieManingat et al.  demonstrThe estimation of the milk yield on days 2, 7, 14, and 21 of lactation was based on the equation described by Hansen et al. . Litter Milk lactose concentration was determined by Lactose/D- galactose (Rapid) Assay kit . Prolactin, progesterone, insulin (1μIU/mL = 1 ng/mL× 21.2), and IGF-1 content in milk infranatant were determined by radioimmunoassay (RIA) method using commercial kits   as described by Foisnet et al. .http://medgen.ugent.be/~jvdesomp/genorm/) as the reference genes for the qPCR. Gene specific forward and reverse primers were designed using Primer Premier 5 . All the primers of the target gene and reference gene for qPCR were shown in Table Total RNA was isolated from the mix of milk fat and TRIzol reagent  according to the manufacturer’s instructions. Total RNA concentration and purity were measured using Nano Drop spectrophotometer . The rate of A260/280 was from 1.95 to 2.10; A260/230 was from 1.78 to 2.11. The genomic DNA elimination and the cDNA synthesis was performed according to the instructions of a Prime Script RT reagent kit . The quantitative real-time PCR was executed on the ABI Prism 7500 Sequence Detection System  in a volume of 20 μL. SYBR Green Real-Time PCR Master  was the reaction mix. GAPDH, TBP, MRPL39, β-actin, and SDHA was selected by geNorm 3.5 . The mRNA expression was calculated using the 2t method .Primary antibodies  for GLUT1 , HK1 , UGP2 , PGM1 , and B4GALT1  were from the Abcam Company Ltd. . The primary antibody for SLC35A2  was from Santa Cruz Biotechnology . The secondary antibodies  were from Cell Signaling . The protein concentrations were measured using Pierce BCA protein assay kits . The Western blot analysis procedure was conducted according to Lv et al. . The milP < 0.05 were considered statistically significant. Data were expressed as means ± SEM.Statistical evaluation of milk yield, lactose and hormones concentration in milk, transcription, and western blot was performed using the ANOVA and LSD procedure of SPSS 19.0 software . Data were analyzed as a randomized complete block with a linear model that included sow (random), time , sow x time (random error), and among samples within sow (random subsample variance). Differences at P < 0.05), and decreased at D21 of lactation  , reaching a relative plateau, with the maximum concentration of 56.17 g/L at D21 of lactation. Similar to milk yield, lactose yield of sows increased 2-fold from D2 to D14 , and then decreased at D21 (476.66 g/d) in lactation  Fig. . There wion Fig. .Fig. 1ThP < 0.05) with a decline thereafter to D7 (P < 0.05), when it plateaued . The milk prolactin and progesterone concentrations declined progressively from 0 h  and plateaued at D7 (prolactin: 4.09 ng/mL) or D1 (progesterone: 1.12 ng/mL), and then remained relatively constant throughout the remainder of lactation Fig. . Howeverued Fig. . The milP < 0.05), and then decreased and was relatively stable to D14 when it increased to levels comparable to 12 h . However, there was no change in the mRNA expression of SGLT1 throughout lactation , but then declined at D21  from 0 h of lactation.The mRNA expression of GLUT1 increased from 0 h to 12 h  to the maximum value, and then gradually decreased during the remainder of lactation  compared with 0 h. Similarly, the HK1 protein expression increased 1.4-fold (P < 0.05) from 0 h to D7  of lactation. However, the mRNA expression of HK2 was increased from 0 h only at 12 h , there was little difference between 0 h and other times of lactation.The transcription abundance of HK1 increased from 0 h to 12 h , and reached a relative plateau from D3 to D21 or D14. The transcript abundance of LALBA was significantly upregulated by 4.44-fold at D2, 11.76-fold at D3, 13.85-fold at D4, 11.88-fold at D7, the peak value (14.84-fold) at D14, and 11.82-fold at D21 respectively (P < 0.05). Compared with 0 h, the mRNA expression for B4GALT1 was augmented 1.21-fold at 24 h, 1.76-fold at D2, 3.15-fold at D3, 3.34-fold at D4, 2.89-fold at D7, 2.83-fold at D14, and 2.02-fold at D21 respectively (P < 0.05). Furthermore, the protein expression of B4GALT1 also increased 3.3-fold by D2 from 0 h  at 2 h, but then decreased gradually, with a decrease of nearly 50% at D21 compared to 0 h. There was no significant change in the mRNA expression abundance of PGM2 in the whole lactation , and then reached a relative plateau from D7 to D21.The mRNA expression of genes related to glucose-galactose interconversion  and UDP-galactose synthesis  and transportation SLC35A2) in milk throughout the whole lactation is shown in Fig. 5A2 in miion Fig. . At variP < 0.05), and then decreased from D3 to D21 . The protein expression of UGP2 increased slowly to D7  compared to 0 h , and then gradually decreased. There was no significant change in the mRNA expression abundance of GALE during lactation , and decreased at D21 from D14 , reaching a relative plateau thereafter  . However, there was no significant change for PRLR mRNA expression throughout lactation.The mRNA expression of AKT1 increased at D1 (05) Fig. , then deAs a key osmotic agent, lactose synthesis is an important factor in milk production  and has + − coupled glucose transporter 1 (SGLT1), were found to participate in this transport [As the primary substrate for lactose synthesis, glucose contributes approximately 80% of the carbons in lactose in humans  and 85% ransport , 33, 53.ransport , bovinesransport , 35, 54,ransport , 30, andransport . Similarransport , 33. In Once glucose enters cells, it is converted into glucose-6-phosphate through hexokinase (HK). Xiao and Cant  reportedOnce glucose is phosphorylated to glucose-6-phosphate, it is subsequently converted into UDP-galactose in the cytoplasm by UGP2, PGMs, GALT, and GALE, and enters the Golgi bodies via SLC35A2 , 11. In Lactose is synthesized by lactose synthetase from glucose and UDP-galactose when they are transported into Golgi bodies by glucose transporter GLUT1 and UDP-galactose transporter SLC35A2. Lactose synthetase is composed of two subunits (LALBA and B4GALT1), both of which are required for activity , 58. β 1Lactogenic hormones such as prolactin, insulin, and growth hormone are necessary to induce and maintain successful lactation . ProlactReports indicate that both prolactin and growth hormone can regulate milk composition synthesis , and groIn summary, milk and lactose yield gradually increased with the progression of lactation in sows. Lactose synthesis was not significantly influenced by lactogenic hormones . The transcript abundances or protein expressions of GLUT1, LALBA, and B4GALT1 increased significantly along with increased lactose concentration and yield during lactation, indicating that they might be important in critical steps in lactose synthesis in lactating sows.


This observation was further supported by an increased tumour burden in the lungs of −/−Pparβ/δ mice as demonstrated in the same animal model. Conclusion: These results indicated a protective role of PPARβ/δ in melanoma progression and metastasis.Background: Peroxisome proliferator–activated receptor (PPAR) β/δ, a ligand-activated transcription factor, is involved in diverse biological processes including cell proliferation, cell differentiation, inflammation and energy homeostasis. Besides its well-established roles in metabolic disorders, PPARβ/δ has been linked to carcinogenesis and was reported to inhibit melanoma cell proliferation, anchorage-dependent clonogenicity and ectopic xenograft tumorigenicity. However, PPARβ/δ’s role in tumour progression and metastasis remains controversial. Methods: In the present studies, the consequence of PPARβ/δ inhibition either by global genetic deletion or by a specific PPARβ/δ antagonist, 10h, on malignant transformation of melanoma cells and melanoma metastasis was examined using both in vitro and in vivo models. Results: Our study showed that 10h promotes epithelial-mesenchymal transition (EMT), migration, adhesion, invasion and trans-endothelial migration of mouse melanoma B16/F10 cells. We further demonstrated an increased tumour cell extravasation in the lungs of wild-type mice subjected to 10h treatment and in  Melanoma is the deadliest and the most aggressive form of skin cancer that originates from the pigment making melanocytes in epidermis. In contrast to the declining incidence for many types of cancers, the number of annually diagnosed melanoma cases has doubled in the past decade . FurtherPeroxisome proliferator–activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. They regulate many biological processes, including cell proliferation, migration and differentiation, immune response, and energy homeostasis . Upon liAngptl4) and Cpt1 expression  and migrate across the endothelium ,30. To u−/−Pparβ/δ mice as compared to that in wild-type controls , tumour associated macrophages (TAMs), tumour-infiltrating lymphocytes (TILs) and pericytes in tumour progression . FurtherPparβ/δ-null mice (mixed genetic background of Sv129/C56BL/6) were kind gifts from Prof. Walter Wahli . Wild type mice (C57BL/6) were purchased from the InVivos Pte Ltd., Singapore. All animal procedures were reviewed and approved by the Nanyang Technological University Institutional Animal Care and Use Committee , Singapore.l-glutamine (Gibco), 100 U/mL of penicillin and 100 µg/mL of streptomycin . Human Pulmonary Microvascular Endothelial Cells (HPMEC) cells were cultured in Endothelial Cell Medium-2 supplemented with endothelial cell growth medium bulletkits . All cell lines were maintained in 5% CO2 atmosphere at 37 °C.Mouse melanoma cell line B16/F10 was cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 2 mM of N-(4-(isopentylamino)-2-methoxyphenyl)sulfamoyl)-thiophene-2-carboxylate; PPARβ/δ antagonist) was synthesised in-house (Shunsuke Chiba) according to as shown in Toth et al. 2012 [Compound 10h  and cDNA was synthesised using qScript cDNA Supermix  according to manufacturer protocols. Quantitative real-time PCR was performed in a total of 20 µL volume containing SYBER green  on a QuantiStudio 6 Flex Real-time PCR system. The primers used were as follows :5 cells/well) was seeded in a well of a 6-well plate and serum starved in serum free DMEM for 8 h before treatment with DMSO (0.05% v/v) as control or 10h (10 µM). After 72 h, the cells were harvested for western blot analysis to detect EMT marker proteins, MITF and MMP-9 expression or seeded for migration and invasion assays.The B16/F10 cells  containing 10% FBS. The cells were cultured with α-MSH at 0.1 µM  treatment in the absence or presence of 10h for 3 days. The cultured cells or media were harvested, and pellets were dissolved in 1N NaOH  containing 10% DMSO (Sigma) at 80 °C for 1 h. The melanin content was measured at 475 nm wavelength using an ELISA plate reader .4 cells/well) were seeded in a fibronectin coated 96-well tissue culture plate and incubated for 30 min at 37 °C in a 5% CO2 incubator. Non-adherent cells were aspirated, and adherent cells were washed thrice with PBS. The remaining cells were then fixed with ethanol and stained with crystal violet. Excess dye was removed by washing with PBS and the dye absorbed by adherent cell nuclei was extracted with 0.5% Triton X-100. Quantification was by measuring the absorbance at 570 nm using an ELISA plate reader (BioTek).For cell adhesion assay, B16/F10 cells (6 × 104) were seeded onto a monolayer of HPMEC and incubated for 15 min at 37 °C in a 5% CO2 incubator. Non-adherent cells were aspirated, and adherent cells were washed 3 times with PBS. Remaining adherent cells were fixed using 1% paraformaldehyde (PFA) for 15 min and the adherent cancer cells tagged with green signal were counted under an inverted fluorescence microscope .For tumour-endothelial assay, B16/F10 cells were pre-treated with 0.05% DMSO or 10 µM 10h and pulsed with 25 µM Cell Tracker Green CMFDA  dye for 30 min and harvested to obtain a cell suspension. B16/F10 cells (2 × 105 cells per well) were seeded into the upper chamber in serum free DMEM, while conditioned medium from NIH-3T3 was applied to the lower chamber. After 4 h incubation, migrated cells on the bottom surface of the membrane were fixed with 1% PFA, non-migrated cells on the top of the membrane were gently removed by cotton bud while the migrated cells at the bottom of the membrane were stained with DAPI. Migrated cells were counted in five random microscopic fields . The cell invasion assay was carried out using a transwell system with pore size of 8 µm , coated with Matrigel . Similarly, the cells (1 × 105 cells per well) were seeded into the upper chamber in DMEM supplemented with 1% FBS, while medium with 10% FBS was applied to the lower chamber. After 24 h, invading cells were quantified as described above.The cell migration assay was carried out using a transwell system with pore size of 8 µm . The cells ; fresh medium was added at day 2). Pre-treated B16/F10 cells with 0.05% DMSO or 10 µM 10h were pulsed with 25 µM CellTracker Green CMFDA dye (Invitrogen) for 30 min before being detached by 0.25% trypsin. 0.8 × 105 of pre-treated B16/F10 cells were seeded onto the endothelial monolayer and allowed to migrate for 18 h at 37 °C in 5% CO2. Conditioned NIH-3T3 medium was used as a chemoattractant. The transwells were then fixed in 4% PFA and cells on the apical side of each insert were scraped off, and the transwell membrane mounted onto glass slides. Migration to the basolateral side of the membrane was visualized with a immunofluorescent microscope at 10× magnification. An average of five random fields were quantified using the NIH ImageJ analysis software (http://rsbweb.nih.gov/ij/). For trans-endothelial migration for HPMEC treated with 10h, the experimental design is similar to the above with the exception that the endothelial monolayer was treated with 0.05% DMSO or with 10h up to 4 days to confluence and green signal tagged B16/F10 cells were seeded onto the endothelial monolayer.1 × 105 B16/F10 cells were injected into the tail vein of 6- to 8-week-old WT and Pparβ/δ−/− mice as described [http://rsbweb.nih.gov/ij/).For experimental metastasis assays, pre-treated 5 × 10escribed . After 26 B16/F10 cells labelled with CellTracker Green CMFDA (Invitrogen) were injected into the tail vein of 6- to 8-week-old C57BL/6 female mice. Twenty-four hours later, mice were sacrificed, and lungs were harvested. Lung tissues were subjected to fixation using 4% PFA for 3 h and were cryoprotected in 15% sucrose and then 30% sucrose in PBS overnight. The lungs were then frozen using Shandon™ Cryomatrix™ embedding resin  and cryostat-cut in 6-μm thick sections. Immunofluorescent staining for blood vessels was conducted with anti-CD31 RFP conjugated primary antibody (1:100 dilution)  and anti-DAPI (1:1000 dilution). The specimens were then examined using an upright widefield fluorescence microscope .1 × 10Pparβ/δ/−− mice. After 30 min, the mice were euthanized before perfused transcardially with 5 mL of PBS. The lungs were then excised, washed with PBS and allowed to dry at 37 °C for 48 h. The dry weights were recorded and 500 µL of formamide  was added. The samples were then incubated for 24 h at 55 °C. The amount of extravasated evans blue was measured by the absorbance of 620 nm using an ELISA plate reader (BioTek).For evans blue extravasation assay, 200 µL of 0.5% Evans Blue Solution was injected into the tail vein of 6- to 8- week-old WT and t-test was used to determine whether the experimental samples were significantly different from the control samples. Differences were considered statistically significant at * p < 0.05; ** p < 0.01; *** p < 0.001.One-way ANOVA followed by Turkey’s post hoc analysis or two-tailed, unpaired Student’s


The high-pressure gas (HPG) was the significant factor in spray drying process, which affect the BET surface area and average particles size of the spray-dried powders. The detailed influences of calcined steps and calcination temperature in the microstructure and average particles size of final W particles were investigated. The size distribution of as-synthesized nanoscale W particles with hollow superstructure was from 40 to 200 nm, and the average size was about 100 nm. The as-synthesized W powder shows good sintering properties. It should be noted that the powder technology in this study can be used to synthesize other powders with high-performance requirements.Nanoscale tungsten (W) powder is used in some special materials. In this study, a hollow superstructure W powder consisting of nanoparticles was synthesized by spray drying combined with two-step calcination from commercial (NH. Tungsten (W) metal is widely used in daily life and modern industry because of its outstanding properties involving high melting point, high thermal conductivity, excellent mechanical properties at elevated temperatures, low sputtering yield, good mechanical strength, low tritium retention, and high erosion resistance –4. The aCompared with micro-sized W particles, nanoscale W particles have excellent superiorities due to their higher surface area and smaller grain, which can be sintered to W compacts with high relative density in relatively low temperature and low pressure –12. SomeSpray drying technique is developed to be a clean, rapid, reproducible, low-cost, and easily scaled-up method to produce spherical nanoscale particles due to instantaneous transition between liquid (solvent) and solid (solute) phases –22. Spra4)6W7O24·6H2O] as raw material. The influences of process parameters, including high-pressure gas of spray drying, heating rate of ignition, and reduction temperature, were discussed. The W compacts prepared by nano-sized W particles were also studied. To the best of our knowledge, there is no report on the synthesis of nano-sized W particles with hollow superstructure. The purpose of this research is to acquire the synthesis mechanism of nanoscale spherical particles with hollow superstructure and determine the best parameters by the investigation of parameters of spray drying and calcination process.In this work, a systematic investigation was presented on the synthesis of nano-sized W particles with hollow superstructure by spray drying method combined with hydrogen reduction using ammonium paratungstate [(NH4)6W7O24 ·6H2O powder was purchased from Xiamen Tungsten Co. Ltd. . The deionized water was used as the solvent, and all reagents and solvents were used as received without further purification. The (NH4)6W7O24·6H2O powder as a solute was dissolved in deionized water, and the solution was heated from room temperature to 80 °C for promoting dissolution. Then, the (NH4)6W7O24 solution with a certain concentration was obtained. The solution was atomized to micro-spherical particles by spray drying equipment with the optimizing parameters. Subsequently, the spray-dried (NH4)6W7O24 powder was ignited by two different calcined processes . Spray-dried (NH4)6W7O24 particles were heated to 800 °C for 60 min with the heating rate of 5 °C/min in N2/H2 (vol 5:5), which obtained cubic polyhedron W particles in the one-step calcination. The nanoscale near-spherical W particles with hollow superstructure were synthesized by the two-step calcination. The temperature went up to 650 °C for 20 min followed by cooling. Then, the temperature was reheated up to 700 °C for 120 min (heating rate of 5 °C/min in N2/H2 (vol 5:5)). As-synthesized W powder  was uni-axially compacted to form pellets  under a pressure of 120 MPa without adding any external lubricant. The compact was sintered under H2 atmosphere for 2 h at 1400 °C (heating rate 20 °C/min).Commercially available (NH4)6W7O24·6H2O particles. The solid phases in different stages were characterized by X-ray diffraction . The microstructure of (NH4)6W7O24·6H2O particles and thermally decomposed products were observed by field emission scanning electron microcopy  and transmission electron microscopy . The average particle size, size distribution, and specific surface area of the as-prepared (NH4)6W7O24 powder were measured by the laser particle size analyzer  and surface area instrument , respectively. The density of the sintered specimen was measured by Archimedes method. The hardness of as-sintered W specimen was measured by Vickers hardness tester .A commercial spray drying equipment  was used to synthesize micro-spherical (NH4)6W7O24·6H2O powder is presented in Fig. 4)6W7O24·6H2O. So, the modification of microstructure of (NH4)6W7O24·6H2O is quite essential for the next step. The XRD pattern of untreated (NH4)6W7O24·6H2O is shown in Fig. 4)6W7O24·6H2O [The microstructure of commercial (NH4)6W7O24 in the exterior of fogdrops. The volume of the fogdrops ceased to change when the concentration of solute at fogdrops surface reached saturation. After that, a shell formed on the surface of the fogdrops where the interior solute constantly formed into solid (NH4)6W7O24. The internal water vapor escaped from the shell to produce numerous nano-sized capillary pores which were hard to be observed. Finally, the micro-sized hollow spherical particles were obtained.The detailed schematic of particles formed in spray drying is shown in Fig. 4)6W7O24 particles atomized by spray drying method are shown in Fig. 4)6W7O24·6H2O is shown in Fig. The microstructures of the (NH4)6W7O24 particles in different high-pressure gas (HPG) are shown in Fig. 4)6W7O24 particles are observed. The BET surface area and average particle size of as-prepared (NH4)6W7O24 powders are also presented in Fig. 4)6W7O24 particles without any agglomeration were obtained , the concaves on the exterior of particles were noticeably reduced 6W7O24 solution at the nozzle was not sufficiently crashed into micro-sized fogdrops by HPS owing to the low relative speed between solution and HPG. As a result, the dried powders had larger average particles size and lower BET surface area 6W7O24 was completely transformed into ultrafine fogdrops because of a high relative speed between them, so the spray-dried particles had a small average size (0.9 μm) and a large BET surface area (6.8 m2/g), as shown in Fig. 4)6W7O24 particles with lower BET surface area could be acquired by using a small HPG. The result could be the opposite when the HPG were larger.The HPG is one of the most important factors in atomization process, which can influence the BET surface area and average particle size of spray-dried particles. When the HPG was 150 L/min, the NH6W7O24 so4)6W7O24 particles (HPG was 300 L/min) were calcined by two different reduction calcination processes. The one-step direct calcination was carried out, as shown in Fig. In this work, spray-dried 6W7O24 decomposed to NH3, H2O, and intermediates . The newborn W had low activity and energy because of cooling, which had a restriction on the growth of W particles. When the intermediates were reheated to 650 °C, the W particles started to increase slightly because of a few WOx sublimated, and the XRD patterns of intermediate products are shown in Fig. 2/H2) were easy to enter into the pores between W nanoparticles, which could increase the reaction rate and contaction between gases and intermediates. The sublimated WOx decreased obviously at 700 °C due to the sublimation of WOx at 720 °C or higher temperature [In the two-step calcination process, the detailed formation mechanism of hollow W nanoparticles is shown in Fig. perature ; thus, t3, was obtained, and this corresponds to a relative density of 98.55% based on the tungsten theoretical density. Moreover, Vickers hardness of as-fabricated W compact was 384 ± 10 Hv. It is indicated that the W powder prepared by spray drying and two-step reaction has good sintering property.As-synthesized W powder  was sintered at 1400 °C. The microstructure of W compact is shown in Fig. 4)6W7O24·6H2O. The three-dimensional irregular polyhedral (NH4)6W7O24·6H2O was successfully turned into the micro-sized near-spherical particles by spray drying method, from which the high-pressure gas (HPG) was one significant factor to synthesized near-spherical (NH4)6W7O24 powders. When the HPG was 300 L/min, well near-spherical particles with a small average size (0.9 μm) and large BET surface area (6.8 m2/g) could be obtained. Different reduction calcination process had great influence on the microstructures of W particles. The cubic polyhedron W particles with average size of 1.6 μm were synthesized by one-step reduction calcination. In the two-step reduction calcination process, the hollow superstructure W composed of nanoparticles were obtained. The particle size distribution of as-synthesized W powders by two-step reduction calcination ranged from 40 to 200 nm, and the average particle size was about 100 nm. W target was acquired by sintering hollow superstructure W powders. The relative density and Vickers hardness were 98.55% and 384 ± 10 Hv, respectively. The as-synthesized W powder showed good sintering properties. The powder technology in this study can be applied to synthesize other powders with high-performance requirements.The hollow superstructure W powders composed of nanoparticles were synthesized by spray drying combined with two-step calcination from commercial (NH