Much evidence indicates receptor for advanced glycation end products (RAGE) related inflammation play essential roles during aging. However, the majority of studies have focused on advanced glycation end products (AGEs) and not on other RAGE ligands. In the present study, the authors evaluated whether the accumulation of RAGE ligands and binding intensities between RAGE and its ligands differ in kidney, liver, and skeletal muscle during aging.In C57BL/6 N mice aged 12 weeks, 12 months, and 22 months, ligands accumulation, binding intensities between RAGE and its ligands, activated macrophage infiltration, M1/M2 macrophage expression, glyoxalase-1expression, and signal pathways related to inflammation were evaluated. The RAGE ligands age-associated accumulation patterns were found to be organ dependent. Binding intensities between RAGE and its ligands in kidney and liver increased with age, but those in skeletal muscle were unchanged. Infiltration of activated macrophages in kidney and liver increased with age, but infiltration in the skeletal muscle was unchanged. M1 expression increased and M2 and glyoxalase-1 expression decreased with age in kidney and liver, but their expressions in skeletal muscle were not changed.These findings indicate patterns of RAGE ligands accumulation, RAGE/ligands binding intensities, or inflammation markers changes during aging are organs dependent.The online version of this article (doi:10.1186/s12979-017-0095-2) contains supplementary material, which is available to authorized users. The aging process can be described as a universal, intrinsic, progressive accumulation of deleterious changes in cells and tissues that increase morbidity and lead to death . AccordiSeveral studies have focused on the role played by receptor for advanced glycation end products (RAGE) on aging, because RAGE is known inducer of inflammation and oxidative stress.RAGE belongs to the immunoglobulin superfamily of cell surface molecules and has an extracellular region containing one V-type immunoglobulin domain and two C-type immunoglobulin domains , 4. The RAGE has several ligands, such as, advanced glycation end products (AGEs), proinflammatory S100/calgranulin family members, and high motility group box 1 protein (HMGB1) , 6. RAGEAfter binding these ligands, RAGE activates an inflammation-related signaling cascade involving nuclear factor-(NF)κB, ERK  1/2, p38 MAPK (mitogen-activated protein kinases), JNK , PKC (protein kinase C), Rac/Cdc42, and TIRAP and MyD88 (adaptor proteins for TLR 2 and 4) , 6.Because AGEs accumulates in organs, such as, kidney , liver , brain , and skeAlthough much evidence indicates that RAGE-related inflammation and oxidative stress participate in the aging process, the majority of studies on the topic have been focused only AGEs and not on other RAGE ligands –15. In fTherefore, we sought to determine whether the accumulation patterns of the RAGE ligands which are AGEs, HMGB1, and S100β and the binding intensities between RAGE and its ligands in kidney, liver, and skeletal muscle are age-dependent.n = 6), middle-aged , and old  mice. The skeletal muscle was collected from adductor profundus of mouse hind limb. All animals were examined daily for signs of injury or illness by trained persons. When tissue samples were collected no abnormality was evident. This study was approved by Lee Gil Ya Cancer and Diabetes Institute of Gachon University, and conducted in strict accordance with the guidelines issued by our Institutional Animal Care and Use Committee .A total of 18 male C57BL/6 N mice were used and kidney, liver, and skeletal muscle were extracted from young . All tissues were homogenized with lysis buffer plus proteinase inhibitor and phosphatase inhibitor and briefly sonicated 10 times for 10 s in a cold bath sonicator. After centrifuging at 14,000 x g for 20 mins at 4 °C, supernatant phases were collected and protein concentrations were determined using a Bicinchoninic acid assay kit .PrimeScript 1st strand cDNA Synthesis Kit .Total RNA in kidney, liver, and skeletal muscle from young, middle-aged, and old mice were isolated using a Trizol reagent  according to the manufacturer’s instructions. All tissues were homogenized in ice using a disposable pestle in 1 ml of Trizol reagent. Homogenized tissues were added to 0.2 ml of chloroform , mixed, and centrifuged at 12,000 x g for 15 mins at 4 °C. Aqueous phases were collected, placed in cleaned tubes, mixed with 0.5 ml of isopropanol, and centrifuged using the same conditions. Isolated RNA was then washed with 70% ethanol, and dissolved in 30 μl of DEPC treated water. To perform quantitative real time polymerase chain reaction (qRT-PCR), 1 μg of total RNA was subjected to complementary DNA (cDNA) synthesis using the Mice were anesthetized with Rumpun (2.5 mg/kg) and Zoletil (500 mg/kg). Kidney, liver, and skeletal muscle tissues from young, middle-aged, and old mice were fixed in 4% paraformaldehyde  overnight and placed in an automatic dehydration machine (Leica ASP-300 S). Tissues were dehydrated in a series of steps, that is, with 90% ethanol for 3 × 1 h, followed by 100% ethanol for 2 × 2 h. Tissues were then cleared with 100% xylene for 3 × 1.5 h, embedded in warmed paraffin, and paraffin embedded tissue blocks were sectioned at 10 μm.Macrophage infiltration was identified by the expression of Iba1 in kidney, liver and skeletal muscles. Paraffin was removed by zylene and washed three times for 10 mins each in phosphate-buffered saline (PBS). Endogenous peroxidase was blocked by 0.3% hydrogen peroxide in PBS, followed by three times rinses with PBS and we then blocked for 1 h with normal serum. The tissues sections were incubated with anti-Iba1 antibody .AGE, HMGB1, and S100β levels in kidney, liver and skeletal muscle tissues in young, middle-aged and old mice were measured by an Indirect ELISA. Coating solution mixture (0.6% sodium bicarbonate and 0.3% sodium carbonate in distilled water) were incubated onto 96-well plates overnight at 4 °C. Protein samples were mixed with 5% skim milk containing 0.1% Triton x-100 in phosphate-buffered saline (TPBS) and then incubated overnight at 4 °C. Unbound proteins were removed by washing with TPBS and incubated with anti-AGE, anti-HMGB1, and anti-S100β antibodies for 6 h at room temperature  level, we used quantitative real time polymerase chain reaction (qRT-PCR). Briefly, 300 ng cDNA, 5 μl ROX plus SYBR green premix (TAKARA) and 0.4 μM forward and 0.4 μM reverse primers , 30 μg of proteins per lane were separated by 4–12% NuPAGE Bis-Tris gel (Thermo Scientific) electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes at 200 mA for 2 h. Non-specific binding was blotted with 5% skim milk. Membranes were then treated with primary antibodies . The Kruskal-Wallis test was used to compare groups, and the Mann-Whitney U test was used for multiple comparison.P values of <0.05 were deemed significant. All results are expressed as means ± standard deviations.The AGEs accumulation in the kidney of young group was significantly lower than in the middle-aged group, and there was difference between middle-aged and old group in the kidney. The accumulation of HMGB1 and S100β in kidney of the three age groups was significantly increased with age Fig. . In live/HMGB1, and RAGE/S100β binding intensities were increased with aging in liver  into tissues increased significantly with age Fig. , b. HoweIn kidney and liver, GLO-1 levels were significantly different in the three groups decreased with age. In addition, there was statistical difference middle-aged and old group Fig. , b. HoweIn kidney and liver, NFκB and IL-1β levels increased with age Fig. , e, but The present study shows; (1) the age-related accumulation patterns of RAGE ligands  are organ dependent; (2) binding intensities between RAGE and its ligands in kidney and liver increased with age, but binding intensities in skeletal muscle were not changed by aging; (3) infiltrations of activated macrophage into kidney and liver were increased with age, but infiltrations into skeletal muscle were not changed; (4) M1 expression was increased and M2 expression was decreased by age in kidney and liver, but M1 expression in skeletal muscle was unchanged; (5) GLO-1 expressions in kidney and liver were decreased by age, but unchanged in skeletal muscle; and (6) the activation of inflammation related signal pathway in kidney and liver increased with age, but in skeletal muscle they remained unchanged.Several studies have shown that the AGEs-RAGE pathway is related to aging of humans and animals , 17. In In the present study, AGEs accumulations in kidney in the middle-aged and old groups were significantly higher than in the young group, however no difference was observed between the middle-aged and old groups. These results suggest AGEs accumulation in kidney reaches a maximum level even in middle-age. AGEs are generated endogenously by glycation, and this process is enhanced by ROS or hyperglycemic conditions or by ingestion of exogenous AGEs in food . PhysiolThe glomerular changes in C57B6 mice begin at 18 to 20 months of age, before recognizable tubulointerstitial changes, and these progressively increase to death at ~30 months , 22. AltWe found that AGEs accumulation in liver was significantly increased by aging, whereas GLO-1activity decreased, which agrees with other studies . Age-relUnlike AGEs, the age-related accumulation patterns of other RAGE ligands, such as, HMGB1 and S100β, have not been in different organs. However, the effects of the accumulations of HMGB1 and S100β during aging have been in the context of brain aging. In particular, the expression of S100 protein is increased in the aging brain . It has In the present study, HMGB1 and S100 β accumulation in kidney increased with age. In liver, there was no difference between the middle-aged and old groups in HMGB1 and S100β accumulation. In skeletal muscle, the accumulations of both ligands were not changed by aging.Although RAGE ligand age-related accumulation patterns differed in kidney, liver, and skeletal muscle, binding intensities between RAGE ligands and RAGE increased with age in kidney and liver. These patterns paralleled age-related inflammation signal pathway activation, activated macrophage infiltration, M1 increases, and M2 decreases.Ligand binding with RAGE triggers ROS increases, activates NADPH oxidase, increases the expressions of adhesion molecules, and upregulates inflammation through NFκB and other signaling pathways , 28. FurIn the present study, we measured binding intensities between RAGE and its ligands rather than RAGE expression. Binding intensities between RAGE and its ligands in the liver and kidney were increased by aging, regardless of age-related ligand accumulation. The patterns of binding intensities between RAGE and its ligands in the liver and kidney matched NFκB and IL-1β activations, which suggests that binding intensity between RAGE and its ligands in the liver and kidney is a more important factor of age-related inflammation and oxidative stress than absolute ligand accumulations.A previous study showed that the RAGE pathway can enhance macrophage migration  and proms, macrophage activation, and M1 polarization were not changed by aging in skeletal muscle, which suggests the RAGE pathway and inflammation induced by RAGE pathway do not play a main role in skeletal muscle aging. In aged animal skeletal muscle, AGEs cross-linking collagen was increased and those collagen made muscle stiff [The patterns of binding intensities between RAGE and its ligands in liver and kidney were similar to patterns of M1 expression increase and M2 expression decrease with age in liver and kidney, which suggests local tissue inflammation was increased by aging in liver and kidney and that the RAGE pathway plays an important role in the aging process. On the contrary, the aging process of skeletal muscle appears to be different from those of liver or kidney. The binding intensity between RAGE and its ligands, activation of inflammatory signal pathwayle stiff , 25. TakThe main limitation of this study was that we did not measure ROS levels in tissues directly, however many studies have been shown that ROS levels increase during aging in many tissues. Our study shows NFκB increased with age in kidney and liver, and the NFκB signal pathway is known to be importantly related to ROS generation.Our study shows binding intensities between RAGE and its ligands increased during aging and that these increases occur in parallel with activated macrophage infiltration, macrophage polarization, and inflammation signal pathway activation in the kidney and liver. However, skeletal muscle did not show any age-related changes in RAGE ligands accumulation, binding intensities, or inflammation marker changes, which suggest aging processes differ in different organs.


Streptomyces from previously phylogenies as a reference. Rarefaction analysis for total Actinobacteria and for Streptomyces isolates were performed to estimate species’ richness in the intermediate lagoon (IL) in the oligotrophic Churince system. A total of 350 morphologically and nutritionally diverse isolates were successfully cultured and characterized as members of the Phylum Actinobacteria. A total of 105 from the total isolates were successfully subcultured, processed for DNA extraction and 16S-rDNA sequenced. All strains belong to the order Actinomycetales, encompassing 11 genera of Actinobacteria; the genus Streptomyces was found to be the most abundant taxa in all the media tested throughout the 3-year sampling period. Phylogenetic analysis of our isolates and another 667 reference strains of the family Streptomycetaceae shows that our isolation effort produced 38 unique OTUs in six new monophyletic clades. This high biodiversity and uniqueness of Actinobacteria in an extreme oligotrophic environment, which has previously been reported for its diversity and endemicity, is a suggestive sign of microbial biogeography of Actinobacteria and it also represents an invaluable source of biological material for future ecological and bioprospecting studies.The phylum Actinobacteria constitutes one of the largest and anciently divergent phyla within the Bacteria domain. Actinobacterial diversity has been thoroughly researched in various environments due to its unique biotechnological potential. Such studies have focused mostly on soil communities, but more recently marine and extreme environments have also been explored, finding rare taxa and demonstrating dispersal limitation and biogeographic patterns for Streptomyces. To test the distribution of Actinobacteria populations on a small scale, we chose the extremely oligotrophic and biodiverse Cuatro Cienegas Basin (CCB), an endangered oasis in the Chihuahuan desert to assess the diversity and uniqueness of Actinobacteria in the Churince System with a culture-dependent approach over a period of three years, using nine selective media. The 16S rDNA of putative Actinobacteria were sequenced using both bacteria universal and phylum-specific primer pairs. Phylogenetic reconstructions were performed to analyze OTUs clustering and taxonomic identification of the isolates in an evolutionary context, using validated type species of  The phylum Actinobacteria are gram-positive bacteria with a high G+C content, and it constitutes one of the largest phyla within the Bacteria domain . ActinobSalinispora , an endangered oasis in the Chihuahuan desert . This isBacillus , Pseudomudomonas  and Exigacterium  have beeacterium . This, tacterium , makes Cacterium . This anThe Churince hydrological system  is locat∘50′53.79″N, 102°08′30.29″W; location B: 26°50′53.53″N, 102°08′31.81″W; location C: 26°50′54.37″N, 102°08′32.96″W; location D: 26°50′55.30″N, 102°08′33.63″W; location E: 26°50′55.63″N, 102°08′35.28″W; location F: 26°50′56.57″N, 102°08′36.03″W. At each site, water and surface sediments (0.2=-1 cm) were transferred to sterile conical tubes (50 ml). Samples were transported to a nearby laboratory in the town of Cuatro Cienegas at room temperature (≤1.5 h) and were used for streaking out primary plates immediately.Sampling took place during 2013–2016 at the following times: February 2013, March 2013, October 2013, October 2014, January 2015, February 2015, July 2015, April 2016. Samples were obtained from water and upper layer sediment from six locations along the shore in the Intermediate Lagoon in the Churince system  in CuatrAIM1 ; AIM2 ; AIM3 ; AIM4 ; AIM5 3, 0.001 g MgCl2, 0.001 g ZnSO4, 18g agar, 25g Reef salt mix); AIM6 [per liter]: 10g starch, 1g K2HPO4,1g H14MgO11S, 2g H8N2O4S, 1g NaCl, 2g CaCO3, 0.001 g FeH14O11S, 0.001 g MgCl2, 0.001 g ZnSO4, 20g agar, 25g Reef salt mix); AIM7 ; AIM8 ; AIM9 .Nine selective Actinobacterial Isolation Media (AIM) were designed for this work to enhance the isolation of actinobacteria of aquatic and sediment environment. All isolation media were autoclave-sterilized and supplemented with 0.20 µm pore size filtered Nystatin (100 µg/ml) to inhibit fungal growth, nalidixic acid (50 µg/ml) to inhibit gram-negative bacteria growth and to favor the growth of slow-growing Actinobacteria.Prepared media were used for primary selective isolation of Actinobacteria by plating 150 µl directly from fresh samples, and using sterile 3 mm glass beads. Inoculated plates were incubated at 27°C for 1–6 weeks. Isolates were selected based on colony morphology and Gram stain, picked and re-streaked several times to obtain pure cultures. Isolates were maintained on AIM1 and AIM6 agar plates for short-term storage, and long-term strain collections were set up in 50% glycerol and preserved at −20°C (sporulated) and −80°C (non-sporulated).To confirm Actinobacteria identity and further phylogenetic analysis of isolates, after testing several techniques, genomic DNA was prepared using a modified phenol/ chloroform method that yielded the best quality DNA for our isolates: colonies of putative Actinobacteria were carefully scraped from agar plates and placed in centrifuge tubes; cell pellets were washed 2× 10 ml of 10% (w/v) with sucrose and resuspended in 400 µl of lysis solution . After resuspension, 400 µl of Phenol/Chloroform and 0.1 mm glass beads were added to lysis mix and this was mechanically disrupted for 2 min. The lysates were centrifuged  and DNA in aqueous phase was precipitated with 2 volumes of ethanol and 1/10 volume of 3M sodium acetate, pH 5.2; after overnight incubation at −20°C, DNA was centrifuged , washed with 70% ethanol and eluted in TE with RNase.Genomic DNA from putative Actinobacteria was sent to Macrogen, Inc., USA, to perform 16S rDNA gene amplification by PCR and sequencing using the universal primers 27F (5′-GAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′), as well as phylum-specific primers: S-C-Act-235-a-S-20 (5′CGCGGCCTATCAGCTTG TTG-3′)  and 23SRhttp: //www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST) to determine genus-level affiliations and are deposited in GenBank, which is associated with this document and are also available as The 16S rDNA gene sequences were edited and assembled using CodonCode Aligner 5.1 software ; assembled contigs were compared to 16S rDNA gene sequences in the NCBI database  and also microorganisms originally isolated from water and sediments from aquatic environments. Other reference strains were added to provide biological interpretation, and were selected from previous work reporting isolation of Streptomyces from deserts  algorithm using MEGA software v. 7  and Tamu deserts ). Model  deserts .Streptomyces. From these early observations, we decided to compare distances between our Streptomyces isolates, to available information from previous studies, so we included a dataset of 667 16S-rDNA sequences of validated species of the Streptomycetaceae family; most of them were selected for a wide phylogenetic analysis within the family .To reconstruct a second phylogenetic tree of the members of family Streptomycetaceae, we used the Maximum-likelihood (ML) method using MEGA software v. 7 and the Tamura–Nei I + G parameter as an evolutionary model. The reliability of nodes was estimated by ML bootstrap percentages  obtainedTo provide support to ML tree, we conducted a Bayesian analysis employing MrBayes v3.2.5  with 10,Streptomyces isolates. The definition of operational taxonomic units (OTUs) was conducted with MEGA software v. 7 at 97% cutoff according to their pairwise distances. Then we conducted the rarefaction curve using the EstimateS 9.1.0 software package  is large  and other clades unique to soil and sediment. These analyses are consistent with our isolation efforts, which yielded abundant and diverse Streptomyces and abundant Arthrobacter isolates. It is possible to speculate that those several putative non-culturable Actinobacteria lineages detected by Illumina in concurrent projects, relate to our great numbers of cultured isolates which were not able to be detected by universal and phylum-specific primers.Morphological and genetic diversity of this phylum in the Churince does not come totally as a surprise since in concurrent studies using Illumina16S rRNA tags  it was observed that Actinobacteria are the most successful lineage in CCB water, with the notable presence of genera Many interesting morphotypes could not be identified using 16S rDNA sequences; in addition, many were lost as the purification of a single colony proceeded. Success at bringing the environment into the laboratory culture is not sufficient for successful cultivability of bacteria. Subsequent culturing of Actinomycetes to obtain axenic (pure) cultures from the Churince, dramatically reduced the total number of unique pure isolates, suggesting obligate mutualism and cross-feeding .Streptomyces individuals among the numerous isolated Actinobacteria  this collection has been isolated from an environment of a diversity and endemicity, that has previously been considered comparable to that in the Galápagos Island  algorithm for representative CCB-isolated Actinobacteria and their closely related type strains. Bootstrap values based on 1000 replicates.Click here for additional data file.10.7717/peerj.3247/supp-3Figure S2Streptomycetaceae family using the neighbour-joining method. Bootstrap values based on 1000 replicates.Phylogenetic tree based on a data set of 667 16S rRNA sequences of the Click here for additional data file.10.7717/peerj.3247/supp-4Data Set S1Complete data set used for phylogenetic analyses.Click here for additional data file.


Eriocheir sinensis) during the stage of the fifth shelling to maturity were investigated in the present study. The culture density of crabs was 600 per 667 m2, and they were continuously fed 120.4 mg Se from Se-biofortified corn per 667 m2 every two days for 90 days. The results showed that the total muscle Se levels in the crabs were significantly increased (p < 0.05). Activities of hemolymph supernatant enzymes including alkaline phosphatase (AKP), lysozyme (LZM), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were also enhanced (p < 0.05). The protein and crude fat levels at maturity were higher than those at the fourth molt. The levels of total essential amino acids (∑EAAs) and total delicious amino acids (∑DAAs) were significantly increased (p < 0.05). We demonstrate that the feeding of Se-biofortified corn could significantly improve total muscle Se concentrations and hemolymph supernatant antioxidant enzyme activities in Chinese mitten crab, and slow down the rapid decline of ∑EAAs and ∑DAAs at maturity, thus improving the nutritional quality of Chinese mitten crab.The effects of selenium (Se)-biofortified corn on the total Se contents, the antioxidant enzyme levels, and the amino acids composition in Chinese mitten crab ( Eriocheir sinensis) plays an important role in the freshwater fishery in China, with a total annual yield of 700,000 tons in 2012 [Eriocheir sinensis) [The aquaculture of Chinese mitten crab (inensis) . It is t2SeO3) was 63.9% [Ictalurus punctatus) to show that the organic Se had higher bioavailability than inorganic Se [Seriolalalandi) [Apostichopus japonicus) [As one of the essential nutrient elements, the proper addition of selenium (Se) may significantly improve the anti-hypoxia ability and immunity of aquatic creatures . It has as 63.9% . Wang etganic Se . Le and lalandi) . Moreoveponicus) .−1 in corn) with SeMet and Se-methylselenocysteine (MeSeCys) as the major Se-containing compounds, thus being used as a new source of Se [Eriocheir sinensis) to evaluate the application of grain-biofortified Se in aquaculture.Biofortification technology has been widely applied in animals/agriculture ,16,17,18ce of Se . The preEriocheir sinensis)  were provided by Jiangxin Island Chinese Mitten Crab Aquaculture Base in Zhangjiagang, Suzhou, Jiangsu. Se-biofortified corn  was provided by Suzhou Setek Co. Ltd., Suzhou, Jiangsu, China. Crab feedstuff was purchased from Jiangsu Jinkangda Group. Normal corn  was purchased from the market.Chinese mitten crabs  consisted of the crabs in the other four ponds, which were given the same diet as that used before the fourth shelling.The experiments were carried out in eight aquaculture ponds with a size of about 13,340 mCrab samples were collected on 29 July 2015 ), 30 August 2015 (right after the fifth shelling), and 30 September 2015 (at the harvest stage), respectively. Crab samples were randomly chosen and contained six male and six female crabs in each pond every time. Those crab samples were transported with ice to the lab to collect the hemolymph and meat samples within 3 h.For each crab, 8 mL hemolymph was collected by leg shearing and centrifuged . The supernatant was stored at −80 °C. The crabmeat sample was taken from the legs and the abdomen and stored at −20 °C after being smashed by a grinder.Since no significant difference was found between the male and the female when detecting the total Se levels in crabmeat, the samples were no longer separated according to gender for supernatant enzymes, amino acids, proteins, and fats measurement so as to reduce cost.About 0.5 g of the crabmeat sample was accurately quantified, put into an Erlenmeyer flask, and combined with 10 mL of a mixed acid . After more than 3 h of digestion at room temperature, the flask was heated for 1 h at 110 °C, 2 h at 130 °C, 1 h at 180 °C, and then at 210 °C till clouds of white smoke were produced and the volume of the solution was reduced to about 2 mL. After cooling to room temperature, the funnel and the inner wall of the flask were rinsed using deionized water. The flask was then heated again at 210 °C till clouds of white smoke were produced and the solution volume was reduced to about 2 mL. After cooling to room temperature, 5 mL of hydrochloride acid (AR) was added into the flask, which was then shaken well and covered with a plastic film. The solution was diluted to 25 mL the next morning to be analyzed by hydride generation-atomic fluorescence spectrometry (HG-AFS).−1) was chosen as standard sample with Relative Standard Deviation (RSD) ≤ 5%.Chinese national standard reference material GSV-2  (Disodium phenyl phosphate-4-Aminoantipyrine-Potassium ferricyanide method), lysozyme (LZM) (Lysozyme assay kit-nephelometry method), glutathione peroxidase (GPx)  method (DTNB)), and superoxide dismutase (SOD) (hydroxylamine method) were measured using a UV/visible-6100 spectrophotometer  as described by the manufacturer’s protocols from the Institute of Biological Engineering of Nanjing Jiancheng . Three replicates for each sample were analyzed.Amino acids, protein, and crude fats in the crabmeat samples were analyzed by the Shanghai Academy of Agricultural Sciences . Types and levels of all free amino acids in the sample were analyzed using an automatic amino acid analyzer . Protein content was determined by a semiautomatic nitrogen determination apparatus following the method GB/T 5009.5-2010. The soxhlet extraction method was applied according to the method GB/T 14772-2008 to measure the crude fat content. The difference between the two results obtained from the same sample was no more than 5% of the average of the two values.t-test in Origin7.0. Statistically significant difference was reported when the probability of the result assuming the null hypothesis (p) is less than 0.05.The results are expressed as mean value ± standard errors (x ± SE). The statistical analysis was performed using the procedure of two-sample −1) and the female crabs (345.8 ± 4.2 μg·kg−1) in the CK samples (p > 0.05). The one-month feeding of Se-biofortified corn increased the total Se levels to 650.9 ± 44.1 μg·kg−1  and 635.9 ± 44.5 μg·kg−1 , which were increased 1.86-fold  and 1.84-fold  compared to those of the CK samples, respectively (p < 0.05). The total Se levels in crabmeat samples taken from the crabs fed normal corn for one month were 369.4 ± 7.2 μg·kg−1  and 343.9 ± 12.4 μg·kg−1 , which were not significantly different than those of the CK samples (p > 0.05).The total Se levels in crabmeat samples are shown in −1 , 356.7 ± 2.79 μg·kg−1 , 697.8 ± 5.6 μg·kg−1 , and 356.9 ± 2.53 μg·kg−1 , which were similar to those of the one-month feeding (p > 0.05). The results suggested that the feeding of Se-biofortified corn for one month could significantly increase the total Se level in crab (p < 0.05), which then kept at a state of balance. No difference in the total Se levels was found between the male and the female crabs (p > 0.05).The two-month feeding of corn increased the total Se levels to 697.1 ± 43.4 μg·kg−1, 127.0 ± 15.4 U·mL−1, and 6.00 ± 0.8 IU·100 mL−1, respectively. The one-month feeding of Se-biofortified corn increased the enzyme activities compared to the CK sample 1.30-fold , 1.24-fold , 1.08-fold , and 1.55-fold , respectively (p < 0.05). Supernatant antioxidant enzyme activities in crabmeat samples taken from the crabs fed normal corn for one month were 339.6 ± 14.5 U (GPx), 172.3 ± 15.9 U·mL−1 (SOD), 127.5 ± 5.4 U·mL−1 (LZM), and 5.78 ± 0.8 IU·100 mL−1 (AKP), which were not significantly different than those of the CK sample (p > 0.05).Supernatant antioxidant enzyme activities are shown in −1 (SOD), 128.8 ± 2.1 U·mL−1 (LZM), and 5.03 ± 0.49 IU·100 mL−1 (AKP) for the normal corn-fed crabs, and 342.5 ± 7.0 U (GPx), 211.3 ± 2.6 U·mL−1 (SOD), 139.2 ± 4.9 U·mL−1 (LZM), and 9.85 ± 0.2 IU·100 mL−1 (AKP) for the crabs fed Se-biofortified corn.The two-month feeding of corn did not result in any difference in hemolymph supernatant antioxidant enzyme activities compared to the one-month feeding, with levels of 328.7 ± 7.1 U (GPx), 160.7 ± 8.94 U·mL−1, which was significantly higher than that of the crabs in the CK sample (7.81 g·kg−1) (p < 0.05), and significantly higher than that of the crabs fed normal corn for one month (8.00 g·kg−1) (p < 0.05). The next one-month feeding of Se-biofortified corn resulted in the continuous increase of the protein content in crabmeat to 8.38 g·kg−1. However, the protein content in crabmeat would significantly decrease to 7.36 g·kg−1 after continuous feeding with normal corns.p > 0.05). The second month of feeding with Se-biofortified corn, however, significantly decreased the crude fat level in crabmeat (0.21 g·kg−1) compared to that from the crabs fed a diet of normal corn (0.23 g·kg−1) (p < 0.05).The crude fat levels in all sample groups were not significantly different in the first month of feeding with corn . The total levels of EAAs (∑EAAs) in crabs fed either Se-biofortified corn or normal corn were similar to those in crabs of the CK sample after the one-month feeding (p > 0.05). The two-month feeding of Se-biofortified corn, however, significantly enhanced the total level of EAAs (∑EAAs) in the crabs to 3.87 g·kg−1, which was increased 1.27-fold compared to that in the crabs fed normal corn (3.06 g·kg−1), and increased 1.52-fold compared to that in the crabs of the CK sample (2.55 g·kg−1) (p < 0.05). The total level of DAAs (∑DAAs) in the crabs fed normal corn significantly decreased with the feeding duration from 3.40 g·kg−1 (the CK sample) to 3.13 g·kg−1 (the August sample) (p < 0.05), and then to 2.85 g·kg−1 (the September sample), whereas the total levels of DAAs (∑DAAs) in the crabs fed Se-biofortified corn were 3.15 g·kg−1 (the August sample) and 3.15 g·kg−1 (the September sample), which were not significantly different from those in the crabs of the CK sample (p > 0.05).In total, 16 types of amino acid were determined in the present study, among which lysine (Lys), phenylalanine (Phe), methionine (Met), threonine (Thr), isoleucine (Ile), leucine (Leu), and valine  are essential amino acids (EAAs). Half essential amino acids (HEAAs) include histidine (His) and arginine (Arg). Glutamic acid (Glu), glycine (Gly), alanine (Ala), aspartate (Asp), tyrosine (Tyr), proline (Pro), and serine (Ser) are non-essential amino acids (NEAAs). Phe, Glu, Gly, Ala, Asp, and Tyr are grouped into delicious amino acids (DAAs) ,21. It w2O2 and lipid peroxide, and SOD is a scavenger of O2−. Some hemolymph supernatant enzymes are helpful in enhancing the immunity and well-being of organisms, e.g., LZM can eliminate pathogenic bacteria, and AKP, as a nonspecific phosphomonoesterase, helps in the absorption of dissolved calcium by crustaceans and the formation of calcium phosphate. AKP can also catalyze the hydrolysis of phosphate monoester and the transfer of phosphoric acid [r = 0.87, p < 0.01), SOD , LZM , and AKP , suggesting that the intake of Se-enriched corn significantly improve the anti-oxidative capability as well as the immunity of Chinese mitten crab (p < 0.05).Evaluation of the hemolymph supernatant biochemical characteristics has become an important method in studying the normal physiology, pathological process, and toxicological mechanism of organisms ,23,24. Fric acid . Figure 2SeO3 and SeMet could significantly enhance the activity of hemolymph supernatant GPx in crucian carp (Carassius auratus gibelio) [Eriocheir sinensis) [Neocaridinaheteropoda) fed Se was significantly higher than those fed the supplemented control diet [p < 0.05). The two-month feeding of Se-biofortified corn resulted in a GPx/SOD ratio of 2.11 ± 0.02, which was significantly higher than that of 1.95 ± 0.03 in the crabs fed normal corn (p < 0.05). The results suggested the anti-oxidative capability of Chinese mitten crab was enhanced by feeding with Se-biofortified corn.GPx is an antioxidant selenoenzyme, functioning in removing free radicals and protecting cells . Wang etgibelio) . Qin et inensis) . Wang etrol diet . It has rol diet . In the Trichogaster trichopterus) was 33.72% higher after two weeks of exposure to sodium selenite [Litopenaeusvannamei could be enhanced by the combined addition of βG and Se [Seriola lalandi) [As one of the non-specific immune factors, LZM functions in the inhibition of Gram-positive bacteria. It was found the concentration of hemolymph supernatant LZM in blue gourami (selenite . Liu et G and Se . LZM actChrysophrys major) [Mytilus galloprovincialis, was not significantly related to the increase of Se levels, but might be due to adaptation or compensatory mechanisms during the chronic exposure to a polluted environment [The increase of the AKP activity can provide more inorganic phosphorus to accelerate the phosphorylation of ADP to produce ATP, thus accumulating energy to promote growth and enhancing the non-specific immunity of organisms . AKP hels major) . Regoli ironment ,37,38.r = 0.81, p < 0.01), the ∑EAA levels , and the ∑NEAA levels . Se levels were not associated with the crude fat levels  or the ∑DAA contents , and positively but not significantly correlated with the ∑TAA contents  and the ∑HEAA contents .4) into triiodothyronine (T3). T3 controls the synthesis of growth hormone as well as the improvement of insulin levels, and promotes protein synthesis in muscle and the growth of animals [2SeO3 was not associated with the protein contents in rainbow trout  [Litopenaeusvannamei, and found that Se could significantly increase body crude protein contents and decrease crude fat contents [Se, as selenocysteine in 5′-deiodinase, can catalyze the conversion of tetraiodothyronine  . Li et acontents . SeMet, contents .Eriocheir sinensis) [p < 0.05) and 18.72% higher than that of the fourth molt sample (p < 0.05). The protein contents, the levels of amino acids, and the crude fat levels in Chinese mitten crab fed Se-biofortified corn were significantly higher than those in the CK samples. The present study demonstrated that Se-biofortified corn could improve the contents of amino acids as well as promote protein synthesis and the accumulation of crude fat in Chinese mitten crab. These findings are consistent with those of Ashouri et al. (2015) on Cyprinus carpio (Cyprinus carpio) [Eriocheir sinensis) [Up to now, few studies have been conducted to evaluate the effects of Se on muscle amino acids in Chinese mitten crab. A previous study revealed that nano-Se could increase the contents of free amino acids in Chinese mitten crab (inensis) . An idea carpio)  and Tianinensis) .−1, which were significantly higher than those of Chinese mitten crab fed dietary normal corn (350.8–360.0 μg·kg−1). The diet of Se-biofortified corn also improved the activities of hemolymph supernatant enzymes including GPx, SOD, LZM, and AKP, which were increased 1.04-fold, 1.31-fold, 1.08-fold, and 1.96-fold, respectively, compared to those of crabs fed normal corn, indicating that the Se-biofortified corn diet could enhance the anti-oxidative capacity of Chinese mitten crab. The contents of protein and amino acids in the muscle tissue of Chinese mitten crab fed Se-biofortified corn were also 7.29% and 26.69% higher than those of Chinese mitten crab fed normal corn, whereas the levels of crude fat in the former were significantly decreased from 0.23 g·kg−1, as determined in the latter, to 0.21 g·kg−1. The present study demonstrated that feeding with Se-biofortified grain, e.g., Se-biofortified corn, might be an effective strategy to enhance Se intake levels in aquatic animals, and to improve their anti-oxidative capacity.The total Se contents in the muscle tissue of Chinese mitten crabs fed dietary Se-biofortified corn (with SeMet as the major Se-containing compound) were in the range of 635.9–697.8 μg·kg


Progressive cerebral accumulation of tau aggregates is a defining feature of Alzheimer’s disease (AD). A popular theory that seeks to explain the apparent spread of neurofibrillary tangle pathology proposes that aggregated tau is passed from neuron to neuron. Such a templated seeding process requires that the transferred tau contains the microtubule binding repeat domains that are necessary for aggregation. While it is not clear how a protein such as tau can move from cell to cell, previous reports have suggested that this may involve extracellular vesicles (EVs). Thus, measurement of tau in EVs may both provide insights on the molecular pathology of AD and facilitate biomarker development. Here, we report the use of sensitive immunoassays specific for full-length (FL) tau and mid-region tau, which we applied to analyze EVs from human induced pluripotent stem cell (iPSC)-derived neuron (iN) conditioned media, cerebrospinal fluid (CSF), and plasma. In each case, most tau was free-floating with a small component inside EVs. The majority of free-floating tau detected by the mid-region assay was not detected by our FL assays, indicating that most free-floating tau is truncated. Inside EVs, the mid-region assay also detected more tau than the FL assay, but the ratio of FL-positive to mid-region-positive tau was higher inside exosomes than in free solution. These studies demonstrate the presence of minute amounts of free-floating and exosome-contained FL tau in human biofluids. Given the potential for FL tau to aggregate, we conclude that further investigation of these pools of extracellular tau and how they change during disease is merited. Aggregation and deposition of the tau protein is a common feature of Alzheimer’s disease (AD) and a range of neurodegenerative disorders, collectively referred to as tauopathies ,2. In reTo date, the mechanisms underlying how tau is transferred between neurons, and the forms of tau released from neurons, remain unknown. Tau lacks a secretory signal and thus its release must occur by an unconventional mechanism. Possible release mechanisms include tunneling nanotubes, trans-synaptic transfer, and release of tau due to synaptic, neuritic, or neuronal compromise ,13,14,15EVs consist of a lipid bilayer encapsulating cytosol and are released by all cells . The loaPrevious studies have reported that certain forms of tau are present in EVs ,32,33,34We previously found that small amounts of tau are present in exosomes isolated from conditioned media (CM) of human and rodent neurons . In thosInitial experiments focused on CM from iPSC-derived neurons. Human neurons were generated from a control iPSC line  using a Having detected FL tau in iN CM, we searched for tau in CSF samples from six healthy controls using both our mid-region and FL ELISAs A. In accValues detected in CSF supernatants by the mid-region assay averaged around 200 pg/mL, and all of the values fell within the normal range expected for neurologically healthy individuals . ImportaMindful of the low and variable levels of tau present in CSF exosomes, we developed an ultrasensitive Simoa-based immunoassay to search for FL tau. Simoa (named for single molecule array) technology is based upon the isolation of individual immunocomplexes on magnetic beads using standard ELISA reagents . The maiAs with individual CSF samples A, tau waFL]/[tauFL + tauMR])  B,C.Having detected FL tau in CSF-derived exosomes, we next searched for tau in blood-borne, neuron-derived exosomes. The most widely used method for isolating neuronal exosomes from blood involves depleting plasma of clotting factors, precipitating EVs using ExoQuick, and immunoprecipitating neuronal exosomes using an anti-L1CAM antibody  34,35,5,51.Before analyzing precious clinical samples, we tested a number of modifications designed to further optimize this protocol. In the Goetzl/Kapogiannis protocol, clot formation was induced by the addition of thromboplastin D derived from rabbit brain , but we recent report  and indiWith the optimized protocol, we proceeded to isolate exosomes from plasma samples obtained from a small cohort of clinically well-characterized human donors . NeuronaPrevious studies reported detection of tau in neuronally-derived exosomes isolated from plasma ,56,57,58As seen with iN and CSF exosomes, the highest tau signal in neuron-derived exosomes isolated from plasma was detected with our mid-region assay . All 40 Two prior reports indicated that the levels of p181-tau in blood-derived neuronal exosomes were elevated in mild cognitive impairment (MCI) and AD relative to healthy control subjects ,35 whileSince the efficiency of exosome isolation could vary between samples and this might mask the differences in the amount of tau measured, we normalized measurements of tau in each sample relative to that of the established exosomal marker CD81 D–F. To eUntil relatively recently, tau was considered to be an exclusively intra-neuronal protein, and the presence of tau in human CSF was thought to be a product of axonal damage or neuronal death . It was Since exosomes have been suggested as potential vehicles by which small tau aggregates may be passed from neuron-to-neuron and the MTBR domains of tau are required for aggregation, we developed sensitive immunoassays to search for full-length (FL) tau both free-floating in solution and contained within exosomes. Our experiments are the first to unambiguously demonstrate that a small fraction (~1%) of extracellular tau in iN CM and human CSF is full-length. A number of studies using Western blotting have claimed to detect FL tau in human CSF and in CM from untransfected cells ,71,72,73The few studies that concentrated large volumes of CSF prior to Western blotting have each detected fragments of tau in the range of ~35–40 kDa, but did not detect FL tau ,40,78. TWhile the levels of free-floating tau are much higher than those found in EVs, FL tau contributed more of the mid-region signal inside exosomes than was the case for extra-vesicular tau. This relative enrichment of FL tau in exosomes was detected in CSF and plasma exosomes, and, for yet unknown reasons, was particularly strong in iN exosomes. At least a portion of tau is degraded by autophagy , and higIn contrast, there is an urgent need for blood-based biomarkers that can replace or supplement current CSF markers. Given the demonstrated utility of quantifying tau in CSF, there is much interest in measuring tau in blood, but, unlike CSF, the contents of blood are influenced by many organs. Therefore, levels of blood tau may not be sensitive to minor pathobiological changes in brain. On the other hand, measurement of tau in brain-derived, blood-borne EVs should better reflect alterations occurring in brain. Recent evidence suggests that it is possible to isolate neuronal exosomes from blood plasma and that measurement of certain forms of tau in neuronal exosomes can be used as diagnostic and prognostic biomarkers ,35. HoweRegardless of the precise forms and sources of tau detected by the ExoQuick/L1CAM isolation method, a future question to address will be whether such exosomes contain aggregated tau capable of seeding monomeric tau in recipient cells. A corollary of such a finding might be the possibility of transmitting aggregation-competent tau by using blood products and would necessitate testing the infectious potential of exosomes containing tau aggregates. Similarly, it will also be important to examine if other diseases linked to altered tau metabolism exhibit changes in the amount and/or forms of tau found in neuronal exosomes.2 for viral infection. Ultrapure lentiviral titers were obtained from Alstem  and used at the following concentrations: Tet-O-Ngn2-puro: 0.1 µL/50,000 cells; Tet-O-FUW-EGFP: 0.05 µL/50,000 cells; FUdeltaGW-rtTA: 0.11 µL/50,000 cells. To induce neurogenin 2 expression, doxycycline was added on iN day 1 (D1) (2) or 10 cm dishes (300–50 cells/mm2) and maintained in media consisting of Neurobasal, Glutamax, 20% dextrose, MEM-NEAA, B27  and 10 ng/mL BDNF, CNTF, GDNF . Half of the culture media was replaced every four days with fresh complete media. On iN D21, cells were characterized using live cell imaging to monitor neurite development (The YZ1 iPSC line  was usedy 1 (D1) A at a coelopment B and useelopment C–E.2 in opaque-wall 96 well plates  were either live-imaged or fixed and stained for mature neuron-specific markers. Briefly, cells were washed extensively with filtered, warm artificial cerebrospinal fluid , and live-imaged in 50 µL using brightfield and FITC filters with an IN Cell Analyzer 2200 . Thereafter, adjacent wells were fixed with cold 4% paraformaldehyde/4% sucrose in PBS for 10 min at 4 °C, washed twice with PBS, quenched in 0.1 M glycine/PBS for 5 min, washed 4× with PBS, and transferred to 4 °C overnight. The following day, plates were warmed to room temperature for 30 min prior to immunostaining to minimize MatriGel interference. Cells were permeabilized for 5 min with 0.5% Triton-X 100, washed 3× with PBS, blocked in 3% BSA/PBS for 1 h, washed 3x and immunostained for MAP2 , β-III-tubulin  or tau  for 2 h in 3%BSA/PBST. Thereafter, cells were washed 6× with PBST and incubated with Alexa647 conjugated donkey-anti chicken (MAP2), rabbit (β-III-tubulin) or mouse (tau) secondary antibodies  and Alexa488 conjugated phalloidin  for 1 h in 3%BSA/PBST. Plates were then washed 3× with PBST, incubated with DAPI (1:1000 in PBS) for 5 min, washed 3× with PBST, 2× with PBS and imaged using DAPI, FITC and Cy5 filters with an IN Cell Analyzer 2200 (20× 0.75 NA objective lens). Images were extracted and analyzed with IN Carta .On iN D21, iNs plated at 150 cells/mm2), pooled and then processed in 30 mL batches as described in g and 4 °C for 10 min to pellet floating cells. Then, the upper 97% was recovered  and stored at −80 °C. When required, S2 CM was thawed, pooled, and further diluted in NBM to a final volume of 30 mL/experiment. Diluted S2 was centrifuged at 10,000× g and 4 °C for 30 min to pellet microvesicles and cell debris. Finally, the upper 97% was recovered (S3) and centrifuged at 100,000× g and 4 °C for 70 min to pellet exosomes. A portion (~0.25 mL) of each fraction was stored at −80 °C. Finally, the upper 97% of S4 was removed and stored at −80 °C. The 100,000 g exosome pellet was washed in 1 mL DPBS  containing protease and phosphatase inhibitors , and re-pelleted at 100,000 g for 70 min. Exosomes were lysed in 75 µL M-PER  containing 1× phosphatase and protease inhibitors and subjected to two rounds of freezing on dry ice and thawing in a room temperature water bath. All of the dilution and concentration steps were considered when calculating tau concentrations.Media was collected on iN D23-33 from cells plated on 10 cm dishes  and transferred onto 0.2 μM nitrocellulose membranes at 400 mA for 2 h. Thereafter, blots were blocked in Odyssey blocking buffer  and membranes cut to allow the analysis of the same samples for Alix , Tsg101 (clone EPR7171(B), 1 μg/mL; Abcam, Cambridge, MA, USA), Flotillin-1 , PrP , and TOM20 . Membranes were incubated overnight at 4 °C in primary antibody solution, followed by 6 × 10 min washes with Tris buffered saline with 0.05% Tween-20 (TBST). Then, membranes were incubated with infrared-labeled goat anti-mouse 800 or donkey-anti-rabbit 680 secondary antibodies  for 1 h at room temperature, followed by 6 × 10 min washes with TBST. Immunoreactive bands were visualized using a Li-COR Odyssey CLx infrared imaging system .2 weighting using MasterPlex ReaderFit . Lower limit of quantitation (LLoQ) was determined by calculating the average + 9 standard errors and 100 ± 20% recovery for each standard. For the experiments shown, the LLoQ of the mid-region assay was 7.81 pg/mL.This assay was performed essentially, as described previously  and FiguC. Half-aThis assay was performed as with the mid-region assay except different capture and detection antibodies were used. Here, Tau12  was used for capture and TauAB (a generous gift from Andy Billinton and Mike Perkinton of MedImmune) for detection B. For thAll of the samples were obtained and used under IRB approval . Individual CSF samples were from six healthy female volunteers participating in the Harvard Biomarker Study (HBS) and CSF pools were obtained from Crimson Clinical Discards Partners (CCDP) Biobank. CSF pool A was the product of CSF collected from four individuals and pool B from three subjects. Under the terms of the IRB, demographic information is available for the samples obtained from either the CCDP biobank or the HBS. For the six HBS samples, 10 mL aliquots of CSF were centrifuged exactly as described for iN CM in n = 10), donors with mild cognitive impairment , and with mild or moderate AD (n = 20). Demographic and clinical data are provided in Forty, 500 µL aliquots of plasma were obtained from the Harvard NeuroDiscovery Center Biomarker Study. Samples came from donors free of cognitive impairment   containing 3x phosphatase  and protease  inhibitors. Thereafter, the samples were centrifuged at 6000× g for 20 min at 4 °C and the supernatant transferred to a new tube. Next, 252 µL ExoQuick  was added to the supernatants, samples mixed by inversion and left to stand at 4 °C for 1 h. Vesicles present in the serum were recovered by centrifugation at 1500× g for 20 min at 4 °C and resuspended by vortexing in 500 µL MilliQ water containing 3× phosphatase and protease inhibitors. To ensure the complete resuspension of the ExoQuick pellets, samples were mixed overnight at 4 °C on a vertical rotating mixer.Neuron-derived exosomes were isolated from plasma using a slightly modified version of the procedure pioneered by the Goetzl/Kapogiannis group . Five µLg for 10 min at 4 °C and washed once with 3% BSA/DPBS-/- before elution in 200 µL 0.1 M glycine (pH 3.0). Resin was removed by centrifugation at 4500× g for 5 min at 4 °C and 5 µL of the neuronal exosome-containing supernatant was diluted in 995 µL filtered DPBS for NanoSight analysis. The remaining 195 µL supernatant was neutralized with 15 µL of 1 M TrisHCl (pH 8.0) and exosomes were lysed by adding 360 µL M-PER , 25 µL 3% BSA/DPBS containing 1× phosphatase and protease inhibitors, and 2 freeze-thaw cycles before downstream analysis.To isolate neuronal exosomes from the heterogenous suspensions, 4 µg biotinylated anti-CD171/L1CAM antibody  in 42 µL 3% Bovine Serum Albumin in DPBS (BSA/DPBS) was added to the resuspended vesicles and incubated for one hour at 4 °C with rotation. Thereafter, 15 µL of pre-washed Streptavidin-Plus UltraLink Resin  in 25 µL 3% BSA/DPBS was added to each sample and incubated for 4 h at 4 °C with rotation. Neuronal exosomes bound to the antibody/resin complex were recovered by centrifugation at 200× The ELISA kit from Cusabio  was used to measure CD81. Briefly, 100 μL of sample or standard prepared in glycine/M-PER buffer  was added to pre-coated wells and incubated for 2 h at 37 °C with shaking at 300 rpm. Thereafter, 100 μL of biotinylated secondary antibody was added and incubated for 1 h at 37 °C with shaking at 300 rpm. Wells were washed three times before addition of 100 μL HRP-avidin per well. Plates were incubated for 1 h at 37 °C with shaking at 300 rpm, and then washed five times before 90 μL TMB substrate was added and incubated at 37 °C. Plates were developed for 15 min, stopped by the addition of 50 μL stop solution, and absorbance measured at 450 nm in a CLARIOstar plate reader . For the experiments shown, the LLoQ was between 0.31 and 1.25 ng/mL.Tau phosphorylated at threonine 181 was measured using the Innotest ELISA . Briefly, 75 μL sample (diluted 1:2 in assay buffer) or standards was added to each well together with 25 μL of Conjugate Working Solution 1 provided by the manufacturer. Plates were agitated overnight at 4 °C and 300 rpm. After 5 washes, 100 μL Conjugate Working Solution 2 was added to each well and plates were incubated for 1 h at 25 °C. Next, wells were washed five times and 100 μL of Substrate Working Solution was added to each well and left for 30 min at 25 °C in the dark. Thereafter, 50 μL of Stop Solution was added to each well and absorbance determined at 450 nm. Standard curves were fitted and LLoQs determined as with the mid-region assay. CSF samples provided by the manufacturer were added as internal controls and used to monitor plate-to-plate variability. Values for the internal controls were within the defined concentration range and the plate-to-plate variance was low (70.2 and 83.4 pg/mL). For the experiments shown, the LLoQ was 24.5 pg/mL.For sample types in which the levels of FL tau were too low to measure by ELISA , tau was measured using a Simoa -based version of our FL assay and a Simoa HD-1 platform . The C-terminal antibody, TauAB (a generous gift from Andy Billinton and Mike Perkinton of MedImmune), was used for capture and the N-terminal Tau12-biotin antibody  for detection. Beads were activated with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide  and coated with TauAB. Tau12 was biotinylated using N-hydroxysuccinimide-polyethylene glycol (NHS-PEG4)-biotin . Standards  and blanks were prepared using sample buffer and assayed in triplicates while samples were assayed in duplicate. Standard curves were fitted and LLoQs determined as with the mid-region and p181 tau assays. For the experiments shown, the LLoQ of the FL Simoa assay was 0.74 pg/mL. Full validation of this assay will be described in a separate publication .To enable the measurement of Tsg101 in exosome lysates, we developed an in-house Meso Scale Discovery (MSD)-based assay. The wells of MSD GOLD 96-well streptavidin plates  were blocked with 150 µL of 5% Blocker A  in TBST for 1 h at room temperature and gentle agitation on an orbital shaker (300 rpm). Plates were washed three times with 1× Tris wash buffer  and 25 µL/well of biotinylated mouse anti-Tsg101 (clone 4A10—2 µg/mL) capture antibody  was added in 1% Blocker A/TBST. Coating was carried out for 1 h at room temperature with shaking at 300 rpm, and plates were washed three times with 1× Tris wash buffer before adding 25 µL/well standards or samples in triplicate. Standards were prepared by diluting recombinant Tsg101 protein  in glycine/M-PER buffer: 400,000 ng/mL, 100,000 ng/mL, 40,000 ng/mL, 20,000 ng/mL, 10,000 ng/mL, 5000 ng/mL, and 2500 ng/mL. After incubation for 1 h at room and gentle agitation (300 rpm), plates were washed three times with wash buffer and incubated for 1 h at room temperature and 300 rpm with rabbit SULFO-tag anti-Tsg101 detection antibody (1 µg/mL)  diluted in 1% Blocker A/TBST. Plates were then washed 3× with wash buffer before addition of 1× Read Buffer  to allow for electrochemiluminesence detection (150 µL/well). A Sector imager was used to measure the intensity of emitted light, thus allowing the quantitative measurement of analytes present in the samples. The LLoQ for this assay, calculated as described for the other tau immunoassays, was between 2.5 and 5 ng/mL.Blood-derived neuronal exosomes diluted 1:200 in filtered DPBS were injected into a NanoSight LM10  to assess vesicle size and concentration. Each sample was measured in duplicate, with 5 × 1 min nanoparticle tracking analysis videos captured at a constant flow rate of 40 μL/min. Extracellular vesicles were detected at a camera gain of 10 and threshold value of 5. Resultant concentration and size distribution data were averaged within-sample using 10 measurements . To determine global distribution of all vesicles measured, particle counts were binned into 5 nm intervals and each interval expressed as a percentage of total particles analyzed within a given sample.


Vibrio gazogenes ATCC 43942 has the potential to synthesize a plethora of metabolites which are of clinical and agricultural significance in response to environmental triggers. The complete genomic sequence of Vibrio gazogenes ATCC 43942 is reported herein, contributing to the knowledge base of strains in the Vibrio genus. Vibrio gazogenes is an estuarine Gram-negative bacterium that is known for its ability to synthesize industrially relevant proteins, such as amylases and proteases  that were able to inhibit aflatoxin synthesis in the aflatoxin producer Aspergillus parasiticus . Quiver was subsequently used to polish the assembly. The finished genomic sequences were annotated with NCBI’s Prokaryotic Genome Annotation Pipeline. A high-quality finished version of the V. gazogenes genome is reported here as two circular chromosomes and one circular plasmid, with a mean coverage of 185×, with features as follows: (i) chromosome 1 (denoted as Chr_1), size, 3,471,064 bp; GC content, 45.5%; 2,988 proteins; 25 rRNAs; 87 tRNAs; 4 noncoding RNAs (ncRNAs); 3,153 genes; and 49 pseudogenes; (ii) chromosome 2 (denoted as Chr_2), size, 1,303,572 bp; GC content, 44.9%; 1,102 proteins; 4 tRNAs; 1,138 genes; and 32 pseudogenes; and (iii) a plasmid (denoted as P_1), size, 11,916 bp; GC content, 45.2%; 22 proteins; 23 genes; and 1 pseudogene.Genomic DNA extraction (10 to 20 μg) was performed using the PureLink genomic DNA minikit (Invitrogen). The extracted DNA was quantified using a NanoDrop 1000 (Thermo Scientific), and the quality of the DNA was assessed by running a 1% agarose gel with the DNA gel stain SYBR safe  and visualized in a ChemiDoc MP system (Bio-Rad). DNA sequencing was performed on the Pacific Biosciences RSII platform. A single-molecule real-time (SMRT) cell, yielding 73,434 postfiltered polymerase reads and having an V. gazogenes ATCC 43942, coupled with its ability to produce unique antibiotics and mycotoxin inhibitors under custom-designed environmental settings makes this strain “chemically gifted.” In this context, its finished genomic sequence provides a necessary point of comparison with other V. gazogenes strains and bacterial species within the Vibrio genus for elucidation of the molecular factors that govern its unique metabolic profile.The utility of prodigiosins that are synthesized by V. gazogenes ATCC 43942 has been deposited in NCBI GenBank under the accession numbers CP018835, CP018836, and CP018837.The sequence of


While the safety risk of methanol and tough process control for methanol induction usually cause problems especially in large-scale fermentation. By testing the functions of trans-acting elements of PAOX1 and combinatorially engineering of them, we successfully constructed a methanol-free PAOX1 start-up strain, in which, three transcription repressors were identified and deleted and, one transcription activator were overexpressed. The strain expressed 77% GFP levels in glycerol compared to the wide-type in methanol. Then, insulin precursor (IP) was expressed, taking which as a model, we developed a novel glucose-glycerol-shift induced PAOX1 start-up for this methanol-free strain. A batch phase with glucose of 40 g/L followed by controlling residual glucose not lower than 20 g/L was compatible for supporting cell growth and suppressing PAOX1. Then, glycerol induction was started after glucose used up. Accordingly, an optimal bioprocess was further determined, generating a high IP production of 2.46 g/L in a 5-L bioreactor with dramatical decrease of oxygen consumption and heat evolution comparing with the wild-type in methanol. This mutant and bioprocess represent a safe and efficient alternative to the traditional glycerol-repressed/methanol-induced PAOX1 system.The  Pichia pastoris is a widely used expression system beyond Escherichia coli for production of heterologous proteins1alcohol oxidase 1 promoter (PAOX1) to produce foreign proteins at high levelsAOX1; ability to grow high cell-density with rapid growth rate and almost protein-free medium; elimination of negative effects of endotoxin and bacteriophage; diverse posttranslational modifications like polypeptide folding, glycosylation, methylation, acylation, proteolytic adjustment, and targeting to subcellular compartments146589101112The methylotrophic yeast P. pastoris, the use of alternative promoters, such as PGAP, PICL1 and PPHO89, have been proposed1415AOX1, mainly because of their weak expression ability or uncontrolled behavior. Rewiring the PAOX1 has led to various activations of PAOX118cis-acting approach on rewiring PAOX1 has been reported by several groups, trans-acting approach has not been well studied currently. As reported currently, several trans-acting elements have been reported to regulate methanol induction, glucose or glycerol derepression in P. pastoris. Three transcription factors, Mit1, Mxr1, and Prm1, which positively regulated PAOX1 in response to methanol2021AOX1 at different sites and did not interact with each otherAOX1 through a cascadeAOX1 in response to glucose. Nrg1 suppressed PAOX1 in glucose and glycerol by directly binding to five sites of PAOX1, including two binding sites of transcriptional activator Mxr1In order to avoid the indispensable of methanol in AOX1 in P. pastoris, there is still a long way to go, especially for the industrial use. In this work, we are committed to engineer a new methanol-free P. pastoris by modifying transcription factors of AOX1 promoter and developing its accessional bioprocess control for protein expression. Accordingly, an efficient glucose-glycerol-shift induction in the newly constructed strain was established to replace the traditional glycerol-methanol induction in the wild-type for protein expression. It exhibited an economic, safe and environment-friendly pattern comparing with the traditional methanol-dependent expression system, and so that holds great potential for industrial application.Although many efforts have been made to facilitate methanol-free heterologous protein expression driven by PS. cerevisiae, ScMig1, ScMig2 and ScMig3 are known to repress the transcription of genes involved in other carbon source metabolism in the presence of glucose26P. pastoris genome2His2 proteins, which located close to the N-terminus . Accordingly, high levels of AOX were detected in wild-type and the mutants in response to 0.5% methanol, no AOX activity was detected in any of the cells in the presence of 1% glucose Candida boidinii (glucose repressed activity of the AOD1 promoter even with methanol existed)In order to elucidate their roles in the regulation of P glucose . The expP. pastoris. As for glucose repression, it has been widely reported and is mediated by several transcriptional repressors including Mig1, Mig2, and Nrg130etc30GAL1, SUC2, MAL) and recruit a co-repressor, the Ssn6-Tup1 complex to repress their transcription30SUC2, HXT2, PCK1)30P. pastoris.It seemed that glucose repression was more strict than glycerol repression on AOX expression in AOX1, which was proved to be closely related with different repression effects on AOX expression by glycerol in P. pastoris and H. polymorphaH. polymorpha Mpp1, but it shows low identity to Mpp1 and other proteins with undetermined function. Interestingly, expressing Mpp1 in Δmit1 cells showed remarkable AOX expression in the presence of glycerol in P. pastoris. Thus we analyzed the functions of different domains in Mit1 and verified that the structural dissimilarity between Mit1 and Mpp1 contributed to the differential repression of AOX1 promoter and MOX promoter in the presence of glycerol, respectively, in P. pastoris and H. polymorpha. As complementing Mpp1 in Δmit1 cells derepressed AOX expression in glycerol in P. pastoris, Mig1, Mig2 and Nrg1 should not function for various glycerol repressions in different strains. Besides, as Mig1, Mig2 or Nrg1 functioned widely in catabolite repression, they might regulate Mit1 expression by binding to promoter of MIT1 or interact with Mit1 protein, which further functioned for glycerol repression. They may also bind to promoters of other genes involved in methanol utilization. For catabolite repression, the glucose repression is typically and widely existed in various organisms, thus Mig1, Mig2 and Nrg1 may regulate genes involved in methanol utilization more finely and strictly when cells grown in glucose than in glycerol. However, it still needs substantial works to clarify the different repression mechanisms mediated by glucose and glycerol.However, few reports were on glycerol repression mediated by Mig1, Mig2, and Nrg1. In our previous work, we have identified a transcription activator Mit1 of PAOX1 in response to methanol. We also identified an activator Mit1 participating in PAOX1 regulationAOX1 at different sites and did not interact with each other. However, these factors cooperatively activated PAOX1 through a cascadeMXR1 and PRM1 remained similar in all the cells , cells cultured in glycerol achieved 22% and 36% of AOX activity compared to wild-type cells cultured in methanol. As the AOX activity in Δmig1Δmig2Δnrg1 cells cultured in glycerol only reached 9% and 17% of wild-type cells in methanol, overexpression of Mit1 had a clear impact on the expression of AOX. To evaluate the potential utility of expressing recombinant proteins in MF1, we measured GFP expression controlled by the AOX1 promoter. As shown in AOX1 was more strict than that of glycerol. It seemed that the relative expression level of GFP was much higher than that of AOX in the recombinant strains when grown on glycerol. As is known, methanol usually acts as a strong peroxisome proliferator for Pichia pastorisP. pastoris cells in glycerol may be weaker than that in methanol, which further leads to lower level of AOX content. Differently, the expressed GFP distributed freely in cytoplasm regardless of peroxisome proliferation in various carbon sources.Based on these observations, we overexpressed Mit1 under the  mutants , in accoAOX1 is the tight response of methanolAOX1 controllable to express heterologous protein toxic to hostAOX1 controllable, the intensity of PAOX1 in different concentrations of glucose was determined by AOX activity assay reflected by colorimetrical assayAOX1 in the MF1 could be highly suppressed at glucose concentration over 2%.One major advantage of PP. pastoris grow in a fed-batch manner to a high cell density in order to obtain a high productivityFor protein expression in the wild-type, the typical fermentation process usually follows a three-stage strategyAOX1 in the MF1 cells could be suppressed by high concentration of glucose, derepressed by glycerol or low concentration of glucose. Thus, a novel methanol-independent fermentation process was then designed by a three-stage strategy: (1) a batch phase for biomass growth with initial glucose of 40 g/L, suppressing PAOX1; (2) a fed-batch phase for further biomass enhancement with glucose feeding to maintain residual glucose not lower than 20 g/L, suppressing PAOX1; and (3) glucose used up and glycerol feeding induced the expression of heterologous protein driven by PAOX1 as the carbon source without methanol in a fed-batch mode.According to the results of AOX1 promoter could be turned off/on by glucose-glycerol-shift in our new methanol-independent Pichia system.In order to determine the fermentation process of glucose growth phase, the MF1 producing a secreted insulin precursor (IP) was constructed and cultured in a 5-L bioreactor by different glucose feeding strategies. As shown in Although the novel induction strategy of glucose-glycerol-shift was proved to be feasible, the cell growth and metabolism in MF1 must be different from the WT because genes were reformed and carbon sources during different phases were altered. Thus we optimized the fermentation bioprocess  for this new system .Then, the whole fermentation processes were evaluated between the engineered methanol-free system and the wild type methanol-induced system under their specific optimal culture conditions. As shown in OUR, kLa and viscosity were analyzed. According to OUR of the WT-IP reached 464.7 mmol/Lbroth/h, which reached 211% of that of the MF1-IP. This result accorded with oxygen consumption, mainly ascribed to huge oxygen requirement and heat release of methanol oxidation. Moreover, the kLa of the WT-IP increased faster than that of the MF1-IP during the growth phase because the rotation speed of the WT-IP increased faster. The kLa of the WT-IP was much higher than that of the MF1-IP in induction phase, which was kept between 729.9 1/h and 1128.1 1/h. Interestingly, the kLa of the MF1-IP kept declining rapidly during the induction phase, which decreased from 678.2 1/h to 242.5 1/h despite that the OUR increased. Accordingly, the oxygen concentration in the gas inlet kept increasing to 77% in the late phase of fermentation. Therefore, the oxygen transfer in bioreactor became worse and worse with MF1-IP culture going on. As shown in It is curious that the MF1-IP also needed pure oxygen inlet after 65 h, despite the oxygen consumption of it was only half of that of the WT-IP. To clarify this, Then we need to find out the reason for the sharp increase of viscosity during the late phase. Thus the relationship between broth viscosity and WCW of different batches of the MF1-IP was analyzed. As shown in As for the WT-IP, its fermentation process has been optimized for many years in our group and is the highest IP production reported so far, which has been applied in a biopharmaceutical company. Although the IP production by MF1-IP was lower than by WT-IP here, it is still a high titer and is higher than other reported results even from the wild-type methanol strategies . To furtGFP-MIG1 fusion gene under a constitutive GAP promoter in the wild type strain P. pastoris GS115. Three strains of Δmig1, Δmig2 and Δnrg1 were constructed by double crossover homologous recombination to evaluate the repression roles of the transcription factors Mig1, Mig2 and Nrg1 in regulating PAOX1. Then the Δmig1 and Δmig2 strains were crossed and diploid hybridsmig1Δmig2 double-deletion mutant. Furthermore, a Δmig1Δmig2Δnrg1 treble-deletion strain was constructed by the deletion of the NRG1 gene in the Δmig1Δmig2 double-deletion strain. Two strains overexpressing transcription activator genes MIT1 and PRM1 were constructed to test their promotion effects on PAOX1 and designated as WT-Mit1 and WT-Prm1, respectively. The Mit1 was then overexpressed in the strains of Δmig1Δmig2 and Δmig1Δmig2Δnrg1, respectively, and generated Δmig1Δmig2-Mit1 and Δmig1Δmig2Δnrg1-Mit1 (MF1). The green fluorescent protein was overexpressed in the wild-type, Δmig1Δmig2-Mit1 and MF1, which resulted in WT-GFP, Δmig1Δmig2-Mit1-GFP and MF1-GFP, respectively. A gene encoded for insulin precursor (IP)Standard procedures for the manipulation of plasmid DNA were described beforeAOX1 in WT and P. pastoris MF1 strains was monitored. Cells were cultured in yeast nitrogen base medium (0.67% YNB) containing 1% glucose (YND), 1% glycerol (YNG) or 0.5% methanol (YNM), respectively. Cells of each strain were pre-grown overnight in YND medium and transferred into the YND, YNG and YNM media at an initial OD600 of 1.0. The quantification levels of GFP expression of each strain were the highest during the growth process respectively. For western blot analysis, yeast cells were first grown overnight in YPD medium to stationary phase. Aliquots of the stationary culture were used to inoculate an appropriate volume of YND medium containing any necessary supplements. Cells were grown to an optical density at 600 nm (OD600) of approximately 2~6 and then collected, washed three times by sterile water. For induction purpose, cells were transferred to YNB media with indicated carbon sources and nutritional supplements to a final optical density at OD600 of 1.0. The cells were grown with vigorous shaking at 30 °C for 10 h, cell pellets were harvested and then processed for cell extract preparation and western blot analysis as described previouslyMIT1, MXR1 and PRM1 in WT, Δmig1, Δmig2, Δnrg1, Δmig1Δmig2 and Δmig1Δmig2Δnrg1 were verified by RT-PCR. Cells were prepared as described in western blot analysis except that cells were induced in glycerol for 6 hours. RT-PCR was carried out as described previously−ΔΔCT methodACT1) as the endogenous reference genesg, and supernatant was discarded; weight the total weight of the tube and precipitation; the weight of the tube was subtracted from the weight of the tube and precipitation; the weight left was the WCW, g/L. The IP titer was determined by HPLCEnzyme assays of alcohol oxidase and fluorescence measurement were adapted from previous studies23P. pastoris MF1-IP cells (OD600 = 7.0–8.0) stored in −80 °C were inoculated directly into 500-mL shake flasks containing 100 mL YND medium as the first seed culture. After cultivated at 30 °C and 200 rpm for 36 h, 37.5 mL first seed culture was inoculated into a 1 L shake flask containing 300 mL YND to obtain the second seed culture. In 5 L bioreactor, culture was started with a glucose growth phase with 3.5 L basal salt medium, which contains (per liter): 13 mL H3PO4 (85%), 0.82 g CaSO4, 18.2 g K2SO4, 14.9 g MgSO4·7H2O, 10.6 g KOH, 13.2 g (NH4)2SO4, 40.0 g glucose, 0.33 mL antifoam, and 4.5 mL PTM1 trace salts. The PTM1 trace salts stock solution contains (per L): 6.0 g CuSO4·5H2O, 0.08 g NaI, 3.0 g MnSO4·H2O, 0.2 g Na2MoO4·2H2O, 0.02 g H3BO3, 0.5 g CoCl2, 20.0 g ZnCl2, 65.0 g FeSO4·7H2O, 0.2 g biotin, and 5.0 mL of H2SO4. During glucose growth phase, 50% (w/v) glucose containing 12 mL PTM1 per liter was fed with a feeding rate of 50 mL/h. After wet cell weight (WCW) reaching about 200 g/L and glucose was almost exhausted, glucose growth phase was finished and then switched to glycerol fed-batch phase. Feeding medium was 50% (w/v) glycerol containing 12 mL PTM1 per liter. During the fermentation process, the temperature was maintained at 30 °C. The pH was kept at 5.0 by adding 25% ammonia solution, and the dissolved oxygen (DO) was kept above 30%. The WT-IP strain was cultured as a control following the previously described method3PO4 (85%), 0.82 g CaSO4, 18.2 g K2SO4, 14.9 g MgSO4·7H2O, 10.6 g KOH, 13.2 g (NH4)2SO4, 300 g glycerol, 0.33 mL antifoam, and 4.5 mL trace salt stock solution. The continuous culture was performed for at least five resident times to reach steady-state conditions. The working volume was kept constant by balancing medium feeding and broth removing.For fed-batch culture, two milliliters of 2 and O2 concentration of in-gas and off-gas were determined by exhaust gas analyzer , while the mass flow of in-gas and off-gas was monitored by a mass air flow meter.The COThe oxygen consumption rate was calculated by the equation below:oc is oxygen consumption rate, L/h/L fermentation broth; F is volume flow under standard state  of the gas, L/h; O2in, O2out is volume content of the in-gas and off-gas, %, respectively; Vfer is volume of the fermentation broth, L; N2in, N2out is the nitrogen concentration in the gas inlet and gas outlet, respectively.where νThe specific oxygen consumption to WCW and the specific oxygen consumption to IP were calculated by the following equation:μox is specific oxygen consumption to WCW, L/h/g wet cell; WCW is wet cell weight, g/L; μop is specific oxygen consumption to IP, L/h/g IP; p is concentration of IP in supernatant, g/L fermentation broth;where The total heat released was calculated by the following equationQgr is total heat released, kcal/L fermentation broth; ∆Hf = 0.11 kcal/mmol O2 is a proportionality constant; Qox is total oxygen consumption (mmol/L fermentation broth).where The rate of the heat evolution was calculated by the following equationQH is specific rate of heat evolution, cal/g wet cell/h; −∆HO* = 106 cal/mmol O2 is heat of combustion per available electron equivalent; 2/g wet cell/h.where OTR were assumed to equal OUR and were calculated as follows:OTR is the oxygen transfer rate, mmol/L/h; OUR is the oxygen uptake rate, mmol/L/h; Vm is the molar volume of gas, 22.4 L/mol; F is volume flow under standard state  of the gas, L/h; Vfer is the volume of fermentation broth; O2in, O2out is the oxygen concentration in the gas inlet and gas outlet, %, respectively; N2in, N2out is the nitrogen concentration in the gas inlet and gas outlet, respectively.where KLa was determined by the equation below:CO2,S is the oxygen concentration at saturation in the liquid phase, mmol/L; CO2,L is the oxygen concentration in the liquid phase, mmol/L; KLa is the mass-transfer coefficient, 1/h.where Oxygen concentration at saturation under fermentation conditions varies with the fermenter overhead pressure and oxygen concentration in gas, and it can be calculated using Henry’s lawP is the fermenter overhead pressure, mbarg; H is Henry’s law proportionality constant, 4.75 × 104 atm/mol/mol (water at 30 °C).where How to cite this article: Wang, J. et al. Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris. Sci. Rep.7, 41850; doi: 10.1038/srep41850 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Imprinting disorder during somatic cell nuclear transfer usually leads to the abnormality of cloned animals and low cloning efficiency. However, little is known about the role of donor cell imprinting in the development of cloned embryos. Here, we demonstrated that the imprinting (H19/Igf2) in porcine fetus fibroblasts derived from the morphologically abnormal cloned fetuses  was more hypomethylated, and accordingly, significantly higher H19 transcription and lower Igf2 expression occurred in comparison with those in fibroblasts derived from morphologically normal cloned fetuses  or donor fetus fibroblasts (the control group). When these fibroblasts were used as donor cells, the abnormal imprinting group displayed an even lower imprinting methylation level, in correspondence to the significantly downregulated expression of Dnmt1, Dnmt3a and Zfp57, and a markedly reduced blastocyst rate, while the normal imprinting group took on the similar patterns of imprinting, gene expression and embryo development to the control group. When 5-aza-dC was applied to reduce the fibroblasts imprinting methylation level in the normal imprinting group, cloned embryos displayed the more severely impaired imprinting and significantly lower blastocyst rate. While the upregulated H19 transcription in the abnormal imprinting group was knocked down, the imprinting statuses were partly rescued, and the cleavage and blastocyst rates significantly increased in cloned embryos. In all, donor cell imprinting disorder reduced the developmental efficiency of cloned embryos. This work provides a new insight into understanding the molecular mechanism of donor cells regulating the cloned embryo development. Somatic cell nuclear transfer (SCNT) has achieved in many species, owning a broad application prospect in the basic research, agriculture, biomedicine, etc . HoweverIt is generally believed that the developmental abnormalities of cloned animals and low cloning efficiency are largely due to the imprinting disorder . ImprintNaturally, genomic imprinting is erased and established during gametogenesis and faithfully maintained throughout the subsequent embryo development in the normal reproduction . Indeed,At present, H19/Igf2, representing genomic imprinting, is widely studied and critical for the normal embryo development . Igf2 paNumerous studies have demonstrated that H19/Igf2 imprinting problems constrain the cloning efficiency –17, and Our previous study has shown that 4 of 6 porcine cloned fetuses were morphologically abnormal and their imprinting levels were hypomethylated [P < 0.05, Figure To investigate whether donor cell imprinting plays a critical role in the development of cloned embryos, PFFs a2 , and disposable and sterile plasticware was obtained from Nunclon , unless otherwise stated. All experiments were approved by the Animal Care Commission of Qingdao Agricultural University according to animal welfare laws, guidelines and policies. All surgery was performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.in vivo fertilized or cloned fetuses were obtained from sows after anaesthetized and sacrificed at day 35 of pregnancy, then PFFs were isolated from the fetuses under sodium pentobarbital anaesthesia. After the removal of fetal head, internal organs and limbs, the remaining tissues were finely minced into pieces, digested with 0.25% trypsin-0.04% ethylenediaminetetraacetic acid solution (GIBCO), and dispersed in high glucose enriched Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin (GIBCO). The dispersed cells were centrifuged, resuspended and cultured in DMEM. Until confluence, PFFs were digested, centrifuged, resuspended in FBS containing 10% dimethyl sulfoxide and stored in liquid nitrogen until use. Prior to SCNT, PFFs were thawed, cultured and used in 2–3 passages.Donor cell culture has been described previously . BrieflyFor 5-aza-dC treatment , PFFs we2O to the concentration of 20 μM. Before transfection, PFFs were cultured in 400 μl Opti-MEM (GIBCO), 1.5 μl Lipofectamine 2000  was added into 50 μl Opti-MEM and incubated at room temperature for 5 min, 20 μM H19 siRNA was diluted into 500 nM with Opti-MEM, and, 100 μl siRNA-Lipo 2000 complexes was obtained through a mixture of 50 μl Opti-MEM with 500 nM siRNA and 50 μl Opti-MEM with 1.5 μl Lipo 2000, incubated at room temperature for 30 min and added into each 24-well culture plate with PFFs and 400 μl Opti-MEM. After 6 h, the medium including siRNA-Lipo 2000 complexes was replaced by DMEM containing 10% FBS and 1% penicillin-streptomycin. The interference efficiency was examined at 6 h, 12 h, 24 h, 36 h, 48 h or 72 h posttransfection, respectively. The negative siRNA with the same amount was transfected as a control.For H19 knockdown, methods of siRNA design, synthesis and transfection have been reported in our study . AccordiAfter 5-aza-dC treatment or H19 siRNA transfection, PFFs were harvested at 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h or 120 h, respectively, and the cell number at every time point was determined with a hemacytometer. Then, the manner of 5-aza-dC treatment with the obvious imprinting hypomethylation or siRNA transfection with H19 significant knockdown but no notable effect on cell proliferation was applied in the subsequent experiment.Oocyte maturation has been reported . BrieflyThe procedure for SCNT has been described in our reports , 31. BriFor blastocyst cell number, cloned embryos at 156 h postactivation were treated with acidic Tyrode's solution to remove zona pellucida, fixed in 4% paraformaldehyde for 30 min, and stained with 10 μg/ml Hoechst 33342 for 5 min in the dark. After staining, cloned blastocysts were washed and mounted on slides. Then, blastocyst cell number was examined under ultraviolet light from a fluorescence microscope.4 PFFs or 50 pooled embryos at each stage using an RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions, and the elution volume was 50 μl. Reverse transcription was performed using a PrimeScript RT Reagent Kit (TaKaRa). The 100 μl reaction volume contained 20 μl 5 × PrimeScript Buffer, 5 μl PrimeScript RT Enzyme Mix I, 5 μl Oligo dT Primer (50 μM), 5 μl Random 6 mers (100 μM), 50 μl Total RNA and 15 μl RNase Free dH2O. The reaction condition was 37°C for 15 min and 85°C for 5 sec, and the cDNA was stored at −20°C until use. For quantitative real-time PCR, reactions were performed in 96-well optical reaction plates (Applied Biosystems) using SYBR Premix ExTaq II (TaKaRa) and a 7500 Real-Time PCR System (Applied Biosystems). Each reaction mixture (20 μl) contained 2 μl cDNA solution, 10 μl 2 × SYBR Premix Ex Taq II, 1.6 μl PCR primers (10 μM), 0.4 μl ROX Reference Dye II (50×) and 6 μl dH2O. Thermal cycling conditions were 95°C for 30 sec, 40 two-step cycles of 95°C for 5 sec and 60°C for 34 sec, and finally a dissociation stage consisting of 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. For each sample, the cycle threshold (CT) values were obtained from three replicates. The primers used for the amplification of target and internal reference genes were presented in −ΔΔCT method.Measurement of gene expression with quantitative real-time PCR has been applied in our studies , 32. BriTM Kit (Zymo Research) according to the manufacturer's protocol. For PFFs, a Universal Genomic DNA Extraction Kit (TaKaRa) was used to extract genomic DNA. For samples of 50 4-cell or 10 blastocyst stage pooled zona pellucida-removed cloned embryos in each group, digestion was performed in M-Digestion Buffer plus with Proteinase K at 50°C for 20 min. After treatment, a cytosine to thymine conversion was carried out at 98°C for 10 min and 64°C for 2.5 h. Then, the samples were desalted, purified and diluted with M-Elution Buffer. Subsequently, nested PCR was carried out to amplify DMR3 of H19/Igf2 using the previously reported primers as described in TM Polymerase (TaKaRa) with a profile of 94°C for 5 min, 40 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min, followed by 72°C for 10 min. Products from the first amplification reaction were used in the second PCR reaction, and the optimal annealing temperature of inner primers was 50°C. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa), and the purified fragments were cloned into a pMD18-T Vector (TaKaRa) and subjected to sequence analysis.Bisulfite sequencing has been reported . Brieflyt-test when two groups were compared. For all analyses, differences were considered to be statistically significant when P < 0.05.Differences in data (mean ± SEM) were analyzed with the SPSS statistical software. Statistical analyses of data concerning embryo development, gene expression and cell proliferation were performed with one-way ANOVA or


Jasmonic acid deficiency in the lodicule leads to scattered floret opening time in cytoplasmic male sterile rice by retarding lodicule expansion through reduced accumulation of osmotic regulation substances and water. OsAOC expression. Applying JA restored scattered FOT in ZS97A by promoting ORS and water accumulation, and inducing the expansion of the lodicules. Taken together, JA deficiency inhibited lodicule expansion by retarding the accumulation of ORSs and water, leading to scattered FOT in CMS rice ZS97A.Cytoplasmic male sterile (CMS) rice has been widely used for hybrid rice seed production in China. However, CMS rice suffers from undesirable flowering habits including scattered floret opening time (FOT), which causes different FOTs among parental rice plants and greatly reduces hybrid rice seed production. Little is known about the mechanism of scattered FOT in CMS rice. Our results demonstrate that scattered FOT in CMS rice Zhenshan 97A (ZS97A) resulted from the lack of a driving force to open florets, which was directly caused by retarded lodicule expansion. Our results indicate that retarded lodicule expansion in ZS97A was caused by reduced water accumulation due to retarded accumulation of osmotic regulation substances (ORSs). Further, the retardation in accumulation of ORSs and water were caused by jasmonic acid (JA) deficiency, resulting from down-regulation of  Oryza sativa L.) is a major staple food that feeds more than half of the human population  and its derivative jasmonates are important plant hormones involved in plant responses to biotic and abiotic stresses, root growth, sex determination, leaf senescence, and fruit ripening , and its isonuclear maintainer line, Zhenshan 97B (ZS97B) (Oryza sativa L. indica).These results suggest that scattered FOT in CMS rice may result from the lack of a driving force to open the florets, which may be due to JA deficiency in CMS rice florets. However, this suggestion lacks strong evidence, and the mechanism of scattered FOT of CMS rice remains elusive. In order to explore this mechanism, we investigate the lack of a driving force to open the florets, the cause of JA deficiency, and the underlying mechanism of JA deficiency on FOT using an elite CMS rice, Zhenshan 97A (ZS97A)  were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. They were dissolved in a small amount of ethanol and diluted to the desired concentration with deionized water. Control water received the same amount of ethanol alone, but without JA or IBU. The pH values of all treatment solutions were adjusted to 6.5 with 0.1 M HCl or NaOH.Panicles having florets nearing anthesis, in which several florets had flowered before the experimental date, were selected for tests. The selected panicles were carefully immersed in 100-ml flasks containing different treatment solutions for 30 s. The effects of the treatments on lodicule volume, lodicule fresh and dry weights, lodicule water content, filament length and potassium and total soluble sugar (TSS) contents were monitored.V=4/3πabc; where V is volume, π is 3.14, a is half-length, b is half-width, and c is half-height.The lemma and palea were peeled away with forceps before photographing the filament and lodicule using a stereoscopic microscope (Leica S8 APO). The length of the filament, and the length, width and height of the lodicule were measured with ImageJ. Lodicule volume was calculated using the ellipsoid volume formula: Samples with >50 pairs of lodicules were removed and collected from the florets, and the fresh weight of each sample was measured immediately using an analytical balance with readability of 0.1 mg. The samples were oven dried at 105 °C for 30 min and then dried at 80 °C to constant weight. The dry weight of each sample was measured, and the fresh and dry weights of each pair of lodicules were calculated.Each sample with 100 pairs of lodicules was removed and collected from the florets. The potassium content of each sample was determined by atomic absorption spectrometry following the method of Lodicules were collected and stored at −80 °C. The samples were prepared using a modified crude extraction procedure originally reported by Total RNA was extracted from lodicules using an RNAprep Pure Plant Kit  following the instructions in the user manual. First-strand cDNA was synthesized from 1 μg of total RNA using a FastQuant RT Kit .OsLOX genes, four OsAOS genes, one OsAOC gene, ten OsOPR genes, and three OsACX genes in rice have been reported previously (OsLOX3 (LOC_Os03g49260), OsAOS3 (LOC_Os02g12680), and OsAOS4 (LOC_Os02g12690) are not involved in JA biosynthesis in rice  and quantitative RT-PCR (qRT-PCR).As shown in 13JXB online. The rice OsACTIN (NM_001057621) gene fragment was used as an internal control in the RT-PCR analysis. The OsACTIN fragment was amplified at 94 °C for 3 min, followed by 24 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s, and an extension at 72 °C for 10 min. The OsLOX1, OsLOX2, OsLOX4, OsLOX5, OsLOX6, OsLOX7, OsLOX8, OsLOX9, OsLOX10, OsLOX11, OsLOX12, OsLOXL-2, OsOPR2, OsOPR3, OsOPR4, OsOPR5, OsOPR6, OsOPR8, OsOPR9, and OsOPR10 fragments were amplified at 94 °C for 3 min, followed by 38 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s, and an extension at 72 °C for 10 min. The OsAOS2, OsOPR1, OsOPR7, OsACX1, OsACX2, and OsACX3 fragments were amplified at 94 °C for 3 min, followed by 36 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s, and an extension at 72 °C for 10 min. The OsAOS1 fragment was amplified at 94 °C for 3 min, followed by 32 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s, and an extension at 72 °C for 10 min. The OsAOC fragment was amplified at 94 °C for 3 min, followed by 27 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s, and an extension at 72 °C for 10 min.The expression levels of JA biosynthetic genes were analysed by RT-PCR using the primers listed in CT) method, with OsACTIN as the internal control.The JA biosynthetic gene expression levels were also determined by qRT-PCR analysis on an iQ5 Real-Time PCR Detection System . The primers used for qRT-PCR are listed in t-test, or one-way ANOVA followed by Duncan’s multiple range test with at least three replicates. Values of P<0.05 were considered statistically significant. All data were expressed as means±SE.Statistical analysis was performed using an independent samples ZS97B florets opened from 09.30 to 14.00 h . FloweriImmediately before the floret opening, the filaments of ZS97B florets extended rapidly, and the fully elongated 10.34 mm-long filament pushed the anther out of the glume , D. HoweFilaments extended and the lodicules swelled rapidly and simultaneously immediately before floret opening in ZS97B , D, E, sThe driving force for floret opening was investigated in ZS97B florets to test whether scattered FOT was caused by retarded filament extension and/or lodicule expansion in ZS97A. As shown in −1, respectively, at the beginning of the investigation , whereas their fresh weights increased to 445.70 and 569.50 μg pair−1, respectively at FOT  between lodicule fresh weight, lodicule volume, and water weight in the lodicule (Fresh weights of the ZS97A and ZS97B lodicules were 36.03 and 35.31 μg pairy at FOT . During lodicule . Thus lo−1) in ZS97B lodicules 98 h later, whereas water weight in ZS97A lodicules was only 99.47 μg pair−1 at the same time. The ZS97A lodicules continued to accumulate water for another 26 h. Moreover, the maximum water weight of the ZS97A lodicules was less than that of ZS97B at FOT  between ZS97A and ZS97B lodicules, expression of OsAOC was significantly down-regulated in ZS97A lodicules  in lodicule cells, and that potassium (K) and soluble sugars are the main ORSs in lodicule cells , B. ApplApplying JA strongly and quickly induced ZS97A florets to open, whereas applying IBU significantly inhibited opening of ZS97B florets , B. The Scattered FOT in CMS rice leads to low rates of cross-pollination and low yields of hybrid rice seed production. However, until now, the mechanism of scattered FOT in CMS rice has remained elusive. Here, we provide evidence that scattered FOT in CMS rice ZS97A was caused by retarded expansion of lodicules via reduced water accumulation, which resulted from retarded ORS accumulation caused by JA deficiency in the lodicules.spw1-cls rice mutant are normal, but the lodicules are transformed homeotically to lodicule–glume mosaic organs, thereby engendering cleistogamy  and OsAOC knockdown transgenic rice (AOC-RNAi) are severely reduced compared with that of control rice  analysis.Table S2. Primers used in the quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis.Supplementary_Tables_S1_S2Click here for additional data file.