MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we constructed a transgenic yeast strain harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR). OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, metabolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT and ara2Δ cells under oxidative stress. Furthermore, the survival of OsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. High OsMDHAR expression also improved the fermentative capacity of the yeast during glucose-based batch fermentation at a standard cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing transgenic yeast during fermentation was approximately 25% (0.18 g·g-1) higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsMDHAR expression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity.Monodehydroascorbate reductase  is an important enzyme for ascorbate recycling. To examine whether heterologous expression of  In addition, abiotic and biotic stimuli can lead to further ROS accumulation [In aerobic organisms, oxygen is a double-edged sword; it is absolutely required for normal metabolic growth, yet continuous exposure to oxygen can cause cellular damage. This is because molecular oxygen is continually reduced within cells to reactive oxygen species (ROS), such as superoxide anions , and salicylic acid in B. rapa [2 and O3 in conifer needles [Ascorbate (AsA)/D-erythroascorbate and glutathione are important antioxidants that are maintained in their reduced forms by enzymes in the AsA-glutathione cycle in higher eukaryotes, especially plants. The enzymes in this cycle, including AsA peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), are considered critical for minimizing and/or protecting cells from ROS induced by abiotic stressors . MDHAR b mustard –12. The  mustard . Transcr B. rapa ; strong  B. rapa ; SO2 and needles ; drought needles ; and int needles .Saccharomyces cerevisiae is a eukaryotic unicellular microfungus that is widely distributed in natural environments and in association with various insects, animals, and plants. It plays a critical role in food chains and the carbon, nutrient cycles such as nitrogen and sulfur, and is used to produce food, beverages, chemicals, pharmaceuticals, biocontrol agents, and industrial enzymes as well as in agriculture [S. cerevisiae is the main yeast responsible for biomass-based alcoholic fermentation using sugars derived from rice, wheat, barley, corn, and grape in a commercially important agriculture sector [iculture . S. ceree sector . During e sector . Increase sector . Specifie sector ,23. BecaMDHAR from the rice plant Oryza sativa (OsMDHAR) was cloned, and its function was analyzed in a genetically modified S. cerevisiae strain. Herein, we report that heterologous OsMDHAR expression improves tolerance to ROS-induced oxidative stress and fermentative capacity in S. cerevisiae. Furthermore, these findings improve our understanding of the development of ethanol-tolerant yeast strains and demonstrate the potential application of transgenic yeast in the fermentation field for industrial bioethanol production using glucose-based biomass.Unlike in plants, little is known about the stress response involving MDHAR in yeast. In this study, a cDNA encoding the cytosolic O. sativa seedlings, and the cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR). The OsMDHAR coding region was amplified from the cDNA by PCR using Taq and Pfu polymerases . The reaction conditions were as follows: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 7 min. The primer set used for PCR cloning of the OsMDHAR gene is shown in ® TOPO-TA cloning vector . After sequence confirmation and digestion with EcoRI, the DNA fragment containing the OsMDHAR gene was ligated into the yeast expression vector p426GPD . The construct was transformed into S. cerevisiae BY4741 cells . RT-PCR was performed using Maxime RT-PCR Premix  according to the manufacturer’s instructions. The oligonucleotide primers used were OsMDHAR-F and OsMDHAR-R . PCR ampeference .6 cells per mL) grown overnight at 28°C were inoculated into YPD medium  containing various concentrations of H2O2. The cells were cultured for 16 h at 28°C with shaking (160 rpm). After culture, the optical density at 600 nm (OD600) was measured. To monitor thegrowth kinetics, cells (starting concentration adjusted to 1 × 106 cells per mL) were inoculated into YPD medium supplemented with 5 mM H2O2, and the OD600 was measured at 2-h intervals for the indicated times. For the spotting assay, mid-log phase cells (OD600 ≈ 2.0) were exposed to 20 mM H2O2 for 1 h at 28°C and serially diluted (100 to 10−4). Then, a 5-μL aliquot of each dilution was spotted onto YPD agar plates and incubated for 2–3 days at 28°C. Additionally, to determine the stress sensitivity of mutant cells  grown at 28°C overnight were inoculated into YPD medium containing 10 mM AsA and 10 mM IAA in the absence or presence of 5 mM H2O2. The OD600 was measured at 2-h intervals for the indicated time.Yeast cells in mid-log phase were treated with 10 mM AsA and 10 mM isoascorbate (IAA) for 1 h at 28°C, washed twice with cold phosphate-buffered saline (PBS) to remove residual antioxidants, and suspended in fresh YPD medium. Resuspended cells were exposed to 20 mM H2O2 for 1 h at 28°C and harvested by centrifugation at 4°C. Crude protein extracts were prepared using glass beads. Cells were washed three times with cold PBS and resuspended in lysis buffer  with an equal volume of glass beads . After vigorously vortexing 4 times for 1 min each at 2-min intervals on ice, the lysates were centrifuged at 13,000 × g for 20 min at 4°C, and the supernatants were used as protein extracts. Protein concentration was determined by using Protein Dye Reagent .Mid-log phase cells were incubated in the absence or presence of 20 mM H−1cm−1. One unit is the amount of enzyme that oxidizes 1 nmol of NADH per min at 25°C [MDHAR activity was assayed spectrophotometrically. The assay was performed at 25°C with a reaction mixture containing 50 mM potassium phosphate, pH 7.2, 0.2 mM NADH, 2 mM AsA, 1 unit of AsA oxidase (Sigma-Aldrich), and crude extract. MDHAR activity was measured by monitoring the decrease in absorbance at 340 nm resulting from NADH oxidation. Activity was calculated using an absorbance coefficient of 6.2 mM at 25°C .6 cells per mL were inoculated into YPD medium and cultured for 6 h at 28°C until the cells reached mid-log phase and were then exposed to 20 mM H2O2 for 1 h at 28°C with shaking. The cells were harvested and lysed with glass beads as described above. Total protein (20 μg) was resolved by SDS-PAGE and electrophoretically transferred to a PVDF membrane. The membrane was blocked with 5% non-fat skim milk in Tris/HCl-buffered saline containing 0.05% Tween 20 (TBST), and then incubated at 4°C overnight with each primary antibody appropriately diluted in TBST. The blot was washed four times for 40 min with TBST and then incubated for 90 min at room temperature with appropriately diluted secondary antibody. After washing four times for 40 min with TBST, the bands were visualized using ECL Western Blotting Detection Reagent . The primary antibodies used were against the following proteins: hexokinase (Hxk), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (Ald), alcohol dehydrogenase (Adh), Cu/Zn superoxide dismutase (Sod1), GR, glutathione peroxidase (Gpx), porin [To examine the expression changes in antioxidant proteins and metabolic enzymes, initial cell concentrations with 1 × 10), porin , thiored), porin .2O2 for 1 h at 28°C, harvested by centrifugation, and washed twice with cold PBS. AsA-like content was determined spectrophotometrically. Yeast cells were disrupted with glass beads and 5% m-phosphoric acid. The homogenate was centrifuged at 12,000 × g for 20 min. Total AsA content was determined in a reaction mixture containing 100 μL of supernatant, 500 μL of 150 mM KH2PO4 buffer (pH 7.4) containing 5 mM EDTA, and 100 μL of 10 mM dithiothreitol (DTT) to reduce dehydroascorbate [2O was substituted for DTT and NEM. In both reactions, color was developed by adding 400 μL of 10% trichloroacetic acid, 400 μL of 44% o-phosphoric acid, 400 μL of α, α′-dipyridyl in 70% ethanol, and 200 μL of 30 g·L-1 FeCl3. The reaction mixtures were incubated at 40°C for 1 h and quantified spectrophotometrically at 525 nm. The concentration of DHA was estimated from the difference in the concentrations of total and reduced AsA. The concentrations of AsA and DHA were quantified by generating of a standard curve using AsA from 1 to 10 μM mL-1 [Yeast cells were treated with or without 20 mM Hscorbate  to AsA.  μM mL-1 .2O2 for 1 h at 28°C. The cells were harvested and lysed with glass beads. The intracellular hydroperoxide level was determined by ferrous ion oxidation in the presence of the ferric ion indicator xylenol orange [2O2 for 1 h at 28°C. Treated cells were washed twice with PBS and visualized by fluorescence microscopy  [Cells were cultured for 6 h at 28°C until they reached mid-log phase and then exposed to 20 mM Hl orange . Fifty m 525 nm) . Protein6 cells per mL) grown at 30°C overnight were inoculated into YG medium containing 20% glucose and 1% yeast extract and then fermented under aerobic conditions on a shaker (160 rpm) for 72 h at 30°C. The alcohol concentration was determined based on the percentage (v/v) of alcohol in the distillate after fermentation, which was measured using an alcohol hydrometer . The residual glucose concentration was measured with a hand-held Refractometer N1 . Alcohol and residual glucose were measured after removing cell debris by centrifugation . Growth was monitored for 24 h under the same conditions. To investigate cell survival during fermentation, cells were harvested at three time points  and serially diluted to 10−9 with deionized distilled water, and then 5 μL of each dilution was plated onto YPD agar, incubated for 3 days at 30°C, and then photographed. During fermentation, MDHAR expression was analyzed at various time points by western blotting as described above.Cells . The results of the spotting, growth kinetics, and fermentation assays are representative of at least two independent experiments performed under identical conditions.Significant differences in the measured parameters were identified using Origin Pro 8.0. Means were considered to be significantly different when MDHAR from O. sativa (OsMDHAR) was subcloned into the yeast expression vector p426GPD, which allows constitutive expression of a target gene under the control of the yeast GPD  promoter , whereas no amplification signal was detected in wild-type cells transformed with vector alone  of promoter . To deteor alone . To inveWT cells . There w mM H2O2 . To confhe cells . The strWT cells . These r2O2; however, there was no difference between TC and WT cells cultured under normal conditions  were hypersensitive to oxidative stress (por1Δ cells in growth kinetics and spotting assays was slower than that of WT cells without an empty vector (BY cells) in the presence of H2O2; however, there was no difference between these cells under normal condition , heat shock, metals , heavy metals , acids , and high salinity (NaCl)  . The expy (NaCl) . This hiy (NaCl) . Thus, TOsMDHAR-expressing cells to eliminate free radicals. Since AsA-like content is an indication of the capacity of cells to manage exogenous stressors, we investigated a yeast strain in which the EAA biosynthesis-related gene ARA2 was deleted (ara2Δ). Deletion of ARA2 was confirmed by evaluating empty vector (p426GPD)- and OsMDHAR::p426GPD-transformed ara2Δ cells  by immunoblotting , are antioxidant molecules that act as scavengers of free radicals and are essential for resistance and adaptation to abiotic stresses in yeast . AsA is TA cells . The impnditions . Ara2Δ cBY cells , and theBY cells . These rtic acid .OsMDHAR were more tolerant to oxidative stress than WT cells, these cells were tested in glucose-based batch fermentation in YG medium under aerobic conditions. Distinct differences in alcohol yield and residual glucose content were observed between TC and WT cells at 30°C, which is the temperature that is typically used for industrial fermentation. During fermentation for 72 h at 30°C, the alcohol yield of TC cells was approximately 25% higher than that of WT cells. The final alcohol concentration was approximately 13.5% and 10.2% with TC cells and WT cells, respectively, and the residual glucose concentration was inversely proportional to the alcohol concentration during fermentation , and then exposed to 15% or 20% ethanol for 1 h with shaking. After exposure, the cultures were serially diluted and spotted onto YPD agar plates. TC cells were more tolerant to ethanol than WT cells  than WT cells , which constitutively express OsMDHAR under the control of the GPD promoter, was inhibited by a higher concentration of H2O2, than the concentration that inhibited the viability of WT cells with an empty vector (OsMDHAR-expressing TC cells were more resistant to H2O2 than WT cells. We measured the stress tolerance of TC cells containing recombinant OsMDHAR in the presence of 5 mM or 20 mM H2O2. OsMDHAR expression in yeast enhanced the acquired resistance to H2O2 (t-BOOH), redox-active metals , a non-redox-active metal (cadmium), SDS, and ethanol  enhances acquired resistance to ROS-induced oxidative stresses, including oxidants , ethanol, and lactic acid, by improving redox homeostasis following the reduction of oxidative damage through cross-protection between target proteins and other antioxidant enzymes in S. cerevisiae [2O2 and other antioxidant proteins play a secondary role by protecting ribosomal proteins from stress-induced aggregation through the function of CAT and other peroxiredoxins [glr1-deficient yeast cells and buffer oxidative stress [MDHAR- and Arabidopsis thaliana MDHAR (AtMDHAR1)-overexpressing transgenic plants showed acquired tolerance against abiotic stressors  by improving redox homeostasis through lowering the levels of hydrogen peroxide and lipid peroxidation and increasing the net photosynthetic rate, PSII effective quantum yield (Fv/Fm), fresh weight, and antioxidant’s enzyme activities  by elevating the AsA level compared to in WT plants [2O2, which accelerates aging, auxotrophy, and cell death [por1Δ cells showed increased cytosolic and mitochondrial ROS levels , the Hsp70 family members (Ssa1 and Ssb1), Hsp60, Hsp42, Hsp30, and Hsp26, in TC cells under oxidative stress. . AccumulOsMDHAR expression in TC cells increased the ratio of reduced and oxidized forms following the accumulation of the AsA-like molecule EAA under oxidative stress , EAA induced larval growth almost as well as AsA because AsA can be converted to EAA by a ubiquitin-mediated process [e stress . TC celle stress . These re stress . These rand fish . AsA andand fish . The pree stress . After peductase . In bioa process . BecauseOsMDHAR, the glucose-based batch fermentative capacity of TC and WT cells was compared at 30°C  because Brazil, the United States, and Korea produced approximately 21.6, 50.3, and 0.15 billion gallons of bioethanol, respectively in 2012, which is more than double that produced in 2007 [OsMDHAR was constitutively expressed until the end of fermentation (−1) and 10.2% (0.53 ± 0.03 g·g−1), respectively (−1) higher than that in WT cells. In general, the theoretical ethanol yield from glucose fermentation by S. cerevisiae is 10–11% (0.50–0.55 g·g−1) [−1) higher than of WT cells reported previously [OsMDHAR-expressing TG yeast  was higher than that of CaDHN-  and OsTPX-expressing TG yeasts ; which were under the control of the same promoter) [S. cerevisiae, other yeasts have some limitations, such as low ethanol yield and poor ethanol tolerance in typical glucose-based fermentation [Saccharomyces spp. is most attractive microorganism for fermenting sugars to ethanol. Besides its innate properties, overexpression of stress-related genes could be a useful method for developing ethanol-tolerant yeasts because ethanol toxicity is highly related to ROS generation. For example, overexpression of TRX2 increased fermentation capacity and wine yield and quality by preventing the oxidative damage to glycolytic and fermentation proteins (Ahp1) through cross-protection of transcription factor (Yap1) and antioxidant enzymes  in industrial wine yeast during biomass-based batch- and fed-batch fermentation [G6PDH (ZWF1) [MPR1 [PAD [S. cerevisiae strains that are tolerant of ethanol and different metabolic environments are required, such as the OsMDHAR-expressing transgenic yeast developed herein. As shown, OsMDHAR expression in TC cells increased tolerance to ethanol and sugar-induced osmotic and physiochemical stresses.To further elucidate the functions of  in 2007 . During entation . The alcectively , and the5 g·g−1) . Intereseviously . Survivaromoter) ,63. Currromoter) . The ethentation ,65. Saccentation ,66. UpreH (ZWF1) , MPR1 [61) [MPR1 , and PADPR1 [PAD  conferreOsMDHAR in a transgenic yeast strain increased acquired tolerance to ROS-induced oxidative stress, such as that generated by H2O2, by maintaining balanced cellular redox homeostasis, proteostasis, and the pool of EAA as an AsA-like molecule. It is possible that the expression of OsMDHAR in yeast enhanced tolerance to oxidative stress and that all antioxidant defense mechanisms act synergistically, cross-protecting the cells at different levels and functioning to generate a fast and effective defense response under oxidative stress. In addition, heterologous OsMDHAR expression enhanced tolerance to high concentrations of ethanol, resulting in improved fermentative capacity. The genetically modified yeast used in this study show potential for use in the fermentation industry to improve ethanol yield. Further studies are needed to elucidate the mechanism underlying increased ethanol tolerance and yield in OsMDHAR-expressing S. cerevisiae during fermentation.In conclusion, high expression of S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S2 Methods(DOCX)Click here for additional data file.S3 Methods(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Bovine parainfluenza virus type 3 (BPIV3) isolates are classified into three genotypes (BPIV3a to -c). Here, we report the complete genome sequence of the BPIV3c isolate for the first time in Japan. Our results indicate that new primer sets will be required to detect all genotypes of BPIV3 strains. Respirovirus of the Paramyxoviridae family. BPIV3 causes severe respiratory illness in cattle, associated with bovine respiratory disease complex worldwide. Recent phylogenetic analyses revealed that BPIV3 viruses can be classified as genotypes A, B, and C (–Bovine parainfluenza virus type 3 (BPIV3) is an enveloped, nonsegmented negative-sense RNA virus within the genus B, and C –4. Accor, and C –, 2, 5. T, and C –. However, and C –, was obt, and C –, 9. SimiLC000638).The genome sequence of HS9 has been submitted to GenBank (accession number


Vigna mungo L. For amelioration purposes, different levels and combinations of iron and zinc were supplied to the plants along with 50% TE that is chosen on the basis of prior test under Petri dish culture. Cytotoxic and biochemical analysis and plant tolerance index (PTI) of plant were observed. Mitotic index deceased with increase in effluent concentration whereas abnormality % was increased. The pigments  were decreased with increasing treatment levels of TE at both growth stages. However, carotenoid content increased significantly at all dilution levels of TE after first growth stage. Chlorophyll b was increased significantly after 35 days of growth but decreased after 70 days. The protein contents were also significantly decreased with increase in all TE treatments and increased significantly in zinc recovery treatments. Activities of catalase and peroxidase enzymes were significantly affected and increased significantly with effluent treatments. PTI showed an enhanced tolerance capacity of plant with treatment of iron and zinc. A negative correlation was found (r = −0.97) between plant height and different dilutions of effluent whereas it was positively correlated (r = 0.95) with iron and zinc treatments. The study represents the ameliorative effect of iron and zinc for phytotoxic damage in V. mungo caused by tannery effluent.Different dilutions, that is, 25, 50, 75, and 100%, of tannery effluent (TE) were chosen for the present study to assess the phytotoxic effects on  With the increasing demand for leather and leather products, both for indigenous use as well as for export, a large number of vegetable and chrome tanneries have mushroomed in India, especially in states like Tamil Nadu, Uttar Pradesh, and West Bengal. There are around 3000 tanneries in India with an annual processing capacity of 70,000 tones of hides and skins . To procOn humans and animals, chromium can have carcinogenic and genotoxic effects and may have immunotoxic effects also . However Vigna mungo and to find out ameliorating effects of Fe and Zn on phytotoxicity induced in plants with effluent treatment.Chromium toxicity leads to reduced photosynthesis and hampered chloroplast synthesis . High chVigna mungo L.) were sterilized in 0.1% HgCl2 solution to remove any surface contamination, washed with redistilled water for three changes, and then soaked in water overnight. The soaked and uncontaminated seeds were evenly sown in earthen pots containing soil mixed with cow dung in 3 : 1 ratio under glasshouse conditions. Ten seeds were sown in each pot and five were maintained later. Four different dilutions of tannery effluent (TE) were prepared using tap water. For recovery of plant damage, levels 5, 10, and 15 ppm of zinc sulphate (ZnSO4) and iron sulphate (FeSO4) were also prepared and added in 50% effluent treatment. Different combinations of zinc and iron, that is, 5 ppm Zn + 5 ppm Fe, 10 ppm Zn + 10 ppm Fe, and 15 ppm Zn + 15 ppm Fe, along with 50% effluent concentration were also given to plants. The final treatments were T1 = Control (distilled water), T2 = 25% TE, T3 = 50% TE, T4 = 75% TE, T5 = 100% TE, T6 = 50% TE + 5 ppm Zn, T7 = 50% TE + 10 ppm Zn, T8 = 50% TE + 15 ppm Zn, T9 = 50% TE + 5 ppm Fe, T10 = 50% TE + 10 ppm Fe, T11 = 50% TE + 15 ppm Fe, T12 = 50% TE + 5 ppm Zn + 5 ppm Fe, T13 = 50% TE + 10 ppm Zn + 10 ppm Fe, and T14 = 50% TE + 15 ppm Zn + 15 ppm Fe. Analysis was done in 35- and 70-day-old plants.The seeds of urad  . It contMitotic index (MI) and abnormality percent in root tips of urad plants  were fouThe photosynthetic pigments were decreased with increase in effluent concentration ; Table 3As compared to control, protein content decreased significantly at all levels of TE except 50% TE + 15 ppm Fe and 50% TE + 5 ppm Zn + 5 ppm Fe where an increase of 127 and 117% was observed ; Table 3r = −0.97) was observed for plant height and effluent dilutions whereas a positive correlation (r = 0.95) was observed with recovery treatments  might have reduced the toxicity of effluents by making essential nutrient available to plants for their growth. The results, found in the present study, are quite significant and portray a clear picture that though bioremediation is a widely accepted technology for tannery effluents, scope of ameliorating them by providing additional supply of essential nutrients may be worked out for their use in irrigational water for better plant growth and ultimately a safe environment.


Pichia pastoris has not been clarified although this strain can grow well on rhamnose as a sole carbon source. In this study, four genes, PAS_chr4_0338, PAS_chr4_0339, PAS_chr4_0340, and PAS_chr4_0341, were, for the first time, predicted to be involved in rhamnose metabolism along with the previously identified gene PAS_chr1_4-0075. Moreover, expression of these genes, especially PAS_chr4_0341 and PAS_chr1_4-0075 designated as LRA4 and LRA3, was confirmed to significantly increase and clearly decrease in the presences of rhamnose and glucose, respectively. LRA4 encoding a putative L-2-keto-3-deoxyrhamnonate aldolase, was further confirmed via gene disruption and gene complementation to participate in rhamnose metabolism. Using β-galactosidase and green fluorescent protein as reporters, the promoters of LRA4 and LRA3 performed well in driving efficient production of heterologous proteins. By using food grade rhamnose instead of the toxic compound methanol as the inducer, the two promoters would be excellent candidates for driving the production of food-grade and therapeutically important recombinant proteins.The rhamnose utilization pathway in  Escherichia coli, Listeria monocytogenes, and Scheffersomyces (Pichia) stipitis, was strictly induced by L-rhamnose and strongly repressed by D-glucose11011E. coli rhaPBAD promoter performed excellently in the production of heterologous proteins in the presence of rhamnose413L-rhamnose can be utilized for growth by multitudinous microorganisms as a sole carbon and energy source12345679Pichia pastoris, a methylotrophic yeast, is an outstanding host for heterologous protein production. Several strong methanol-inducible promoters of genes related to the methanol utilization pathway, especially the well-characterized alcohol oxidase 1 promoter (PAOX1), have been frequently used to express recombinant proteins1516P. pastoris.P. pastoris can grow on rhamnose as a sole carbon source, but the genes associated with L-rhamnose metabolism have not been identified. We hypothesized that the promoters of L-rhamnose metabolism-related genes would also perform well in realizing massive production of heterologous proteins. This study aimed to disclose the putative genes involved in rhamnose metabolism pathways in P. pastoris and to exploit the promoters of these genes for controllable production of heterologous proteins. As expected, a series of putative genes involved in L-rhamnose metabolism in P. pastoris was preliminarily identified and the promoters isolated from these genes, especially PAS_chr4_0341 and PAS_chr1-4_0075 designated as LRA4 and LRA3, exhibited obvious inducibility by rhamnose. The two promoters could be exploited to enable efficient production of target proteins, especially food-grade and therapeutically important recombinant proteins, in P. pastoris.P. pastoris grew slightly slower on 2% (w/v) L-rhamnose than on glucose  aldolase, respectively  transcription levels in the presence of an initial concentration of 2% rhamnose (LRA3 promoter (PLRA3) was strongly rhamnose-inducible, while the LRA4 promoter (PLRA4) was weakly rhamnose-inducible.To further investigate the relationships between theses genes and rhamnose metabolism, ecreased . The aborhamnose . AdditioS. stipitis according to amino acid sequence identity. In addition, LRA4 showed high homology when aligned with aldolases from Debaryomyces hansenii, Candida tenuis and Glarea lozoyensis. Furthermore, two amino acid residues (a lysine residue and a tyrosine residue) that are essential active sites in L-KDR aldolase from S. stipitisLRA4 was associated with L-rhamnose metabolism, a LRA4-disrupted strain and a LRA4-complemented strain were developed by gene disruption and gene complementation, respectively. The wild-type strain, LRA4-disrupted strain, and LRA4-complemented strain were grown on glucose or rhamnose as a sole carbon source. As expected, all three strains grew well on glucose, but only the wild-type strain and the LRA4-complemented strain grew well on rhamnose  lacB expression was observed when methanol, glucose, mannitol, sorbitol, and glycerol were used as sole carbon sources, in addition to rhamnose  and production of β-galactosidase driven by PLRA3 and PLRA4  as reporters. Vectors pGHLRA3α, pGHLRA4α, and pGHGAPα based on PLRA3, PLRA4, and PGAP, respectively, were developed, with pGHLRA3α illustrated as an example in lacB, encoding a β-galactosidase, was ligated into the above vectors via the SnaBI and NotI restriction sites to generate pGHLRA3αLacB, pGHLRA4αLacB, and pGHGAPαLacB, in which lacB expression was controlled by PLRA3, PLRA4, and PGAP, respectively, and was then integrated into the gas1 locus of the P. pastoris chromosome after transformation. The β-galactosidase activities in culture supernatants of the three recombinant P. pastoris strains grown in BMRY and BMDY were monitored. It was observed that β-galactosidase activities in supernatants of the different cultures in BMRY increased with incubation duration and reached a maximum at 72 h: 23 U/ml, 18 U/ml, and 6 U/ml for recombinant strains harboring pGHLRA3αLacB, pGHLRA4αLacB, and pGHGAPαLacB, respectively   .gfp), was also monitored. For intracellular expression of gfp, three new vectors, pGHLRA3, pGHLRA4, and pGHGAP, were developed without a protein secretion signal based on vectors pGHLRA3α, pGHLRA4α, and pGHGAPα. gfp was ligated into vectors pGHLRA3, pGHLRA4, and pGHGAP via the EcoRI and NotI restriction sites, and was integrated into the gas1 locus of the P. pastoris chromosome. Green fluorescence was monitored after the recombinant strains were grown in BMRY. Fluorescence was observed in all three recombinant strains, in which gfp expression was regulated by PGAP, PLRA3, and PLRA4. Mean green fluorescence intensity was 235 (PLRA4), 563 (PLRA3) and 2249 (PGAP) respectively, which indicated that the strengths of the three promoters were ranked as follows: PGAP (high), PLRA3 (middle), and PLRA4 (weak)  . AccordiLRA3, a newly discovered strong rhamnose-inducible promoter, could potentially be widely used to drive production of proteins of interest in P. pastoris. To identify the minimal promoter region, 5′-deletion constructs of PLRA3 were derived from pGHLRA3αLacB and were inserted into pGHLRA3αLacB between the AscI and SnaBI sites, generating pGHLRA3(258)αLacB, pGHLRA3(210)αLacB, pGHLRA3(140)αLacB, pGHLRA3(120)αLacB, pGHLRA3(100)αLacB, pGHLRA3(85)αLacB, pGHLRA3(48)αLacB, and pGHLRA3(0)αLacB. These deletion constructs were transformed into P. pastoris and the β-galactosidase activities in culture supernatants of the different recombinant strains were determined. The results showed that a promoter harboring a 5′ flanking region greater than 210 bp upstream of the ATG of the initiating methionine of LRA3 was sufficient for transcriptional activation  and PLRA3 was further determined using β-galactosidase as a reporter gene and PAOX1 as a positive control promoter. The β-galactosidase activities in culture supernatants of recombinant strains harboring pGHLRA3αLacB, pGHLRA3(210)αLacB and pPIC9LacB increased during the induction. At 96 h, activity and production of β-galactosidase in culture supernatants of strain harboring pGHLRA3αLacB and pGHLRA3(210)αLacB were almost same, and were less than half of that of strain harboring pPIC9LacB  and the regulator gene (PAS_chr4_0340) were organized in a gene cluster, while LRA3 was independent. This is not the same organization found in S. stipitis, in which a set of genes correlated with rhamnose catabolism clustered in a rhamnose operon. The difference reflects different evolutionary processes in rhamnose metabolism between S. stipitis and P. pastoris.Identification of genes related to rhamnose metabolism in AOX1, which is strictly and strongly activated by methanol, was the promoter most widely used for production of heterologous proteins in P. pastoris. However, strong promoters that produce heterologous proteins in high quantities are unsuitable for certain situations, such as production of active products that are highly dependent on correct folding or processing in the secretory pathway19AOX1-based fermentation includes the following disadvantages: 1) relatively long fermentation duration and 2) sophisticated feeding strategies including but not limited to, i) initial culture of recombinants in buffered glycerol medium, followed by ii) harvesting and resuspending cells in methanol medium. To broaden the applications of P. pastoris and simplify the process of fermentation, alternative promoters that do not require methanol are in demand, and various promoters have been describedLRA3 and PLRA4 were strictly activated in the presence of rhamnose and almost completely repressed by glucose. PLRA3 in the presence of rhamnose was confirmed to be comparable to the typical strong promoter, PGAP, which has been commonly used to drive efficient expression of target genes in P. pastoris. Due to using rhamnose instead of the flammable and toxic compound methanol as an inducer, PLRA3 could be used for substantial production of recombinant proteins in the food industry and production of therapeutically important recombinant proteins for commercial applications. As for PLRA4, it was much weaker than PLRA3 and is a better candidate for certain academic purposes, for example, controllably expressing potentially lethal or toxic genes that affect cell viability. Few promoters isolated from genes related to sugar metabolism have been reported in P. pastoris and, to our knowledge, the sorbitol dehydrogenase promoter, a constitutively expressed promoter with a transcription strength similar to PGAP, is the only one that had been intensively investigated prior to our study. Compared to the sorbitol dehydrogenase promoter, PLRA3 is disadvantageous in its transcription activity, however PLRA3 is advantageous for controllable expression of heterologous genes, including some genes in which the products might be toxic to the host cell.PE. coli Trans1-T1  and P. pastoris GS115  were used as the hosts for gene cloning and expression, respectively. The pEasy-Blunt Simple Cloning Vector was purchased from TransGen.−5% biotin, 2% dextrose, and 2% agar; BMDY, BMRY, and BMDRY media (pH 6.0) contained 300 mM potassium phosphate, 1.34% YNB, 4 × 10−5% biotin, and 4 × 10−3% histidine, plus either 2% dextrose (BMDY), 2% rhamnose (BMRY), or 1% dextrose and 1% rhamnose (BMDRY).MD medium contained 1.34% yeast nitrogen base (YNB), 4 × 10The primers used for PCR are listed in P. pastoris GS115 was grown in BMDY, BMRY, or BMDRY to OD600 1.2–1.4, and then cells were harvested by centrifugation  at 4 °C and immediately stored at −80 °C. Total RNA from P. pastoris cells was extracted with Trizol reagent (Invitrogen) followed by digestion with DNase I  to remove trace DNA. cDNA was synthesized from each RNA sample (1 μg) using the RevertAid H Minus First Strand cDNA Synthesis Kit . Real-time PCR assays were performed in the IQ5 Real-Time PCR Detection System  using the SuperReal PreMix SYBR Green I Kit . Primers used for real-time PCR are listed in P. pastoris cells using the TIANamp Yeast DNA Kit . Based on the genome sequence of P. pastoris GS115, DNA fragments upstream and downstream of the open reading frame of gas1 were amplified from genomic DNA by two pairs of primers . HIS4, a selectable marker for isolating Pichia recombinant strains, was amplified from plasmid pPIC9 using the primers his4-F and his4-R. The three abovementioned DNA fragments were ligated into a DNA fragment named “GH” via overlap-extension PCR with the primers gas1-LF and gas1-RR. In addition, a DNA fragment designated “OA” containing the origin of replication derived from pBR322 and an ampicillin resistance gene was amplified from plasmid pPIC9 with primers OA-F and OA-R. Finally, the two DNA fragments, GH and OA, were ligated into a circular molecule by homologous recombination using the CloneEZ Kit , generating the recombination vector pGH01. pGH01 was integrated into the gas1 locus of the P. pastoris chromosome via homologous recombination.The genomic DNA was isolated from LRA4, which were used as homologous arms for integration of target genes into the LRA4 locus, were amplified from genomic DNA of P. pastoris GS115. The expression cassette of a zeocin resistance gene was obtained from the plasmid pPICzα. The three PCR fragments were fused by overlap-extension PCR to generate a gene disruption cassette for disruption of LRA4 in P. pastoris. From the resultant DNA fragments, 500 ng were transformed into P. pastoris GS115 via electroporation. Transformants were first screened on yeast extract-peptone-dextrose (YPD) agar plates containing 100 μg/ml zeocin and then verified by PCR.DNA fragments upstream and downstream of LRA4, the complementation plasmid pGH/LRA4 was developed as follows. A DNA fragment harboring full length LRA4 was cloned from genomic DNA of P. pastoris GS115 and then ligated into the pGH01 vector via the AscI and PacI restriction sites to generate the pGH/LRA4 plasmid. pGH/LRA4 was introduced into P. pastoris GS115 with LRA4-disruption via electroporation after linearization by the restriction enzyme SwaI. Transformants were screened on MD medium and then verified by PCR and DNA sequencing.To complement LRA3, LRA4, and GAPDH were obtained from the genomic DNA of P. pastoris. The DNA fragment comprising the protein secretion signal, multiple cloning site, and transcription termination was cloned from plasmid pPIC9. The two DNA fragments were fused by overlap-PCR to generate the exogenous gene expression cassette in which exogenous gene expression was driven by PLRA3, PLRA4 and PGAP, and then the cassettes were respectively ligated into vector pGH01 via the AscI and PacI restriction sites to generate expression vectors pGHLRA3α, pGHLRA4α, and pGHGAPα. Additionally, three vectors, pGHLRA3, pGHLRA4, and pGHGAP, were developed without the protein secretion signal in the same manner as pGHLRA3α, pGHLRA4α, and pGHGAPα.DNA fragments containing the putative promoter regions of lacB gene (GenBank accession number: AJ431643.1), which was cloned from the Aspergillus candidus chromosome and encodes a β-galactosidase, was ligated into pGHLRA3α, pGHLRA4α, and pGHGAPα and pPIC9 via the SnaBI and NotI restriction sites. The gfp gene (GenBank accession number: JQ733033.1) encoding green fluorescent protein was synthesized by GenScript Biotechnology Co. Ltd. and was ligated into pGHLRA3, pGHLRA4, and pGHGAP via the EcoRI and NotI restriction sites.The LRA3 constructs were generated by PCR using pGHLRA3αLacB as a template. The DNA fragment harboring PLRA3 and partial lacB in pGHLRA3αLacB was replaced by the 5′-serially deleted promoters and partial lacB between restriction sites AscI and SnaBI to generate plasmids pGHLRA3(256)αLacB, pGHLRA3(210)αLacB, pGHLRA3(140)αLacB, pGHLRA3(120)αLacB, pGHLRA3(100)αLacB, pGHLRA3(85)αLacB, pGHLRA3(48)αLacB, and pGHLRA3(0)αLacB.The 5′-serially deleted PLRA3, PLRA4 and PGAP, a single colony of different recombinant Pichia cells was inoculated into in YPD medium and gown overnight at 28 °C with vigorous shaking (200 rpm). The cultures were inoculated into fresh BMRY medium at 1% (v/v) and grown at 28 °C with vigorous shaking (200 rpm).To evaluate the strength of PLRA3, PLRA3(210) and PAOX1, a single colony of different recombinant Pichia cells was inoculated into in 50-ml YPD medium at 28 °C for 48 h in shaking flasks. The cells with lacB expression under the control of PLRA3 and PLRA3(210)were harvested and then grown in 50-ml BMRY medium, and the cells with lacB expression under the control of PAOX1 were harvested and then grown in 50-ml BMMY medium with addition of methanol to a final concentration of 1.0% [v/v] at a interval of 24 h. β-galactosidase activity and production in culture supernatant were monitored at intervals.To evaluate the strength of Pet al.oNPG) in pre-incubated (60 °C for 5 min) phosphate-citrate buffer  was incubated at 60 °C for 15 min, followed by sequential addition of 1 ml of 10% trichloroacetic acid and 2 ml of 1 M Na2CO3. The absorbance at 420 nm was measured. One unit of β-galactosidase was defined as the amount of enzyme that released 1 μmol of o-nitrophenol per minute under standard conditions .The β-galactosidase activity was assayed as described by Nie Pichia cells was inoculated into in YPD medium and gown overnight at 28 °C with vigorous shaking (200 rpm). The cultures were inoculated into fresh BMRY medium at 1% (v/v) and grown for 48 h at 28 °C with vigorous shaking (200 rpm). Recombinant Pichia cells were then collected, washed three times, and resuspended in 0.9% NaCl. Green fluorescence intensity in recombinant cells was determined using a laser scanning confocal microscope  with 2s per image to collect a 1024 × 1024 pixel image.A single colony of recombinant How to cite this article: Liu, B. et al. Discovery of a rhamnose utilization pathway and rhamnose-inducible promoters in Pichia pastoris. Sci. Rep.6, 27352; doi: 10.1038/srep27352 (2016).


Objective: To assess the effect of amlodipine and atorvastatin on intercellular adhesion molecule (ICAM)-1 and tumor necrosis factor (TNF)-α expression, as endothelial function and inflammation indicators, respectively, in hypertensive patients with and without prediabetes.Methods: Forty-five consecutive patients with hypertension, diagnosed according to JNC7, were divided into two groups based on the presence  or absence  of prediabetes, diagnosed according to 2010 ADA criteria, including impaired glucose tolerance (IGT) and fasting glucose tests. All patients simultaneously underwent 12-week treatment with daily single-pill amlodipine besylate/atorvastatin calcium combination . Serum isolated before and after treatment from overnight fasting blood samples was analyzed by ELISA.Results: In the HD and H groups after vs. before 12-week amlodipine/atorvastatin treatment, there were significantly  lower levels of ICAM-1  and TNF-α , with more pronounced reductions in HD vs. H group , independent of the blood pressure (BP) and cholesterol level reduction.Conclusions: Amlodipine/atorvastatin improved endothelial function and inflammation, as reflected by lower circulating levels of ICAM-1 and TNF-α, more prominently in hypertensives with than without prediabetes. Starting statin treatment before overt diabetes in hypertensives might thus improve cardiovascular outcomes. Atherosclerosis, the leading cause of death worldwide, is associated with endothelial dysfunction and inflammation Libby, , with hyStatins competitively inhibit HMG-CoA reductase and exhibit various pleiotropic effects which attenuate inflammation and improve endothelial function as reflected by decreased levels of TNF-α and ICAM-1 independent of blood lipid level reduction  ≥160 mg/dl; total cholesterol (Tc) ≥240 mg/dl or triglycerides >200 mg/dl; recent acute illness; neuropathy; or participation in other clinical trials. Overall, age range was 45–67 years, and no subject had been treated for CVD or other risk factors. Twenty-two hypertensive subjects (H group) and 23 hypertensive prediabetic subjects (HD group) were studied. Hypertension was defined as stage 1 hypertension, i.e., systolic blood pressure (SBP) ≥140 mmHg and <160 mmHg, and diastolic blood pressure (DBP) ≥90 mmHg and <100 mmHg . All subjects received dietary instructions, and liver function and creatine kinase level were monitored for potential side effects of statins. After 12 weeks of treatment, all subjects underwent blood testing. The study primary outcome was the changes of circulating levels of ICAM-1 and TNF-α after 12 weeks’ treatment compared with before. The study conformed to the guidelines set forth in the Helsinki declaration, and the protocol was approved by the Medical Ethics Committee of the first affiliated hospital of Wenzhou Medical University. All patients provided written informed consent.All laboratory experiments were carried out after an overnight fast. Blood samples were drawn from an antecubital vein and collected in ethylene diaminetetraacetic acid tubes. Blood specimens were centrifuged at 3000 rpm for 10 min, and the serum was separated and stored at −80°C until analysis. Serum levels of fasting glucose (Glu), glycosylated hemoglobin (HbA1c), 2 h-glucose (2 h-Glu), Tc, LDL-c, alanine amino transferase (ALT) and creatinine (Cr) were determined in the laboratory of the First Affiliated Hospital of Wenzhou Medical University. Serum levels of ICAM-1 and TNF-α, serving as indicators of endothelial function and inflammation, respectively were assessed using commercially available enzyme-linked immunoassay kits in accordance with the manufacturer’s instructions (Bio-Swamp and Rapidbio).T-test and independent samples T-test. Bivariate correlation analysis was performed to explore the relation among ICAM-1, TNF-α, BP, Tc and LDL-c. The 0.05 level of probability was set as being statistically significant. All analyses were performed with the SPSS (version 20.0) statistical package.Normal distribution of the data was tested using Kolmogorov–Smirnov test. All the data were normally distributed. Continuous data are presented as mean ± SD, and were compared using paired samples As shown in Table P < 0.01) BP and blood lipid levels in H and HD groups  decreased by 14.44% in the H group (from 3.81 ± 0.60 pg/ml to 3.26 ± 0.32 pg/ml), and by 24.82% in the HD group (from 4.07 ± 0.70 pg/ml to 3.06 ± 0.34 pg/ml). TNF-α level also significantly (P < 0.01) decreased after treatment by 20.47% in the H group (from 101.79 ± 11.72 pg/ml to 80.95 ± 9.33 pg/ml), and by 29.05% in the HD group (from 110.94 ± 10.71 pg/ml to 78.71 ± 9.19 pg/ml).As shown in Figure P = 0.037; TNF-αΔ: 20.84 ± 14.89 pg/ml to 32.23 ± 14.33 pg/ml, P = 0.011, respectively, Figure Comparative analysis of the changes in ICAM-1 (IACM-1Δ) and TNF-α (TNF-αΔ) levels between the H group and the HD groups revealed a more pronounced decrease in the HD group  patients with hypertension either with or without prediabetes appeared to benefit from treatment with single-pill combination of amlodipine 5 mg and atorvastatin calcium 10 mg, as reflected by significant reductions in ICAM-1 and TNF-α levels; and (2) said reductions in ICAM-1 and TNF-α levels were more pronounced in hypertensives with than without prediabetes, and independent of BP or cholesterol level reductions.Our previous study had already shown that hypertensive prediabetics experienced unfavorable shifts toward higher levels of serum levels of ICAM-1 and TNF-α when compared with patients with isolated hypertension, suggesting a higher risk for CVD and warranting investigation of appropriate treatment interventions. The results of the present study are consistent with those of a study using an animal model, in which amlodipine plus atorvastatin significantly reduced TNF-α levels (Kawai et al., Interpretation of the results of the present study is limited by its single center, nonrandomized design with a relatively small sample size. Further larger, multicenter, randomized studies are warranted.In the present single center, non-randomized, small study, combined treatment with amlodipine and atorvastatin appeared to have a beneficial effect on endothelial function and inflammation, as reflected by reductions in serum ICAM-1 and TNF-α levels in hypertensives with or without prediabetes, with the effect being more pronounced in the former. Based on the latter findings, one could posit that starting statin treatment before overt diabetes becomes apparent in hypertensive patients with prediabetes might result in better cardiovascular outcomes, which needs to be tested in larger studies.WH and ZH designed experiments; CC and FK carried out experiments; SL and PS analyzed experimental results. CC and ZH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TP and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Cynoglossus semilaevis. Cloning and sequence analysis revealed that the cDNAs of FSHβ, LHβ and CGα were 541, 670 and 685 bp in length, and encode for peptides of 130, 158 and 127 amino acids, respectively. The number of cysteine residues and potential N-linked glycosylation sites of the flatfish GTHs were conserved among teleosts. However, the primary structure of GTHs in Pleuronectiformes appeared to be highly divergent. The FSHβ transcriptional level in the pituitary remained high during the vitellogenic stage while plasma levels of FSH peaked and oocyte development was stimulated. The LHβ expression in the pituitary and ovary reached the maximum level during oocyte maturation stages when the plasma levels of LH peaked. The brain GTHs were expressed at the different ovarian stages. These results suggested that FSH and LH may simultaneously regulate ovarian development and maturation through the brain-pituitary-ovary axis endocrine system in tongue sole.The endocrine regulation of reproduction in a multiple spawning flatfish with an ovary of asynchronous development remains largely unknown. The objectives of this study were to monitor changes in mRNA expression patterns of three gonadotropin hormone (GTH) subunits  and plasma GTH levels during ovarian maturation of half-smooth tongue sole  The two gonadotropin hormones (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), secreted by the pituitary gland, are key modulators of gametogenesis and gonadal steroidogenesis in almost all vertebrates, including fish. These GTHs contain a common glycoprotein α subunit (CGα) that forms a heterodimer with a hormone-specific β subunit (FSHβ or LHβ) ,2. The hGTH subunits suggested the different regulatory mechanisms for endocrine control of oogenesis. Expression patterns of GTHs in female fish spawning single batches of eggs, have shown that the FSHβ transcription increased during the vitellogenic stage and decreased during final maturation of gametes. However the LHβ transcription increased rapidly during final ovarian maturation, correlating the fluctuations of plasma FSH and LH levels in rainbow trout (Oncorhynchus mykiss)  × 100) was calculated. The midsection of each ovary from an individual was taken and fixed in Bouin’s solution for hematoxylin and eosin (H&E) staining in order to identify the developmental stages of the ovary. The brains were removed following anesthetization, snap-frozen in liquid nitrogen, and stored at −80 °C until total RNA extraction. The pituitaries were carefully detached from the brains tissue, snap-frozen in liquid nitrogen and stored at −80 °C before proceeding to RNA extraction. Ovaries were dissected and one portion was snap-frozen in liquid nitrogen. Similarly, different tissues and organs of female tongue sole were removed, immediately frozen in liquid nitrogen and finally stored at −80 °C, until later use for total RNA extraction. The treatment of fish adhered to the guidelines of animal experiments set by the Yellow Sea Fisheries Research Institute.The adult female tongue soles  were reared in a fish farm of Rizhao, Shandong China. Before the start of experimental sampling, they were reared in natural sea water under controlled conditions  and stained with haematoxylin , counter stained with eosin (Sigma-Aldrich), and then photographed by light microscope . Five phases of ovarian development were identified accord to the method applied to the Senegalese sole : PreviteSamples stored at −80 °C were immediately transferred to cold RNAiso reagent  to preserve RNA quality. Total RNA was extracted following the manufacturer’s protocols. The quality of RNA was measured at A260 nm/A280 nm . Only RNAs with A260 nm/A280 nm ratios of 1.6–2.0 were used. RNA concentration of each sample was determined, and an agarose gel was applied to check RNA integrity. Then, the first-strand cDNA for isolation of three GTH subunits cDNA fragments was synthesized with 1 μg total RNA of pituitary gland by using PrimeScript 1st Strand cDNA Synthesis Kit  in a 10 μL reaction.GTH subunits fragment of the tongue sole, three pairs of degenerate primers  , listed in FSHβ), 58.2 °C (LHβ) and 55.1 °C (CGα) for 30 s, and an extension temperature of 72 °C for 30 s, followed by a final extension of 72 °C for 10 min. All PCRs were performed in a thermal cycler . The PCR products were separated through 2% agarose gel and purified using the E.Z.N.A Gel Extraction Kit  and then cloned into the pEASY-T1 Cloning Vector , and three clones of each gene were sequenced using the ABI3730XL sequencer .In order to clone FSHβ, LHβ and CGα sequences, three gene specific antisenses, FSHβGSP2 (for FSHβ), LHβGSP2 (for LHβ), CGαGSP2 (for CGα), were designed and used in 3'-RACE cDNA amplification system. The 5' end of cDNAs was amplified by two rounds of PCR. Primers FSHβNGSP1 (for FSHβ), LHβNGSP1 (for LHβ) and CGαNGSP1 (for CGα), were prepared for touchdown PCR as described by manufacturer’s instructions. Both 5' and 3' RACE PCRs were performed in 50 μL using Clontech Advantage 2 Polymerase Mix according to the manufacturer’s protocol. The PCR products which had the corresponding predicted length were excised, purified and cloned into vector, then sequenced as described above. The list of primers used is presented in SMART™ RACE cDNA Amplification Kit  was used for 5' and 3' ends RACE-PCR. To isolate 3' ends of FSHβ, LHβ and CGα cDNAs were analyzed in silico with the SignalP 3.0. and NetNGlyc 1.0 programs (http://www.cbs.dtu.dk/services/NetNGlyc), to predict signal peptide sequences and putative N-linked glycosylation sites, respectively. Multiple protein sequence alignment was performed using the CLUSTAL_X program. MEGA 4.0 software package was used to construct and analyze phylogenetic tree using the Neighbor-Joining method with 1000 bootstrap trials. Protein sequences used for analysis and their GenBank Accession Nos. are as follows: Cynoglossus semilaevis , Solea senegalensis , Hippoglossus hippoglossus , Paralichthys olivaceus , Dicentrarchus labrax , Epinephelus coioides , Channa maculate , Trichogaster trichopterus (Q9PW99), Morone saxatilis , Pagrus major , Oreochromis mossambicus (AAK83079), Oreochromis niloticus , Acanthopagrus schlegelii (ABQ96863), Takifugu niphobles (BAJ12081), Monopterus albus (AAN77069), Odontesthes bonariensis , Fundulus heteroclitus , Danio rerio , Carassius auratus , Cyprinus carpio , Ictalurus punctatus , Clarias gariepinus , Silurus meridionalis (AAY42268), Oncorhynchus mykiss , Oncorhynchus masou , Brachymystax lenok , Coregonus autumnalis (P48250), Plecoglossus altivelis , Oncorhynchus keta (P10256), Anguilla Anguilla , Anguilla japonica , Conger myriaster , Acipenser baerii , Acipenser gueldenstaedtii (AAS92716), Gasterosteus aculeatus (CAD59185), Gadus morhua (ABD62882), Sebastes schlegelii (AAU14140), Rana japonica (BAD16757), Mauremys reevesii (BAB92948), Coturnix japonica (BAC01164), Homo sapiens .Deduced amino acid sequences of the FSHβ, LHβ and CGα transcript levels at the different tissues of the sexual maturation tongue sole, quantitative real-time PCR (qRT-PCR) was conducted on a Mastercycler ep relplex . Further, the qRT-PCR was used to study the changes of GTH subunits mRNA transcript levels in the whole brain, pituitaries, and ovaries at the different ovarian stages. The qRT-PCR was performed using the primers FSHβF1/R1 for FSHβ, the primers LHβF1/R1 for LHβ, and the primers CGαF1/R1 for CGα . Hormone levels were determined by radioimmunoassay (RIA) according to the manufacturer’s instructions [−1 for FSH and 0.9 mIU·mL−1 for LH, respectively. The inter- and intra-assay coefficients of variation for FSH and LH were 5.4%–5.5% and 7.5%–8.7%, respectively. The cross-reactivities of antibody for FSH to TSH, LH, HCG and PRL were 3.0% × 10−3, 1.1% × 10−2, 1.6% × 10−2 and 1.4% × 10−3, respectively. The cross-reactivities of antibody for LH to TSH, FSH, HCG and PRL were 2.0% × 10−3, 2.7% × 10−2, 5.3% × 10−1 and 1.3% × 10−3, respectively.The tongue soles were anesthetized with 3-aminobenzoic acid ethyl ester . The blood was collected from the caudal vasculature by heparinized disposable syringes. Blood samples were put in ice-cold heparinised tubes and held on ice before centrifugation at 12,000× ructions ,70. The p value of <0.05.Statistical analyses were performed with SPSS 13.0 software . All assays were performed in triplicate. All data were expressed as mean ± SE and analyzed by one-way ANOVA followed by Duncan’s multiple comparison tests. Statistical significance was considered as CGα, FSHβ and LHβ subunits were first isolated and characterized in half-smooth tongue sole. The qRT-PCR and RIA analysis first suggested that GTH subunits played an important role through the brain-pituitary-ovary axis endocrine system in this flatfish. In addition, the recombinant GTHs, in combination with pharmacological studies of the tongue sole GTH receptors and their precise cellular localization, in the future, will hopefully help to understand the specific physiological function of GTH subunits to enable the design of novel methods to overcome reproductive dysfunctions of the cultured female tongue sole.In summary, three cDNAs encoding the


Atmospheric carbon dioxide levels have increased by∼25% over the last 50 years. While more carbon dioxide can initially stimulate plant photosynthesis, we found that long-term (12 years) exposure of sweetgum trees to elevated carbon dioxide resulted in no stimulation of photosynthesis. The loss of initial increases in photosynthesis was due to low leaf nitrogen levels, which suggests other limiting resources may moderate future impacts of elevated carbon dioxide on photosynthesis. 2 (eCO2) often increases photosynthetic CO2 assimilation (A) in field studies of temperate tree species. However, there is evidence that A may decline through time due to biochemical and morphological acclimation, and environmental constraints. Indeed, at the free-air CO2 enrichment (FACE) study in Oak Ridge, Tennessee, A was increased in 12-year-old sweetgum trees following 2 years of ∼40 % enhancement of CO2. A was re-assessed a decade later to determine if the initial enhancement of photosynthesis by eCO2 was sustained through time. Measurements were conducted at prevailing CO2 and temperature on detached, re-hydrated branches using a portable gas exchange system. Photosynthetic CO2 response curves (A versus the CO2 concentration in the intercellular air space (Ci); or A–Ci curves) were contrasted with earlier measurements using leaf photosynthesis model equations. Relationships between light-saturated photosynthesis (Asat), maximum electron transport rate (Jmax), maximum Rubisco activity (Vcmax), chlorophyll content and foliar nitrogen (N) were assessed. In 1999, Asat for eCO2 treatments was 15.4 ± 0.8 μmol m−2 s−1, 22 % higher than aCO2 treatments (P < 0.01). By 2009, Asat declined to <50 % of 1999 values, and there was no longer a significant effect of eCO2 . In 1999, there was no treatment effect on area-based foliar N; however, by 2008, N content in eCO2 foliage was 17 % less than that in aCO2 foliage. Photosynthetic N-use efficiency (Asat : N) was greater in eCO2 in 1999 resulting in greater Asat despite similar N content, but the enhanced efficiency in eCO2 trees was lost as foliar N declined to sub-optimal levels. There was no treatment difference in the declining linear relationships between Jmax or Vcmax with declining N, or in the ratio of Jmax : Vcmax through time. Results suggest that the initial enhancement of photosynthesis to elevated CO2 will not be sustained through time if N becomes limited.Elevated atmospheric CO Results suggested substantial eCO2 stimulation of photosynthesis (2 induced significant down-regulation and loss of photosynthetic capacity (e.g. in Arctic tundra grasses and shrubs (2 enrichment (FACE) technology  and net primary productivity is common across woody plant species exposed to FACE eCO2 treatments  as displayed across C3 woody plant species exposed to elevated CO2  is a common temperate North American tree species, and is the dominant canopy species at the ORNL-FACE site in TN, USA, and the primary mid-canopy species at the Duke-FACE site in NC, USA. Prior results from these sites indicate a strong and consistent enhancement of light-saturated photosynthesis (Asat) by elevated CO2; the ∼40 % increase in atmospheric CO2 resulted in 45 % enhancement of Asat at the ORNL-FACE site after 3 years of treatments (Asat at the Duke-FACE site through 6 years of treatments (Amax), maximum electron transport rate (Jmax) or maximum carboxylation rate (Vcmax) when foliage from both treatments was measured at the same CO2 concentrations . Finally, we evaluated treatment effects on coupling between stomatal conductance and C-assimilation.The main goal of this project was to determine if the initial enhancement of photosynthesis by eCOL. styraciflua L.) plantation forest in Oak Ridge National Environmental Research Park in eastern TN, USA . The soil was an Aquic Hapludult with a silty clay-loam texture. A free-air CO2 enrichment system ), median tree diameter was 14.6 ± 4.1 cm (±1 SD) and peak leaf area index (mid-July) was 4.1 ± 0.2. Mean growing season (April–October) temperature was 19.6 °C in 2008 and 19.1 °C in 2009, and growing season precipitation was 440 mm in 2008 and 511 mm in 2009 ; n = 29–31 (2009)). Conditions in the gas exchange cuvette were set to approximate ambient outside temperature: relative humidity was ∼50–80 %, photosynthetically active radiation was 1800 µmol m−2 s−1 and CO2 concentration was initially set at 400 ppm prior to the A–Ci curve measurements (see below). Foliage was retained for the analysis of N, chlorophyll and LMA. Similar raw datasets were available from previous work performed at the site in late July–August 1999 (For each measurement campaign (2008–09), branches were collected early in the morning from mid-canopy (2009) and fully exposed upper canopy  dominant or co-dominant trees within each treatment plot. Canopy access was achieved via the FACE infrastructure towers, which provided access to trees within ∼8 m from the towers. Trees were selected primarily from within the treatment plots (i.e. >2.5 m from the ring edge); however, in ambient plots, several buffer trees were also sampled. On measurement days, 1–2 m long branches were cut from the mid- and upper canopy of selected trees with a pole pruner and quickly placed into plastic bags with wet paper towels to minimize desiccation. Branches were re-cut under water to remove potential embolism induced during removal from the trees. Most Asat) at the CO2 concentration in which the trees were growing, response curves of assimilation versus the CO2 concentration within the intercellular spaces of the leaf (Ci), or A–Ci curves, were measured in all years. These response curves were used to estimate maximum electron transport rate, Jmax, and maximum Rubisco activity, Vcmax, using a consistent set of leaf photosynthesis model equations . This contrasts slightly with curves conducted in 1999 using a declining CO2 regime . To contrast gas exchange responses at growth CO2, results were assessed at 400 or 550 ppm for aCO2 or eCO2, respectively. Since those CO2 concentrations were not part of the 1999 campaigns, values at 400 or 550 ppm were interpolated from the linear portion of each response curve (R2 > 0.99) for comparison. Atmospheric CO2 varied between 1998 and 2008 due to incremental ambient CO2 increases and FACE performance and management; range (384–405 or 528–560 ppm CO2) for aCO2 or eCO2, respectively. Relationships between Jmax, Vcmax, stomatal conductance (gs), foliar N, chlorophyll content and CO2 treatments were assessed.In addition to measurement of the light-saturated photosynthetic rate  and extracted at 4 °C in the dark until analysis. Following extraction total chlorophyll, chlorophyll a and chlorophyll b were determined based on spectroscopy at 647 and 665 nm  + 0.7013 (mg cm−2); n = 28; R2 = 0.66; based on 2008 foliage). All N content is provided on a disc basis.To assess foliar chlorophyll and N concentrations, and LMA, nine leaf discs (9 mm diameter) were collected from the same area of each leaf (avoiding midrib) used for gas exchange measurements (2008–09). For chlorophyll analysis, two leaf discs were immediately placed in scintillation vials containing 5 mL of d 665 nm , and resVcmax and Jmax were analysed using t-tests and analysis of variance techniques. There were no significant plot (ring) effects on chlorophyll, N, Jmax or Vcmax measured in 1999 or later years (0.1 < P < 0.9). As such, individual leaves were considered as the (pseudo-replicated) experimental unit (df = 37–60 per treatment per year) as opposed to use of treatment rings (df = 1–2). This is common for analyses of these large, expensive studies where true replication is limited, and results should be considered in this context; i.e. there is a greater chance to detect spurious treatment effects (Type I errors)  are presented; P < 0.05 indicates statistical significance.Regression slopes and treatment differences in photosynthesis, chlorophyll content,  errors) . Data ma2 over time. In 1999, light-saturated photosynthesis (Asat) was significantly greater in eCO2 than in aCO2 foliage, but when re-measured in 2008, Asat was similar for the two treatments. In 1999, Asat was 15.4 µmol m−2 s−1 for eCO2 foliage, 22 % greater than for aCO2 foliage  ; P = 0.17; df = 56 (2009))  and 12 % lower when both were measured at 400 ppm CO2 (P = 0.09)  and 22 % lower when both were measured at 400 ppm CO2 (P = 0.12). In 2009, measured Asat was 24 % lower for eCO2 foliage than for aCO2 foliage when both were measured at 550 ppm CO2  and 23 % lower when both were measured at 400 ppm CO2 (P = 0.35) . Mass-based foliar N content in eCO2 leaves was significantly lower than for aCO2 in 2008 and 2009 (P < 0.001), but not in 1999 (P = 0.22) , but not in 2009 (P = 0.08) or in 1999 (P = 0.47) , there was a strong linear relationship with chlorophyll content, with a statistically steeper regression slope for the aCO2 foliage , but were significantly lower for eCO2 (0.022 mg cm−2) than for aCO2  foliage in 2008–09. Leaf mass per area also declined through time for both treatments, and was always greater for eCO2 foliage. In 1999, LMA was 8.6 % greater for eCO2 foliage (128.9 g m−2) than for aCO2 foliage . In 2008, LMA was not significantly greater for eCO2 foliage (115.9 g m−2) than for aCO2 foliage . In 2009, LMA was 6.6 % greater for eCO2 foliage (104.5 g m−2) than for aCO2 foliage .Many of the measured photosynthetic parameters were strongly related to N content, which dropped significantly over the course of the experiment. In 1999, the N content of foliage from both the treatments ranged from ∼0.2 to 0.4 mg cmAsat, particularly at lower values of N. Values of Asat plateaued as N content increased  slopes and coincided at 0.057 mg cm−2, which is approximately the N content of senescent tissue (regression not shown).Nitrogen content also had a significant impact on sed Fig. B. The liJmax and Vcmax were not related to N content above ∼0.2 mg cm−2, but declined strongly with declining N content  to late (0.075–0.197 mg cm−2) years, which introduces a potential confounding effect of N × year, and which prohibited further refinement of the ∼0.2 mg cm−2 N response threshold. Even so, annual measurements throughout the study confirm the N differences as foliage displayed an incremental decline in N content through time  from ∼1.6 in 1999 to ∼1.0 in 2009, indicating a decline in electron transport rate with respect to carboxylation rate through time. There was no relationship between Jmax : Vcmax and N content (R2 = 0.02).Values of ent Fig. A and B. age Fig. C. While ime Fig. , the sloAsat : gs) in 2008–09 was ∼35 % greater for eCO2 foliage than for aCO2 foliage  was similar to aCO2 foliage measured earlier (1998–2000) during different seasons and across years, but not for eCO2 foliage measured earlier  and photosynthetic capacity of sweetgum trees declined through time as the stand matured; these responses were correlated with increased nutrient  sequestration in long-term biomass or soil organic matter and reduced availability and uptake of N . Consistent with our first hypothesis, we found a significant reduction in overall photosynthetic capacity across treatments as leaf N content declined. However, in contrast to our second hypothesis, the reduction in foliar N was so acute that the benefit of eCO2 in generating enhanced photosynthetic efficiency was lost. As such, the initial CO2 stimulation (∼45 % greater) of net photosynthesis observed after 3 years  was in excess of photosynthetic requirements during the early years of the experiment. Total canopy N content in aCO2 plots declined from ∼1.8 % prior to the experiment in 1996 to 1.6 % by 2004 (−1 urea), which boosted upper canopy leaf N concentrations from ∼1.65 % (control) to 2.04 % (fertilized) by late July, and substantially increased canopy leaf area and stem growth (2) and 1.16 % (aCO2), which was significantly lower for the eCO2 treatment.There was no relationship between foliar N and m growth . The swe−2 in 1999, similar to the values in 2008–09 (0.014–0.041 mg cm−2), despite much lower N content. Actual chlorophyll levels at the ORNL-FACE site in 1999 were thus likely significantly higher than indicated here. Indeed, chlorophyll extracted from similar aged sweetgum trees at the Duke-FACE site that were not N limited indicated late season values from 0.047 to 0.071 mg cm−2 . A similar pattern of N saturation of Vcmax has been suggested for pine at the Duke-FACE site, with saturation of the carboxylation rate at tissue contents >1.4 % N . At lower foliar levels of N (0.075–0.2 mg cm−2), eCO2 leaves utilized N more efficiently (greater PNUE) compared with aCO2 leaves  such that the processes are co-limiting  with eCO2 , likely as a consequence of the enhanced root exploration. Foliar content of other essential mineral nutrients such as P and K did not decline through time in litter collected from 1998 to 2002, nor in wood tissue collected in 2009.The decline in photosynthetic capacity and resultant reduction in eCO2 foliage than for aCO2 foliage; eCO2 : aCO2gs ranged from 0.75 to 0.81. Similarly, under dry field conditions, whole canopy conductance (gc) based on sap flow remained lower for eCO2 foliage; eCO2 : aCO2gc was ∼0.7 and declined to ∼0.4 as stomatal aperture declined during extreme drought  regulation of photosynthesis has been studied in detail, knowledge of longer-term shifts in the photosynthetic apparatus in response to eCO2 has been generally limited to observations at the FACE studies and a few long-term open-top chamber studies  restricted carbon assimilation due to photosynthetic biochemical limitations. This study indicates that atmospheric enrichment of CO2 may result in an initial ‘fertilization’ effect, directly increasing photosynthesis and productivity, but this may later be offset by the declining soil N availability, which could completely eliminate the initial positive effects of eCO2. Overall, we suggest caution in extrapolating shorter-term eCO2 responses to longer-term ecosystem processes, which are confounded by other controlling factors such as soil nutrient availability, water availability or air temperature that can change through time.Photosynthesis is the predominant mechanism that removes COUS Department of Energy, Office of Science, Office of Biological and Environmental Research, under contract DE-AC05-00OR22725.This article is based upon work supported by the J.M.W. designed and conducted the experiment. A.M.J. performed laboratory work. B.E.M. analysed the data. All authors contributed to the writing and editing of the manuscript.None declared.


To clarify the relationship between the probability of prostate cancer scaled using a 5-point Likert system and the biological characteristics of corresponding tumor foci.The present study involved 44 patients undergoing 3.0-Tesla multiparametric MRI before laparoscopic radical prostatectomy. Tracing based on pathological and MRI findings was performed. The relationship between the probability of cancer scaled using the 5-point Likert system and the biological characteristics of corresponding tumor foci was evaluated.n = 3; score 2: n = 3; score 3: n = 16; score 4: n = 11 score 5: n = 22), while 47 were not pointed out on MRI. The tracing study revealed that the proportion of >0.5 cm3 tumors increased according to the upgrade of Likert scores . The proportion with a Gleason score >7 also increased from scale 2 to scale 5 . On using score 3 or higher as the threshold of cancer detection on MRI, the detection rate markedly improved if the tumor volume exceeded 0.5 cm3 .A total of 102 tumor foci were identified histologically from the 44 specimens. Of the 102 tumors, 55 were assigned a score based on MRI findings (score 1: Each Likert scale favobably reflected the corresponding tumor’s volume and Gleason score. Our observations show that “score 3 or higher” could be a useful threshold to predict clinically significant carcinoma when considering treatment options. In the treatment of prostate carcinoma, one of the major disadvantages of preoperative MRI is the lack of a standardized reporting system. Sometimes, the report may be written in a narrative manner, making it difficult to compare studies from different centers. Recently, the European Society of Urogenital Radiology recommended the use of the MR Prostate Imaging Reporting and Data System (PI-RADS) , and theIn the present study, in order to gain further insight into the association between tumor-foci characteristics and the 5-point Likert score, we performed a detailed comparison between all small cancerous foci on whole-mount histopathology and radiological findings according to 5-point Likert scaling using the recent 3.0-Tesla multiparametric MRI.The study protocol was approved by the institutional review board of Hokkaido University Hospital, and patient consent was not deemed necessary because data were obtained from medical charts and patient identifying information was anonymized before analysis.n = 5), susceptibility artifacts on diffusion- weighted imaging (DWI) (n = 4), or sequence differences (n = 3), the final study population consisted of 44 patients. No prior radiation therapy and/or androgen deprivation therapy was performed in our cohort.Between June 2009 and April 2013, 80 patients with localized prostate carcinoma underwent laparoscopic radical prostatectomy (LRP). Of these, 56 patients underwent 3.0-Tesla multiparametric MRI before surgery. Excluding 12 patients due to a lack of dynamic contrast-enhanced (DCE) MRI  in the remaining 38 patients.Patients underwent MRI with a 3.0-T scanner  and a pelvic phased-array coil (32-channel SENSE Torso/Cardiac Coil). No endorectal coil was used. The slice thickness of all the sequences was 3 mm. The following MR sequences were obtained: axial T1-weighted image (T1WI), axial T2-weighted image (T2WI), axial, sagittal, and coronal fat-suppressed T2WI, axial DWI, and DCE MRI. DCE MRI was performed in the axial plane with one pre-contrast and four post-contrast dynamic series after the intravenous administration of gadolinium-based contrast material at a dose of 0.1 mmol per kilogram body weight and subsequent flushing with standardized 20-mL saline. The apparent diffusion coefficient (ADC) values were calculated from two DWI scans acquired with b = 0 and 2,000 s/mm2, and ADC maps were then rebuilt by calculating the ADC values in each pixel of each slice. In the present study, we did not perform any bowel preparation, or administer any agent to suppress bowel peristalsis.2 of DWI, with a low focal ADC value compared to that of the adjacent normal parenchyma on ADC mapping. On DCE-MRI, it was defined as an early enhancing area in the prostate compared to adjacent normal parenchyma. In the case of biopsy–related hemorrhage, we tried to identify an enhanced lesion by carefully comparing pre and post images. The likelihood of the presence of prostate cancer was determined based on an overall combination of the findings from T2WI, DWI, and DCE-MRI using a Likert scale between 1 and 5 .For this retrospective analysis, all MR images were reviewed in consensus by two radiologists with 13 and 5 years’ experience in prostate MRI. Both readers were aware that the patients had prostate cancer documented by biopsy and had undergone LRP, but were blind to all other clinical data. On pre-contrast DCE MRI, high-signal-intensity areas were considered hemorrhagic after prostate biopsy. On T2WI, a finding suggesting prostate cancer was defined as a hypointense area in the prostate compared to the adjacent parenchyma, excluding an area of hemorrhage. In the case of a wedge-shaped hypointense area without a mass effect around the adjusting prostatic tissue, we suspected chronic prostatitits rather than prostatic cancer, considering the results of other sequences together. On DWI, it was defined as a focal hyperintense area at b = 2,000 s/mmFollowing LRP, the specimens were fixed overnight in 10 % neutral buffered formaldehyde. After staining of the surface, each specimen was cut into transverse 5-mm-thick slices, vertical to the dorsorectal surface, and the apex and base were sagittally sectioned. After pathological evaluation, all tumor foci were mapped on the macroscopic photographs. For all LRP specimens, the primary and secondary Gleason grades as well as the combined Gleason score were recorded. The T-category was defined according to the 2009 TNM classification.For the present study, tumor areas in the macroscopic photographs were traced and measured using computer planimetry software . If the distance between two tumor foci was greater than 5 mm, they were considered separate. The tumor volume was calculated by multiplying the total tumor surface area by the section thickness (5 mm). For analysis, a shrinkage factor of 1.15 was considered . When thχ2 test to compare categorical characteristics among the groups. Univariate and multivariate logistic regression analyses were used to indentify characteristics associated with the visibility of cancerous lesions on 3-Tesla MRI. A p-value <0.05 was considered significant.Analyses were performed with JMP® version 11 . We used the 3 (range: 0.0023–14.0) after shrinkage correction. The Gleason score distribution was 6 in 49, 7 in 40, and 8–9 in 13.Clinical and pathological characteristics of the 44 patients were as follows: mean age, 67.1 years, median age, 68 years (range: 52–77); median PSA, 6.91 ng/mL (range: 1.75–37.6); median time between MRI and LRP, 10 days (range: 1–164). Thirty-one patients were in stage pT2 and 13 in pT3. Regarding the node status, all patients showed pN0. Table n = 3; score 2: n = 3; score 3: n = 16; score 4: n = 11 score 5: n = 22) and 47 were not pointed out on MRI. Figure 3 tumors increased with the upgrade of Likert scores . The median tumor volume according to each scale was 0.459 cm3 (range: 0.0696–0.597) for scale 1, 0.267 cm3 (range: 0.0558–0.954) for scale 2, 0.816 cm3 (range: 0.0023–5.79) for scale 3, 1.53 cm3 (range: 0.332–7.93) for scale 4, and 2.34 cm3 (range: 0.065–14.0) for scale 5. Figure χ2 test, p = 0.0001).Of the 102 tumors, 55 were assigned a score based on MRI findings  for score 1, 23.5 % (4/17) for score 2, 75 % (15/20) for score 3, 73.3 % (11/15) for score 4, and 95.7 % (22/23) for score 5. The positive predictive value was 82.8 % (48/58) for score >3.3, the detection rate on MRI markedly improved . Overall, the sensitivity for cancer detection was 87.2 % (41/47) for tumors larger than 0.5 cm3. Table Based on our current observations, the assigned scores of 3–5 were treated as visible cancerous lesions on MRI in the subsequent analyses. Figure 3 tumors increased according to the upgrade of Likert scores , and the proportion of those with Gleason score >7 also increased from score 2 to score 5 . Our observations confirmed that a threshold of 3 or higher is very helpful for clinicians when considering the possibility of significant cancer, denoting >0.5 cm3 or Gleason score >7 tumors. Although we did not assess PI-RADS data or inter-observer variability in scoring, Renard-Penna et al. reported favorable interobserver agreement between the Likert scale (κ = 0.80) and PI-RADS system (κ = 0.73) [The usefulness of MRI for imaging prostate carcinoma has already been recognized . On the 3: 6/9, 66.7 %, 1.0 < cm3: 35/38, 92.1 %) when the tumor foci volume exceeded 0.5 cm3. Overall, sensitivity for cancer detection was 87.2 % (41/47) for tumors larger than 0.5 cm3, and the positive predictive value was 82.8 % (48/58) for score >3. Multivariate analysis identified only the tumor volume as being significantly correlated with visibility on MRI. Because 1 cm in diameter represents a sphere of 0.5 cm3, we consider that, although the detection limit of small foci was the same as that using endorectal coil or pelvic phased-array coil 1.5-T MRI, modern 3.0-Tesla multiparametric MRI offers a more sophisticated image of the prostate and can clearly visualize most tumors larger than 0.5 cm3. In other words, 3.0-Tesla can identify clinically significant disease in terms of a tumor volume >0.5 cm3. In contrast, it can barely detect tumors with a volume of less than 0.5 cm3 regardless of the Gleason score. In the current study, an endorectal coil was not used, and we, therefore, did not generate data on how an endorectal coil can aid in tumor depiction. Previously, Park BK et al. and Sosna J et al. reported that 3.0-T pelvic phased-array MRI could produce an image equivalent to 1.5-T endorectal MRI [Regarding detectability on MRI according to the cancer volume, Ikonen S et al. previously reported that, with the use of endorectal coil 1.5-T MRI (T2-weighted), the rate of detecting carcinoma foci smaller than 5 mm was 5 %, but it was 89 % for those larger than 10 mm . Villersctal MRI , 14. Kimctal MRI . In contctal MRI . At presn = 5; 4: n = 4; 5: n = 1) were false-positive findings on MRI. After the analysis, we convened a meeting with the pathologists to discuss the cause of these false-positive findings, and found that the most frequent histological finding was inflammation (n = 7), and abscess formation was also noted in one case (data not shown). It would be optimal to perform MRI before prostate biopsy to minimize these artifacts. It has been established that an interval of at least eight weeks is needed to minimize artifacts due to prostate biopsy [3 which were missed on MRI , an anterior TZ location (n = 1), an apex location (n = 1), and islet distribution of small foci (n = 3) may have compromised visualization on MRI. Regarding the relationship between the tumor location and MRI visibility, previous studies showed the lack of additional benefit of DCE-MRI and the difficulty of cancer detection in the transition zone [As described above, the positive predictive value for a diagnosis of cancer based on MRI findings was 75 % 15/20) for score 3, 73.3 % (11/15) for score 4, and 95.7 % (22/23) for score 5. Conversely, 10 lesions assigned a score >3 (3: e biopsy . Regardiion zone , 19. In 0 for scoOur study has several potential limitations. Firstly, this was a small retrospective study. There was a selection bias in that only patients undergoing prostatectomy were enrolled. As described above, because all MR images were reviewed in consensus by two radiologists during a single session, we do not have data on inter- or intraobserver variability of the Likert scale. The lack of an endorectal coil may have influenced our observations. Furthermore, in cases undergoing MRI evaluation after biopsy, post-biopsy hemorrhage might have resulted in difficulty of tumor detection. It remains unknown whether the results would be reproducible in a different institute with a reader with a different experience level. Further validation studies are warranted to evaluate whether a scoring system including a Likert scale can become a common language among physicians treating prostate carcinoma patients in daily clinical practice.Each Likert scale well reflected the corresponding tumor’s volume and Gleason score. Our observations show that “scale 3 or higher” could be a useful threshold to predict clinically significant carcinoma when considering treatment options.


DBMSCs were isolated and then extensively characterized. Differentiation of DBMSCs into three mesenchymal lineages  was performed. Real-time polymerase chain reaction (PCR) and flow cytometry techniques were also used to characterize the gene and protein expression profiles of DBMSCs, respectively. In addition, sandwich enzyme-linked immunosorbent assay (ELISA) was performed to detect proteins secreted by DBMSCs. Finally, the migration and proliferation abilities of DBMSCs were also determined. DBMSCs were positive for MSC markers and HLA-ABC. DBMSCs were negative for hematopoietic and endothelial markers, costimulatory molecules, and HLA-DR. Functionally, DBMSCs differentiated into three mesenchymal lineages, proliferated, and migrated in response to a number of stimuli. Most importantly, these cells express and secrete a distinct combination of cytokines, growth factors, and immune molecules that reflect their unique microenvironment. Therefore, DBMSCs could be attractive, alternative candidates for MSC-based therapies that treat diseases associated with inflammation and oxidative stress. Mesenchymal stem cell (MSC) therapies for the treatment of diseases associated with inflammation and oxidative stress employ primarily bone marrow MSCs (BMMSCs) and other MSC types such as MSC from the chorionic villi of human term placentae (pMSCs). These MSCs are not derived from microenvironments associated with inflammation and oxidative stress, unlike MSCs from the However, BMMSCs have significant limitations including the invasive harvesting method used for collection, insufficient numbers of stem cells in adult BM (approximately 0.001–0.01%), and reduction in cell numbers and differentiation potential with increasing age of the donor . These lMany MSC-based therapies are directed toward diseases and disorders caused by oxidative stress and associated with increased inflammation, which include atherosclerosis, Alzheimer's disease, Parkinson's disease, neurodevelopmental disorders, angina, thrombosis, and hypertension –14. The BMMSCs in their niche are normally exposed to low levels of oxidative stress and only experience increased oxidative stress following injury or disease . Precond decidua basalis, which is a maternal tissue exposed to increased levels of inflammation and oxidative stress throughout human pregnancy  were assessed. Cytokines were dissolved in complete DBMSC culture medium and then added to the cell culture and incubated for 48 hours. The concentrations used gave an optimal effect as shown in a dose response experiment (data not shown) and these concentrations were consistent with our published results [The effect of selected cytokines on the proliferation of DBMSCs was studied by seeding cells at a density of 2 × 10 results . Cell prμm pore filters in 12-well plates as previously published [4 were added to the upper side of the transwell filter chamber in a 12-well plate and complete DBMSC culture medium with chemotactic factors was added to the bottom chamber. Migration observed in the presence of 30% FBS, and with medium alone, served as positive and negative controls, respectively. After overnight incubation as described above, the upper side of the filters was carefully washed with PBS. Cells remaining on the upper side of the filters were removed. Cells that had migrated to the lower side of the filter were stained with haematoxylin, and then the filter was excised from the insert and placed onto a glass slide. The total number of cells that had migrated to the lower side of the filter was counted using light microscopy at 100x magnification. Cells were blindly counted and data were expressed as numbers of total migrated cells per insert related to that of the negative control. The chemotactic activity of stromal derived factor, SDF (150 ng/mL), platelet derived growth factor, PDGF (10 ng/mL), HGF (50 ng/mL), and MCP (100 ng/mL) was determined. All chemotactic factors used were recombinant factors of human origin . The concentrations used gave an optimal migratory effect as previously published [DBMSC migration in response to selected chemotactic factors was determined using transwell inserts  with 8 ublished . Brieflyublished . The numt-test. These analyses were performed using GraphPad Prism 5. Results were considered to be statistically significant if P < 0.05.Data were analysed using the  decidua basalis attached to the maternal side of human placenta and these cells formed a homogenous monolayer of adherent cells which exhibited a fibroblast-like cell morphology  and HLA-ABC . In addiMSCs secrete many cytokines and growth factors to modulate their functions (autocrine) or other cells' functions (paracrine). Consequently, we screened the conditioned medium collected from the culture of DBMSCs at passage three for the secretion of cytokines and growth factors by ELISA. We detected many cytokines and growth factors as shown in α1, α2, α2b, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8), immunoglobulin superfamily , and selectin L and selectin P ligand as shown in P < 0.05), as shown in Human DBMSCs from passages three to five expressed a broad spectrum of adhesion molecules including integrin (3C Chemokine/Receptor family (CX3CL1 and CX3CR1) as shown in P > 0.05). In addition, DBMSCs lacked the expression of CCR1–CCR10, CXCR1–CXCR4, CXCR7, XCR1, and CX3CR1 , CXC Chemokine/Receptor family , C Chemokine/Receptor family , and CXd CX3CR1 .β, IL8, IL9, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL18, IL20, IL23A, IL26, IL27, IL28A, IL29, IL31, IL32, IL33, IL34, IL1R1, IL2RB, IL2RG, IL3RA, IL6R, IL7R, IL17RC, IL18R1, and IL22RA1 and anti-inflammatory cytokines/receptors IL4, IL5, IL6, IL10, IL19, IL24, IL25, IL27, IL4R, IL10RA, and IL10RB as shown in P > 0.05). In addition, these cells lacked the expression of IL1β and interleukin-7 receptor-α (CD127).Human DBMSCs from passages three to five expressed a broad spectrum of cytokines/receptors including inflammatory IL1A, IL1α and β (PDGFRα and β), fibroblast growth factor receptors 2 and 4 (FGFR2 and 4), interferon gamma receptors 1 and 2 (IFNGR1 and 2), interferon alpha, beta, and omega receptors 1 and 2 (IFNAR1 and 2), nerve growth factor receptor (NGFR), kinase insert domain receptor (type III receptor tyrosine kinase) or KDR, interferon, kappa (IFNK), fibroblast growth factor receptors 1 and 3 (FGFR1 and 3), transforming growth factor beta receptors 1 and 2 (TGFBR1 and 2), formyl peptide receptor 2 (FPR2), opioid growth factor receptor (OGFR), insulin-like growth factor 1 receptor (IGF1R), colony stimulating factor 2 receptor beta (CSF2RB), epidermal growth factor receptor (EGFR), neuroplastin (NPTN), histamine receptor H4 (HRH4), fms-related tyrosine kinase 4 (FLT4), fms-related tyrosine kinase 1 , tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), and met protooncogene (hepatocyte growth factor receptor or MET) as shown in P > 0.05). However, the expression of IFN-γ and TGF-β1 by DBMSCs significantly changed from passages 3–5 (P < 0.05) as shown in Human DBMSCs from passages three to five expressed a broad spectrum of growth factor receptors including platelet-derived growth factor receptors P > 0.05).DBMSCs from passages three to five expressed the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO). The level of mRNA expression did not change significantly between passages. In addition, the flow cytometry analysis revealed that DBMSCs at passage three expressed proteins for some of immune factors examined as shown in P < 0.05) the proliferation of DBMSCs compared with the untreated control, as shown in To investigate whether DBMSCs can proliferate in response to a number of proliferative stimuli, an MTS assay was used. The results revealed that HGF, IL-6, IL-4, Rantes, and IL-17A significantly increased (P < 0.05) in the presence of HGF (329.3 ± 35.49), SDF (213.2 ± 21.91), MCP-1 (238.2 ± 24.80), and PDGF (199.8 ± 20.87), compared with the untreated DBMSCs control (118.2 ± 35.49)  . decidua basalis tissue attached to the maternal side of human placenta. We cultured DBMSCs for five passages. Morphologically, these cells formed a homogenous monolayer of adherent cells that exhibited fibroblast-like cell morphology from passage two onward  superfamily cell adhesion molecules (CAMs), integrins, cadherins, and selectins . In thisd MEDCAM . The rold VCAM-1 , 36.3C chemokines. Chemokine receptors are classified as G protein coupled receptors for CXC, CC, C, or CX3C chemokines [MSCs have a great therapeutic potential in treating human diseases. During the course of stem cell transplantation, transplanted MSCs home to the site of injury in a process known as homing, which involves cell recruitment, migration, and adhesion . Chemotaemokines . Chemokiemokines . In respemokines . We and emokines , 41.3C families , and the immune suppressive enzyme IDO Tables  and 7. TThis is the first study to show that DBMSCs express an extensive range of molecules that are implicated in many cellular functions. The diverse expression of molecules by DBMSCs makes them a worthwhile candidate for future functional studies to determine their therapeutic potential in treating human diseases such as cancers, immune disorders, and liver and cardiovascular diseases.