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We tested the continuity of populations as described by Brandt et al.24 with an absolute frequency of 22–37 mtDNA haplogroups. We performed tests assuming three effective population sizes (Ne = 500; 5,000; 500,000), and compared Avars with all conquest-period Hungarians, and with the southeast group of the latter (n = 45), who lived on the territory of the preceding Avar group. We also compared 10th–12th century and modern-day Hungarians and the culturally isolated minority populations, Szeklers and Csangos, who live in Romania (Supplementary Table S7).
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The shared haplotype analysis was carried out in order to detect and compare the mtDNA haplotypes shared between 21 Eurasian ancient populations, and to observe lineage sharing between the conquerors and 23 modern Eurasian populations. Identical HVS-I sequences and numbers of different lineage types were counted (Supplementary Tables S10 and S12). Asian lineages in the conqueror and Avar datasets were also counted in our database of 64,650 Eurasian sequences (Supplementary Table S11).
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The comparative modern mtDNA datasets with detailed information on geographic origin were used for the GDM. From these datasets, we performed genetic distance calculations in two ways. First, we used high resolution haplogroup frequency tables of 157 populations (n = 49,439 individuals), differentiating 211 sub-haplogroups. We calculated genetic distances of these modern populations from the three Carpathian Basin medieval populations (Supplementary Table S13). Second, we randomly chose maximum 140 sequences per population (n = 18,499 sequences altogether), in order to balance the differences in sample sizes, and calculated FST values between medieval Carpathian Basin and 141 present-day populations. The sequence length was uniform, ranging np 16068–16365 (Supplementary Table S14). The analysis was performed in Arlequin software, using Tamura & Nei substitution model47, with a gamma value of 0.177. For the haplotype definition, the original definition was used. FST values between conquerors, Avars, and the contact zone population and each modern population were combined with longitudes and latitudes according to population information in the literature. The FST values and coordinates were interpolated with the Kriging method implemented in Arcmap ArcGIS version 10.3.
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Kruppel-like factors (KLFs) are members of the zinc-finger transcription factors family that are critical in the regulation of cell proliferation, differentiation and inflammation1, 2. So far, the mammalian KLF family has 17 members3, each has 3 contiguous C2H2 (cysteine-histidine) type zinc fingers at the carboxyl terminus4, 5. In particular, KLF2 can modulate expression of genes critical in regulating endothelial homeostasis, vascular tone, thrombosis and inflammation, and has been regarded as an atheroprotective factor2, 6, 7. KLF2 is potently induced by laminar shear stress and confers anti-inflammatory and anti-thrombotic effects on the endothelium7, 8. KLF2 strongly induces endothelial nitric oxide synthase (eNOS) expression, but decreased vascular cell adhesion molecule 1 (VCAM1) and endothelin-1 (ET-1) expression4, 9–11. Endothelial KLF2 expression is regulated by the MEK5/ERK5 (dual specificity mitogen-activated protein kinase 5 (MEK5)/extracellular-signal related kinase 5 (ERK5) pathway requiring myocyte enhancer factor 2 (MEF2) transcription factor, which mediates beneficial effects of laminar flow12–14. Our previous studies showed that KLF2 was also negatively regulated by histone deacetylase 5 (HDAC5)15, 16. In vivo animal experiments demonstrated that endothelial cell-specific deficiency of KLF2 predisposed to atherosclerosis development17. Hemizygous deficiency in Klf2+/−mice on apolipoprotein E deficient (ApoE −/−) background (Klf2 +/−; ApoE −/−) exhibited increased diet-induced atherosclerosis versus wild-type ApoE deficient mice (Klf2 +/+; ApoE −/−)2. In addition, myeloid-specific Klf2 knockout in an atheroprone LDL receptor-deficient background (Ldl −/−) increased atherosclerosis progression18.
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| 99.94 |
Based on these studies, modulating KLF2 expression or function could be a novel strategy for the prevention and treatment of inflammatory related disease including atherosclerosis2, 6, 9. Thus, the aim of this study is to screen small-molecule activators that modulate KLF2 expression. Based on luciferase-based assay, we identified that a small molecule drug tannic acid (TA) was a novel KLF2 activator. TA is a specific commercial form of tannin (a type of plant polyphenol). Previous studies showed that TA reduced aortic lesion formation in Apo E −/− mice19. However, the anti-inflammatory action of TA in ECs has not yet been investigated. In the present study, we provide evidence that TA induces endothelial KLF2 expression and thereby attenuates vascular endothelial inflammation. Collectively, our findings not only identified TA as a novel KLF2 activator but also demonstrated that KLF2 could serve as a promising therapeutic target for the treatment of atherosclerosis.
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COS-7 cells (ATCC, Rockville, MD) were cultured in DMEM (Corning, Cellgro®, USA) containing 10% fetal bovine serum (FBS) (Gibco). HCAECs (#300K-05A, Cell Applications Inc., San Diego, CA) were cultured in Meso Endo Cell Growth Media (Cell Applications Inc.) containing 10% FBS and Growth Supplement (#212K-500, Cell Applications Inc.)20. Passage 4-6 of HCAEC cells were used for experiments. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords from normal pregnancy women in accordance with the University of Rochester human subjects review board procedures that prescribe to the Declaration of Helsinki20. Human umbilical cores are obtained under informed consent from all participants and conform to HIPAA standards to protect the privacy of the donor’s personal health information. HUVECs were cultured in Medium 200 (#M-200-500, Thermo Fischer Scientific, Waltham, MA) containing 10% FBS and low serum growth supplement (LSGS) (#S-003-10, Thermo Fischer Scientific). Passages 3–6 of HUVECs were used for experiments. THP-1 cells (ATCC) were grown in RPMI 1640 containing 10% FBS. Lung ECs were maintained in DMEM containing 20% FBS, penicillin/streptomycin and heparin. All cells were cultured at 37 °C with 5% CO2 in cell incubator.
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The drug library was obtained from University of Rochester Pathway Discovery Resource (NCI Spectrum Compound Library containing 2400 chemical compounds, 1 mM for every compound). The -1.7-kb KLF2-luc promoter-driven luciferase reporter (KLF2-luc) plasmid, KLF2 -221bp promoter wild-type (WT) and KLF2 -221bp promoter mutant plasmids were gifted by Prof. Mukesh Jain21. KLF2 -221 plasmid contains MEF2 binding site while in KLF2 -221 mutant plasmid, the MEF2 binding site was mutated.
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KLF2 luciferase assay was performed as follow. Briefly, COS-7 cells at a density of 80–90% in 100-mm dish were transfected with 5.4 µg plasmid (KLF2-luc or KLF2 -221 WT or KLF2 -221 mutant plasmid) using lipofectamine 2000 (Thermo Fisher Scientific, USA) in Opti-MEM (Gibco) for 6 h. Then the transfected cells were grown in 96-well plates (3.5 × 105 cells/well, 100 µl/well). After 6 h, 1.0 µl /well drugs at a final concentration of 5 µM in 200 µl medium/well were added and incubated for 24 h. The KLF2 luciferase reporter gene activity was detected using Promega dual-luciferase reporter 1000 assay system in a microplate spectrophotometer (BMG, USA). The luciferase activity of the tested compound is as calculated as compound firefly luciferase value/DMSO firefly luciferase value.
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Total RNA extracted from cultured HCAECs or lung endothelial cells (ECs) were performed as previously described20. TA was purchased from Sigma-Aldrich Co. (#MKBV0516V). Cells were grown in 24-well plate and treated with TA (0, 0.1, 1, 10 and 20 µM) for 24 h. Total RNA from cells was extracted using a QIAGEN RNeasy Mini kit (Qiagen) and converted into complementary DNA (cDNA) using a High-Capacity cDNA Reverse Transcription Kit (#4374966, Applied Biosystems, Foster City, CA). Quantitative real-time PCR (qPCR) was performed in a Bio-Rad iQ5 real-time PCR thermal cycler using iQ SYBR Green Supermix (#1708886, Bio-Rad). Relative mRNA expression of target genes was normalized to GAPDH22.
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Western blot was performed as previously described20. Whole cell lysates from cultured cells are were harvested in cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 Mm β-Glycerolphosphate, 50 mM NaF, 1 mM Na3VO4 and supplemented with protease inhibitor cocktail (#P8340, Sigma-Aldrich) for 30 min at 4 °C. Then the cell lysates were centrifuged at 4 °C (12, 000 rpm) for 15 min and protein extract supernatant was collected. Protein concentrations were determined with the Bradford protein assay kit (#500-0006, Bio-rad) using a Beckman DU-800 spectrophotometer (Fullerton, CA). Total cell lysates (20–30 μg) were separated by SDS-PAGE and transferred to nitrocellulose membrane (Bio-rad).The membranes were then blocked with diluted Odyssey® blocking buffer (#927-40000, LI-COR Biosciences, Lincoln, NE) for 1 h. The membranes were incubated with appropriate primary antibodies overnight at 4 °C and then washed with 1 X Tris Buffered Saline with 0.1% Tween-20 (TBST) for 3 times. The primary antibodies include human VCAM-1 (Santa Cruz, #sc-1504), human ICAM-1 (Santa Cruz, #sc-8439), mouse VCAM1 (R&D, #AF643) and GAPDH (EMD Millipore, #AB2302). Membranes were incubated with appropriate second antibodies at room temperature for 30 min. Odyssey Infrared Imaging System (LI-COR) was used to take images. Densitometric analysis of the blots was analyzed with NIH Image J software.
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Mouse lung ECs were isolated as previously described23. Klf2 +/− mice (B6; 129S4-Klf2 tm1.1Hhn/J, Stock# 026926) were obtained from The Jackson Laboratory. Briefly, lungs from three Klf2 +/− or three Klf2 +/+ mice were minced into pieces. The lung pieces were digested using pre-warmed (37 °C) type I collagenase (#9001-12-1, Gibco) in a cell incubator at 37 °C for 45 min. The cell suspension was then filtered and centrifuged at 1200 rpm. The precipitates were resuspended in PBS containing BSA and penicillin/streptomycin and added CD31 (BD Pharmingen™, #553370) coated Dynabeads™ (Invitrogen) to incubate on a rotor at room temperature for 15 min. The cells in EP tubes were put on Magnetic Separation Rack and washed with growth medium (DMEM + 20%FBS + penicillin/streptomycin + heparin) for 4 times. Cells were resuspended in growth medium and grown in 0.1% gelatin coated 6-well plate. When the cells approached confluence, CD102 (BD Pharmingen™, #553326) coated Dynabeads™ sorting were performed. Then, the isolated cells were used to do the experiments. All animal procedures conformed to the Guideline for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center.
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The anti-inflammatory effect assay was performed as follow. HCAECs or lung ECs were plated in 6-well plate and treated with vehicle (DMSO) or TA (10 µM) for 12 h. Recombinant human Tumor necrosis factor (TNFα) (R&D Systems, #210-TA) or murine TNFα (#315-01 A, PeproTech, USA) at a final concentration of 10 ng/ml was added for 3 h for qPCR assay or 6 h for WB assay, respectively. Then the cells were washes with PBS.
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Total RNA were extracted as described in qPCR section. The mRNA expression levels of VCAM-1, ICAM-1, KLF2 and GAPDH were detected. The specific sequences of primers of quantitative real-time PCR were designed as follows: hKLF2: sense, 5′-CACGCACACAGGTGAGAA-3′, antisense, 5′-ACAGATGGCACTGGAATGG-3′; hVCAM-1: sense, 5′-TCAGATTGGAGACTCAGTCATGT-3′, antisense, 5′-ACTCCTCACCTTCCCGCTC-3′; hICAM-1, sense, 5′-GGCCGGCCAGCTTATACAC-3′, antisense, 5′-TAGACACTTGAGCTCGGGCA-3′; hET-1: sense, 5′-AAGGCAACAGACCGTGAAA-3′, antisense, 5′- GTCTTCAGCCCTGAGTTCTTT-3′; mKLF2: sense, 5′-CGTACACACACAGGTGAGAAG-3′, antisense, 5′-TGTGTGCTTTCGGTAGTGG-3′; mVCAM-1: sense, 5′-ACTCCCGTCATTGAGGATATTG-3′, antisense, 5′- TGACAGTCTCCCTTTCTTTGAG-3′; mGAPDH: sense, 5′-AACAGCAACTCCCACTCTTC-3′, antisense, 5′- CCTGTTGCTGTAGCCGTATT-3′.
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Monocyte adhesion to ECs was performed as previously described15. HCAECs seeded in 6-well plates were pretreated with vehicle or TA (10 µM) for 12 h. Recombinant human TNFα at a final concentration of 10 ng/ ml was added for 6 h. Then 0.5 ml (at a density of 5–7 × 106/ml) THP-1 cells were added and incubated for 30 min. Then the cells were gentle washed with Meso Endo Cell Growth Media for 3 times to remove non-adherent THP-1 cells. The pictures were taken by Zeiss Axiovert 40 C microscope (magnification: 10 × ) with a Canon A640 digital camera (Canon USA Inc) with. The numbers of each image adherent cells were counted.
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HUVECs were plated in 6-well plates. The cells were transfected with siControl (control siRNA, 20 nM, #AM4611, Thermo Fisher Scientific) or siKLF2 (KLF2 siRNA, 20 nM, #E006928, GE Dharmacon) in opti-MEM using RNAMAX (Thermo Fisher Scientific). After 6 h, the medium were changed with cell culture medium with 10%FBS and incubated for 48 h. Then the cells were treated with TNFα or TA (10 µM) as described in cell adhesion assay.
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Data were analyzed by GraphPad Prism 5 software (GraphPad Software, Inc). Statistical comparisons and analyses between 2 groups were performed by 2-tailed, paired Student’s t test or one way ANOVA. Data were presented as mean ± SEM. P < 0.05 was considered statistically significant.
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| 94.75 |
KLF2 promoter luciferase assays in COS-7 cells were performed as we described in methods section. Among the positive hit compounds, we observed that TA significantly increased KLF2 promoter luciferase activity at 5 µM (data not shown). The structure of TA is shown in Fig. 1A. Dose dependent assay showed that TA significantly increased KLF2 promoter luciferase activity at 10 and 20 µM (Fig. 1B). Simvastatin (1 µM), which was previously reported as a KLF2 activator21, 24, was used as a positive control. Next, the effect of TA on KLF2 was then confirmed by qPCR assay. KLF2 downstream target gene ET-1 was also examined. As shown in Fig. 2A, TA could increase KLF2 mRNA expression in HCAECs in a concentration-dependent manner. Meanwhile, TA significantly decreased ET-1 mRNA expression at 10 and 20 μM (Fig. 2B). Overall, our data suggest that TA is a KLF2 activator.Figure 1Identification of TA as a KLF2 activator. (A) Chemical structure of the TA. (B) KLF2 luciferase activity was analyzed in COS-7 cells transfected with KLF2-luciferase plasmids treatment with DMSO (vehicle), TA (0.1, 1.0, 10 and 20 μM) or simvastatin (1.0 μM). **P < 0.01, Student’s t test. Values represent mean ± SEM; n = 4. Figure 2TA induced KLF2 expression in HCAECs. (A,B) HCAEC cells were treated with DMSO, TA (0.1, 1, 10 and 20 μM), or simvastatin (1.0 μM) for 24 h, then KLF2 (A) and ET-1 (B) mRNA expression was detected by qPCR. **P < 0.01, *P < 0.05 (TA or vs vehicle), Student’s t test. Values represent mean ± SEM; n = 4.
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Identification of TA as a KLF2 activator. (A) Chemical structure of the TA. (B) KLF2 luciferase activity was analyzed in COS-7 cells transfected with KLF2-luciferase plasmids treatment with DMSO (vehicle), TA (0.1, 1.0, 10 and 20 μM) or simvastatin (1.0 μM). **P < 0.01, Student’s t test. Values represent mean ± SEM; n = 4.
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TA induced KLF2 expression in HCAECs. (A,B) HCAEC cells were treated with DMSO, TA (0.1, 1, 10 and 20 μM), or simvastatin (1.0 μM) for 24 h, then KLF2 (A) and ET-1 (B) mRNA expression was detected by qPCR. **P < 0.01, *P < 0.05 (TA or vs vehicle), Student’s t test. Values represent mean ± SEM; n = 4.
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To further investigate the signaling pathway leading to KLF2 upregulation by TA, the role of the two known upstream signaling molecules in KLF2 regulatory network, namely, ERK5 and MEF2 were assessed7, 25. Previous studies have shown that the transcription factor MEF2 is necessary for the induction of KLF2 by biomechanical and other types of stimuli7, 21, 25, therefore, we first investigated whether MEF2 was required for the TA-mediated increase in KLF2. Using the KLF2 -221 WT Luc promoter fragment which contains a single consensus MEF binding site, the KLF2 inductive effect of TA was totally maintained (Fig. 3A). To assess the importance of this site in TA-mediated induction of the KLF2 luciferase activity, we used the mutant in which MEF2 binding sites was disrupted in the context of the KLF2 -221 WT Luc construct. As shown in Fig. 3A, mutation of the MEF binding site almost completely abolished the TA-mediated induction of the KLF2 luciferase activity. In this experiment, simvastatin was used as a positive control, which could strongly induce KLF2 activity in KLF2 -221 WT Luc promoter plasmid but totally lost in KLF2 -221 mutant Luc promoter plasmid (Fig. 3A). These data implicate that TA induces the KLF2 promoter activity through MEF2 binding.Figure 3The ERK5/MEF2 pathway is involved in TA-induces KLF2 expression. (A) COS-7 cells were transfected with KLF2 -221 WT or KLF2 -221 mutant plasmids, treated with TA (10.0 µM), DMSO (vehicle) or simvastatin (1.0 µM, positive control) for 24 h, and luciferase activities were then detected. *P < 0.05. n = 3. (B–D) HCAECs were pretreated with TA (10.0 µM) for 0, 5, 10, 15, 30 and 60 min, respectively, then protein expression of phospho-ERK5, ERK5 and GAPDH were determined by Western blotting. *P < 0.05. n = 3.
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The ERK5/MEF2 pathway is involved in TA-induces KLF2 expression. (A) COS-7 cells were transfected with KLF2 -221 WT or KLF2 -221 mutant plasmids, treated with TA (10.0 µM), DMSO (vehicle) or simvastatin (1.0 µM, positive control) for 24 h, and luciferase activities were then detected. *P < 0.05. n = 3. (B–D) HCAECs were pretreated with TA (10.0 µM) for 0, 5, 10, 15, 30 and 60 min, respectively, then protein expression of phospho-ERK5, ERK5 and GAPDH were determined by Western blotting. *P < 0.05. n = 3.
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| 100.0 |
MEF2 transcription factor is one downstream target of ERK5, and ERK5 was required for laminar flow-induced expression of KLF2 in HUVECs13. To ask whether ERK5 are involved in TA-induced KLF2 expression, HCAECs were treated with TA and then the levels of ERK5 phosphorylation and total ERK5 were examined. As shown in Fig. 3B–D, TA induced ERK5-phosphorylation in a time-dependent manner without affecting total ERK5 expression. These results suggest that the ERK5-MEF2 dependent pathway is likely to mediate TA-induced upregulation of KLF2 in ECs.
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KLF2 is an anti-inflammatory molecule26, and has an important role in maintaining endothelial function. TNFα-induced monocyte adhesion to ECs was then performed to evaluate the anti-inflammatory effect of TA in HCAECs and HUVECs. As shown in Fig. 4A,B, TNFα significantly induced monocyte adhesion to HCAECs compared with vehicle, while TA treatment significantly reversed TNFα-induced monocyte adhesion (Fig. 4A,B). In order to exclude cell viability affects monocyte adhesion by TA. We performed cell viability assay in HCAECs and HUVECs to evaluate the possible toxicity of TA. We found that TA at 10 µM did not cause obvious toxicity at the same condition with cell adhesion assay (data not shown).Figure 4TA attenuated monocyte adhesion to ECs. (A,B) HCAECs were pretreated with vehicle (DMSO) or TA (10.0 µM) for 12 h, and then exposed to TNFα (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, THP-1 monocytes were added for 30 min. (A) Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering THP-1 monocytes (small, round cells) in the co-culture. (B) THP-1 cells in panel A were counted and statistically analyzed. *p < 0.05, **p < 0.01, n = 4. (C–E) HUVECs were transfected with siRNA (control siRNA) or siKLF2 (KLF2 siRNA) for 48 h. Then the cells were pretreated vehicle (DMSO) or TA (10.0 µM) for 12 h, and then exposed to TNFα (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, THP-1 monocytes were added for 30 min. (C) The levels of KLF2 mRNA in ECs treated with control siRNA and KLF2 siRNA were analyzed by q-PCR. (D) Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering THP-1 monocytes (small, round cells) in the co-culture. (E) THP-1 cells in panel C were counted and statistically analyzed. One way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, n = 3.
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TA attenuated monocyte adhesion to ECs. (A,B) HCAECs were pretreated with vehicle (DMSO) or TA (10.0 µM) for 12 h, and then exposed to TNFα (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, THP-1 monocytes were added for 30 min. (A) Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering THP-1 monocytes (small, round cells) in the co-culture. (B) THP-1 cells in panel A were counted and statistically analyzed. *p < 0.05, **p < 0.01, n = 4. (C–E) HUVECs were transfected with siRNA (control siRNA) or siKLF2 (KLF2 siRNA) for 48 h. Then the cells were pretreated vehicle (DMSO) or TA (10.0 µM) for 12 h, and then exposed to TNFα (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, THP-1 monocytes were added for 30 min. (C) The levels of KLF2 mRNA in ECs treated with control siRNA and KLF2 siRNA were analyzed by q-PCR. (D) Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering THP-1 monocytes (small, round cells) in the co-culture. (E) THP-1 cells in panel C were counted and statistically analyzed. One way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, n = 3.
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To ask whether the inhibitory effect of TA on monocyte adhesion is dependent on KLF2, we used KLF2 siRNA to knockdown KLF2 in ECs and examined its effect on monocyte adhesion. The efficiency of KLF2 siRNA (20 nM) to knockdown KLF2 in endothelial cells was analyzed by qPCR, and the results showed that about 70% KLF2 knockdown was achieved (Fig. 4C). When cells were transfected with control siRNA, TNFα significantly induced monocyte adhesion to HUVECs, and TA treatment significantly attenuated TNFα-induced monocyte adhesion (Fig. 4D,E), which was in consistent with Fig. 4A,B. When cells were transfected with KLF2 siRNA, the inhibitory effects of TA on TNFα-induced monocyte adhesion to endothelial cells was significantly reduced compared that with control siRNA treatment (Fig. 4D,E). Our results suggest that TA represses TNFα-induced monocyte adhesion at least in part via KLF2.
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To investigate the underlying molecular mechanisms by which TA attenuates monocyte adhesion to ECs, we examined the pro-inflammatory vascular cellular adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) mRNA and protein expression. As shown in Fig. 5A,B, TA significantly decreased TNFα-induced VCAM1 mRNA and protein expression, while the effect of TA on TNFα-induced ICAM1 mRNA and protein expression was not significant (5A-B). Meanwhile, TA protection against TNFα-induced monocyte adhesion was associated with a significant increase in expression of anti-inflammatory molecules KLF2 (Fig. 5C). Our results suggest that TA represses TNFα-induced monocyte adhesion via KLF2-dependent VCAM1 downregulation.Figure 5TA decreased TNFα-stimulated inflammatory response in HCAECs. (A) HCAECs were treated as described in Fig. 3 except for the exposure of TNFα for 3 h, then mRNA expression of VCAM1 and ICAM1 were determined by qPCR. Values are mean ± SEM, *p < 0.05, **p < 0.01, n = 5.B.HCAECs were treated as described in Fig. 4, then protein expression of VCAM1 and ICAM1 were determined by Western blot. (C) Quantification of panel B, values are mean ± SEM, *p < 0.05, **p < 0.01, n = 3.
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TA decreased TNFα-stimulated inflammatory response in HCAECs. (A) HCAECs were treated as described in Fig. 3 except for the exposure of TNFα for 3 h, then mRNA expression of VCAM1 and ICAM1 were determined by qPCR. Values are mean ± SEM, *p < 0.05, **p < 0.01, n = 5.B.HCAECs were treated as described in Fig. 4, then protein expression of VCAM1 and ICAM1 were determined by Western blot. (C) Quantification of panel B, values are mean ± SEM, *p < 0.05, **p < 0.01, n = 3.
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| 100.0 |
Next, we asked whether the anti-inflammatory effect of TA is KLF2-dependent. Because systemic KLF2 knockout was embryonically lethal2, Klf2 +/− mice were used to determine KLF2-dependency of TA. Lung ECs from Klf2 +/− and Klf2 +/+ mice were isolated and then stimulated with TNFα in the presence or absence of TA. As shown in Fig. 6, KLF2 expression in lung ECs from Klf2 +/− mice was significantly decreased compared that from Klf2 +/+ mice (Fig. 6A). VCAM1 mRNA and protein expression was greatly induced by TNFα in Klf2 +/− and Klf2 +/+ mouse lung ECs (Fig. 6B,C). TA significantly decreased VCAM1 mRNA and protein expression in KLF2+/+ mouse lung ECs. However, in lung ECs from Klf2 +/− mice, the inhibitory effects of TA on TNFα-induced VCAM1 expression was partially reversed (Fig. 6B,C). These data demonstrate that the anti-inflammatory effect of TA was KLF2-dependent.Figure 6TA inhibited vascular inflammation via KLF2. Mouse lung ECs were isolated from Klf2 +/+ or Klf2 +/− mice as described in methods. Pooled lung ECs from 3 mice with identical genotype were included in each group. (A) The levels of KLF2 mRNA expression in isolated lung endothelial cells from Klf2 +/+ and Klf2 +/− mice were analyzed by qPCR. (B) Lung ECs were treated with or without TA for 12 h and stimulated with mouse TNFα for additional 3 h. qPCR was performed to detect VCAM1 mRNA expression. Statistical comparisons and analyses between 2 groups were performed by 2-tailed, paired Student’s t test. *P < 0.05, **P < 0.01, n = 5. (C) Lung ECs were treated with or without TA for 12 h and stimulated with mouse TNFα for additional 6 h. Western blot assays were performed to examine VCAM1 and GAPDH protein expression.
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TA inhibited vascular inflammation via KLF2. Mouse lung ECs were isolated from Klf2 +/+ or Klf2 +/− mice as described in methods. Pooled lung ECs from 3 mice with identical genotype were included in each group. (A) The levels of KLF2 mRNA expression in isolated lung endothelial cells from Klf2 +/+ and Klf2 +/− mice were analyzed by qPCR. (B) Lung ECs were treated with or without TA for 12 h and stimulated with mouse TNFα for additional 3 h. qPCR was performed to detect VCAM1 mRNA expression. Statistical comparisons and analyses between 2 groups were performed by 2-tailed, paired Student’s t test. *P < 0.05, **P < 0.01, n = 5. (C) Lung ECs were treated with or without TA for 12 h and stimulated with mouse TNFα for additional 6 h. Western blot assays were performed to examine VCAM1 and GAPDH protein expression.
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Taken together, our data indicated that TA had potent anti-inflammatory effects in ECs. The schematic representation of the protective effect of TA on vascular endothelial inflammation is summarized as Fig. 7. TA induced KLF2 expression via the ERK5-MEF2 pathway, then decreased TNFα-induced monocyte adhesion through increased KLF2 expression and decreased VCAM1 expression, thus had an anti-inflammatory effect in ECs. Our study suggests to exploit TA as an effective plant-derived polyphenol that limits endothelial inflammation-associated cardiovascular diseases.Figure 7A working model depicting TA-mediated vasoprotective effects via a KLF2-dependent mechanism. Kruppel-like factor 2 (KLF2), tannic acid (TA), myocyte enhancing factor 2 (MEF2), extracellular-signal related kinase 5 (ERK5), phosphorylation (p-ERK5), vascular cell adhesion molecule 1 (VCAM1), endothelial cells (ECs), tumor necrosis factor alpha (TNFα).
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study
| 99.94 |
A working model depicting TA-mediated vasoprotective effects via a KLF2-dependent mechanism. Kruppel-like factor 2 (KLF2), tannic acid (TA), myocyte enhancing factor 2 (MEF2), extracellular-signal related kinase 5 (ERK5), phosphorylation (p-ERK5), vascular cell adhesion molecule 1 (VCAM1), endothelial cells (ECs), tumor necrosis factor alpha (TNFα).
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study
| 99.94 |
The central finding of this study is that TA induces KLF2 expression and attenuates TNFα-induced inflammation and monocyte cell adhesion in endothelial cells. To the best of our knowledge, our study is the first to demonstrate that KLF2 is critically involved in the effect of TA to prevent TNFα-induced endothelial inflammation.
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| 100.0 |
Mounting evidence support that KLF2 is an anti-inflammatory and anti-atherosclerotic molecule. Thus, modulating KLF2 expression or activity could be a new therapeutic strategy for inflammatory-related disease such as atherosclerosis. Until now, there are some small molecules which have been reported to regulate KLF2 expression or activity. It has been reported that statins induce the KLF2 expression via MEF221. Statins induce eNOS and thrombomodulin and exert endothelial atheroprotective effects dependent on transcriptional regulator KLF2, and thereby provides a novel mechanism for the beneficial effects of statins in cardiovascular disease21, 24. Moreover, it has been shown that the sirtuin 1 (SIRT1) activator resveratrol increases the expression of KLF2 via MEK5/MEF2 pathway in human endothelial cells, which helps us further understand the role of the SIRT1 activators in regulation of endothelial dysfunction-related cardiovascular disease and aging25. In addition, rapamycin, an mTOR inhibitor, could induce the expression and activity of KLF2, which might counteract coronary endothelial dysfunction27, 28. Taken together, these studies demonstrate that KLF2 may be a novel molecular target for modulating endothelial function. In this study, we performed high throughput screening and identified TA is a new KLF2 activator. We further showed that TA inhibited endothelial inflammation through upregulation of endothelial KLF2 expression and hence downregulation of adhesion molecule VCAM1 expression. Our results provide new insight into the mechanisms whereby TA attenuates vascualr inflammation and atherosclerosis.
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study
| 99.94 |
TA is a plant-derived polyphenol that has been of great interest for many years. Polyphenolic compounds as important plant-derived dietary components have antioxidant and/or free radical-scavenging properties in vitro and play an important role in the prevention of coronary artery disease29. Among many studies, extracts from plants were most used. Choi group showed that 0.02% dietary TA has health-promoting effects by alleviating hepatic lipogenesis and atherogenesis in ApoE −/− mice through increasing liver peroxisome proliferator-activated receptor α (PPARα) expression19. In our study, we show that the vasoprotective effects of TA is mediated through activating KLF2, which provide mechanistic insights into vascular benefits of TA. Multiple factors and signaling pathways are involved in the regulation of KLF2 including shear stress, statins, phosphoinositide-3-kinase (PI3K)-dependent/Akt independent pathway and AMPK-dependent MEK5/ERK5/MEF2 signaling pathway7, 30, 31. Jain group has demonstrated that statins induced KLF2 luciferase activity via MEF2 binding site21. In this study, using KLF2 -221 WT and KLF2 -221 mutant plasmids, we found that simvastatin indeed induced KLF2 expression totally dependent on MEF2 binding site. Compared with simvastatin, TA also significantly increased KLF2 expression through MEF2. In addition, we observed that TA increased ERK5 phosphorylation. Therefore, it is likely that TA induced KLF2 expression in part through the ERK5/MEF2 pathway. Further studies are needed to prove the involvement of MEK5/ERK5/MEF2 in TA-induced KLF2 expression and anti-inflammatory effect. In addition, it warrants further investigation to see whether there are some other mechanisms including AMPK activation act synergistically in the process of TA-induced KLF2 expression.
|
study
| 100.0 |
KLF2 can differentially regulate endothelial genes, thus, it was considered as a key “molecular switch” governing endothelial function in vascular health and disease9, 10, 21. The observations presented here reveal that TA is a novel KLF2 activator and suggest that KLF2 could serve as a promising therapeutic target for the treatment of endothelial inflammation-associated cardiovascular disease.
|
study
| 99.94 |
Spermatogenesis is the primary biological process occurring in the testis and produces mature haploid spermatozoa from diploid spermatogonia. This developmental process is complicated and involves a series of cell differentiation and biological events including spermatogonial proliferation, spermatocyte meiosis, and morphological changes of rounde spermatid12. Elucidation of the molecular mechanisms underlying spermatogenesis is important for understanding the genetic regulation of normal male germ cell development. This understanding can also direct strategies for the clinical diagnosis and treatment of male infertility. Therefore, investigation of the molecular mechanisms of testis development and spermatogenesis are prominent areas of research in the field of reproductive biology.
|
review
| 99.75 |
The testis is known as an immunologically privileged organ. Immune tolerance has already been established at birth when testicular germ cells (TGC) contain only stem cells or spermatogonia34. After puberty, they differentiate into spermatocytes and spermatids; the differentiation involves the expression of new molecules as spermatogenesis begins. Therefore, TGC are believed to contain various cell type-specific autoantigens which are recognized as foreign by the immune system34. The blood–testis barrier (BTB), formed by Sertoli cells, protects autoimmunogeneic TGC from any autoimmune attack34. Moreover, testicular cells express and secrete numerous immunoregulatory molecules that have important roles in the regulation of immune responses in the testes. These molecules create a regulatory system called “testicular immune privilege” and include androgens, activin, Fas ligand, protein S, and immunosuppressive cytokines such as interleukin (IL)-10, IL-35 and transforming growth factor (TGF)-β56789101112. When the BTB is functionally damaged, TGC autoantigens can pass beyond the seminiferous epithelium and create a continuous stream of AIs that are exposed to systemic immune system, often for extended periods of time3. For example, damage to BTB of testis due to infection, biopsy, torsion, or surgery in the scrotal area induces orchitis in the contralateral testis131415. Therefore, the AIs in TGC can be considered a critical target of autoimmune damage.
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study
| 75.4 |
Recent studies have demonstrated that testicular inflammatory disorders leading to impairment of spermatogenesis are an important cause for male infertility, and autoimmune orchitis is noticed as one of main reasons161718. Experimental autoimmune orchitis (EAO) is a model of chronic testicular inflammation resulting in male infertility3419202122. The pathological condition is characterized by T-cell-dependent lymphocytic inflammation and damage to the seminiferous tubules involving the shedding and apoptosis of germ cells3419202122232425262728. In rats and mice, EAO is classically induced by immunization with testicular homogenate (TH) plus complete Freund’s adjuvant (CFA) and Bordetella pertussis (BP); it is thought that treatment with the two adjuvants is required to enhance immune responses, resulting in the breakdown of testicular immune privilege272930. We have recently reported that CFA and BP treatment alone augments autoimmune reactions against some testicular autoantigens31. These results indicate that the treatment with adjuvants alone can evoke autoimmune reactions against some AIs irrespectively with exposure to TH31. We have previously established another EAO model induced in both A/J and C3H/He mice with a very high incidence by two subcutaneous injections of viable syngeneic TGC without using any adjuvant21. Our EAO model is unique because serum autoantibodies are only against acrosomal regions of sperm and spermatids, but not Sertoli cells, Leydig cells and seminiferous tubular basement membrane3252632. This model showed that the immunologically privileged state of the testis is easily overcome using only two TGC injections.
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study
| 99.94 |
On the other hand, TGC-specific AIs have also received considerable attention because of their role as cancer/testis antigens (CTs)3334. CTs are protein antigens with expression normally restricted to adult TGCs and yet they become aberrantly activated in and expressed by a proportion of various types of cancer, including melanoma, lung cancer, and pancreatic cancer333435. Hence, CTs are promising candidates for cancer immunotherapy targets and have become a major focus of vaccine-based clinical trials in recent years. Thus, information on the testis-specific proteins and proteins expressed after puberty may reveal additional biomarker candidates for cancer diagnosis/prognosis.
|
review
| 99.8 |
Previous studies using TH + CFA + BP-induced EAO rat and vasectomized mouse models have demonstrated that the proteins endoplasmic reticulum 60, heat shock protein 70, a partial region of D3p domain of Zan with B cell epitope, and others are AIs that are involved in testicular autoimmune response3637. However, there is currently no information available on TGC-specific AIs. The aim of this study is to identify TGC-specific AIs using sera obtained from mice with EAO induced by TGC alone.
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study
| 100.0 |
We induced EAO by immunization using viable syngeneic TGC without using any adjuvant. B cell infiltration and IgG deposit were detected by immunohistochemistry in the testis of EAO. IgG titers in TGC-induced EAO mice were extremely high as detected by enzyme-linked immunosorbent assay (ELISA). Then, we identified TGC-specific AIs by TGC liquid chromatography–tandem mass spectrometry (MS) analysis, followed by two-dimensional gel electrophoresis (2D), which showed serum IgG from EAO mice reaction. The expression pattern of identified TGC-specific AIs was analyzed by the Real time PCR. The recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction.
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study
| 100.0 |
No lymphocyte infiltration was observed in any of the control mice testes (Fig. 1a–c). Conversely, extensive lymphocytic infiltration with spermatogenic disturbances was observed in all TGC-immunized EAO mice testes (Fig. 1e–g). Numerous lymphocytes surrounded the peripheral seminiferous tubules, resulting in aspermatogenesis (Fig. 1e–g). No. inflammation was observed in the epididymis of the TGC-immunized EAO mice (data not shown). Immunohistochemical analysis revealed that a portion of the lymphocytes in the interstitium and in the seminiferous tubules were B220-positive cells (Fig. 1g). Moreover, deposits of immunoglobulin (Ig) G were detected a portion of the lymphocytes that accumulated in the interstitium (Fig. 1h). No deposits of IgG and B220 were detected in any of the negative controls (Fig. 1c,d).
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study
| 100.0 |
Because IgG titers in TGC-induced EAO mice were higher than those of IgA and IgM, IgG was used as a secondary antibody for detecting TGC-specific AIs. Spot numbers were only assigned to spots identified by mass spectrometry (MS) using TGC autoantibody as a primary antibody (Table S1). All spots from EAO and control were detected by MS. However, because the spots in which multiple proteins are mixed together, they cannot be identified by MS, thus spot number was not attached. All proteins that visualized on a silver-stained gel were excised and processed for protein identification using MS (Fig. 3a,d). Altogether, isoelectric point (pI) 4–7 set of 29 protein spots (Fig. 3c and Table S1) and pI 6–9 set of 10 proteins spots (Fig. 3f and Table S1) were identified that reacted with EAO serum sample, whereas pI 4–7 set of 23 protein spots (Fig. 3b and Table S1) and pI 6–9 set of two proteins spots (Fig. 3e and Table S1) were identified that reacted with control sera. Some spots (spot no. 1, 3–9, 11–14, 17–20, 22–23, 25–26, 28–31, and 33) overlapped between the EAO and control serum. Other spots (spot no. 2, 10, 15, 16, 21, 24, 27, 32, and 34–36) in EAO serum were not identified in control sera. In effect, we have identified the spots which reacted only with the serum IgG of EAO as TGC-specific AIs. These spots were summarized in Table 1.
|
study
| 100.0 |
Figure 4 the mRNA expressions of Tubb2c, Pdhb, Hsc70t, Fbp1, Lrrc34, Gapdhs, Pdha2, Dazap1 and the unnamed protein in testes were significantly higher those in the other organs. Atp6v1a mRNA expression in the testis was significantly higher different between the testis and various other organs except for the brain (p = 0.11). Dnpep mRNA expressions in testes were not significantly different from the ones in epididymis (p = 0.57), submaxillary gland (p = 0.33), spleen (p = 0.88), small intestine (p = 0.82), liver (p = 0.28), lung (p = 0.44), while it was significantly higher than heart, pancreas, kidney, muscle, and brain.
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study
| 100.0 |
Figure 5 the mRNA expressions of Tubb2c, Atp6v1a, Hsc70t, Fbp1, Lrrc34, Gapdhs and Dazap1 in testes of 8 week old mice were significantly higher than those of 2 week old mice. However, Pdhb (p = 0.88), Pdha2 (p = 0.071), Dnpep (p = 0.075), and the unnamed protein (p = 0.66) showed no such significant difference.
|
study
| 100.0 |
The mRNA expressions of all AIs were significantly different between TGC and epididymal spermatozoa (ES). The mRNA expressions of Tubb2c, Atp6v1a, Pdhb, Hsc70t, Fbp1, Lrrc34, Gapdhs, Pdha2, Dazap1, and the unnamed protein in TGC were significantly higher those in ES. On the contrary, Dnpep in TGC was significantly lower than that in ES (Fig. 6). We decided to include Tubb2c, Atp6v1a, Hsc70t, Fbp1, Lrrc34, Gapdhs, and Dazap1 from the real-time PCR as candidates for TGC-specific AIs.
|
study
| 100.0 |
Offered commercially plasmid vectors (Tubb2c, Atp6v1a, Pdhb, Hsc70t, Fbp1, Lrrc34, Dnpep, Gapdhs, Pdha2 and Dazap1) were transfected into human embryonic kidney 293 (HEK293) cells. Expression by HEK293 of TGC-specific AI proteins has been confirmed by FLAG antibody (Fig. 7-1). EAO sera (Fig. 7-2) reacted to various TGC proteins, but control sera did not (Fig. 7-3). Presences of Hsc70t (Fig. 7d), Pdha2 (Fig. 7i) and Dazap1 (Fig. 7j) antibody in EAO serum but not in control serum were determined by Western blot. Presences of Tubb2c (Fig. 7a), Atp6v1a (Fig. 7b), Pdhb (Fig. 7c), Fbp1 (Fig. 7e), Lrrc34 (Fig. 7f), Dnpep (Fig. 7g) and Gapdhs (Fig. 7h) antibody in EAO serum and control serum were not determined by Western blot (Fig. 7).
|
study
| 100.0 |
This study identified 11 AIs using serum autoantibodies from TGC-induced EAO mice. AIs related to testicular autoimmunity have been previously identified using various methods. Primakoff et al.38 and Tung et al.39 identified that immunization of male guinea pigs with the sperm surface protein sperm adhesion molecule 1 (SPAM1, PH-20) reproducibly resulted in infertility. They demonstrated that EAO could be induced in guinea pigs immunized with PH-20 + CFA, but they could not induce EAO by the same method in mice3839. Recently, Fijak et al. reported that disulphide isomerase endoplasmic reticulum 60, heat-shock 70 kDa protein 5, heterogeneous nuclear ribonucleoprotein H1, and sperm outer dense fiber major protein 2 are AIs related to rat EAO in a TH + CFA + BP-induced EAO model36. The mice injected with these proteins in CFA had induced EAO at a rate of 25%. PH-20, disulphide isomerase endoplasmic reticulum 60, heat-shock 70 kDa protein 5, and sperm outer dense fiber major protein 2 were expressed in the testis and epididymis in humans and mice40414243. Heterogeneous nuclear ribonucleoprotein H1 was expressed at testicular somatic cells, including Sertoli and Leydig cells, at 0 and 1 week old in mice36. Additional expression was observed in spermatocytes and spermatids at 12 weeks in humans and mice44. Moreover, following mRNA analysis, it was found that sperm outer dense fiber major protein 2 is expressed in the ovary and uterus45, and heat-shock 70 kDa protein 5 is expressed in the lung, pancreas islet, and kidney46. Ten of our 11 detected proteins detected are novel AIs that have not been reported.
|
study
| 100.0 |
We previously investigated AIs relevant to TGC-induced EAO through reacting individual immune serum samples with testes from normal mice of various ages using immunoblotting47. The results showed that the sera obtained from mice with TGC-induced EAO lesions specifically defined testicular antigens with molecular weights of 15 kDa, 40 kDa, 75 kDa, and >200 kDa from 4-week-old mice47. In addition to these, bands of 24–35 kDa and 60–75 kDa were detected in testicular proteins at 8 weeks of age47. Moreover, we incubated immunoblots with EAO serum from various weeks after TGC immunization32. At 0 week of age, a band corresponding to approximately 40 kDa was detected in TGC proteins, indicating the presence of natural autoantibodies against TGC. At 4 weeks of age, in addition to this band, a band of approximately 42 kDa was detected in TGC proteins. At 8 weeks of age, three TGC protein bands (approximately 20, 28, and 60 kDa) were also detected32. Considering the EAO-inducing factors and expression levels in testis at 2 weeks and 8 weeks, the EAO-related proteins were 24–35, 42, and 60–75 kDa. In this study, we identified 11 AIs recognized by serum antibodies from TGC-induced EAO in mice extracted Tubb2c (50 kDa), Atp6v1a (69 kDa), Hsc70t (71 kDa), Fbp1 (37 kDa), Lrrc34 (47 kDa), Gapdhs (48 kDa), Pdha2 (44 kDa), Dazap1 (43 kDa), and the unnamed protein product (55 kDa) from mRNA expression analysis at 2 weeks and 8 weeks of age. Therefore, Atp6v1a, Hsc70t, Fbp1 Lrrc34, Gapdhs, and Dazap1 can reasonably be categorized as EAO-related proteins.
|
study
| 100.0 |
Information from the literature on the 11 identified AIs is presented in Table 248495051525354555657585960616263. Tubb2C, Fbp1, Pdhb, and Gapdhs are all expressed in the sperm tail. Pdha2 is also expressed in haploid germ cells, diploid germ cells, and Sertoli cells. Hsc70t, Gapdhs and Fbp1 are expressed in spermatids. Lrrc34 and Dazap1 are expressed in spermatocytes and spermatids. Thus, Lrrc34, Hsc70t and Dazap1 appear candidate EAO-related AIs based on the present study results and previous literatures. Further, information of TGC-specific AIs by using immunohistochemistry may contribute to elucidate the pathological mechanism of EAO.
|
study
| 100.0 |
We have examined the candidates for TGC-specific AIs from the literature and our experiment. We decided to include Tubb2c, Atp6v1a, Hsc70t, Fbp1, Lrrc34, Gapdhs and Dazap1 as the candidates for TGC-specific AIs from the results of real-time PCR (tissue specificity, age, cell type) in this study. In addition, we selected to include Atp6v1a, Hsc70t, Fbp1 Lrrc34, Gapdhs, and Dazap1 based on molecular weight and age from the literature as potential candidate TGC-specific AIs. Furthermore, we have decided to include Lrrc34, Hsc70t and Dazap1 from testicular localizations, as described in the literature as candidate TGC-specific AIs. Finally, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. The results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs.
|
study
| 100.0 |
In male germ cells, many marked morphological changes occur during spermatogenesis, particularly in haploid spermatids after meiotic division64. In various mammals, male germ cell differentiations proceed actively and continuously in the testis after puberty, and sperms are produced throughout adulthood64. Mice require 1 month for completion of spermatogonial stem cell proliferation and differentiation, meiosis, generation of haploid germ cells, and morphogenesis of the developing testicular spermatozoa in seminiferous tubules64. After meiotic division (during the process of haploid germ cell differentiation, or spermiogenesis), the rounded spermatids undergo marked morphological changes to become testicular spermatozoa: the nucleus assumes a compact shape, the mitochondria are rearranged, the flagellum forms, and the acrosome is generated. During this period of differentiation, which occurs within 5–6 weeks in humans6566 and 2–3 weeks in mice67, haploid germ cells do not divide but morphogenesis occurs, indicating that some regulatory mechanism arrests the cell cycle. Searching for functional changes in genes and gene products involved in male infertility would increase our understanding of the causes of this condition and perhaps lead to new treatments for some cases. The simplest strategy for elucidating the mechanism of spermatogenesis is to identify and characterize differentiation-specific molecules and their associated genes in germ cells. In this study, the mRNA expressions of 10 AIs, excluding Dnpep, were significantly higher in mice at 8 weeks of age compared with those at 2 weeks and in TGC compared with epididymal spermatozoa (ES). Thus, these proteins may be involved in the development of testicular spermatozoa.
|
study
| 100.0 |
CTs, also known as cancer germline antigens, refer to a growing list of antigens that were initially discovered in the 1980 s–1990 s, which are specifically expressed in various tumor types6869. At present, more than 70 CTs are present families encompassing more than 140 individual members with largely unknown functions6970. Because each cancer type is associated with multiple highly expressed CTs, an effective vaccine may require the presence of multiple CTs. CTs are absent in normal humans and rodent somatic cells and are expressed only in male TGC347172737475. In this study, the mRNA expressions of seven TGC-specific AIs (including Tubb2c, Atp6v1a, Hsc70t, Fbp1, Lrrc34, Gapdhs and Dazap1) were significantly higher in testis, particularly in male germ cells, compared with that in other organs in 8-week-old mice than in 2-week-old mice. Generally, CTs can be grouped into two classes based on their chromosomal location: CTs-X are located on the X chromosome and non-X CTs are located on the autosomes. Most CTs-X are unique to primates and constitute several subfamilies of homologous genes, organized in discrete clusters along the X chromosome76. As a result of their restricted expression in an immune-privileged organ, both CTs groups represent attractive immunotherapy targets34. Antigenic CTs-derived peptides that are presented to the immune system with different human leukocyte antigen allospecificities elicit both humoral and cellular immune responses. Spontaneous humoral and cell-mediated immune responses have been demonstrated for several CTs and patients with good antibody titers often present with a better prognosis71. TGC-induced EAO involves both cellular and humoral immune responses to the autoantigen-containing AIs. Therefore, we expect that cellular and humoral immunoreactions to AIs can be identified in TGC-induced EAO. Although emerging evidence has clarified the functions of a few CTs-X in cancer, the majority remains poorly understood. In contrast, non-X CTs are fairly well conserved throughout evolution, with established roles in processes such as transformation76, chromatin remodeling34, transcriptional regulation, and cell signaling72. Thus, non-X CTs are particularly promising targets for the development of small-molecule therapeutics77. In fact, we examined whether the candidate TGC-specific AIs are upregulated in cancer cells or tumors from previous reports (Table 3). These studies suggested that Tubb2c, Pdhb, Hsc70t, Fbp1 and Gapdhs are upregulated in some cancer cells or tumors (Table 3). A list of CTs has been first manually compiled from the literature (http://www.cta.lncc.br) and the resultant CT Database contains 204 genes78. All TGC-specific AIs identified in this study are encoded on an autosomal chromosome.
|
study
| 99.94 |
Using serum autoantibodies from mice immunized with only syngeneic TGCs alone, we have identified 11 proteins as respective testicular AIs. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs (Tubb2c, Atp6v1a, Hsc70t, Fbp1, Lrrc34, Gapdhs, Dazap1) were significantly higher in only mature testis compared to other organs. Three TGC-specific AIs (Hsc70t, Lrrc34, Dazap1) were shown as EAO-related proteins in previous reports and the present study also. Seven TGC-specific AIs (including Tubb2c, Atp6v1a, Hsc70t, Fbp1, Lrrc34, Gapdhs and Dazap1) show human homology. Finally, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Information of AIs have also received considerable attention because of their role as cancer/testis antigens (CTs). Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies. In the future, we are planning to generate some recombinant proteins of TGC-specific AIs.
|
study
| 99.94 |
A/J mice (8-week-old) were purchased from SLC (Shizuoka, Japan) and maintained in the Laboratory Animal Center of Tokyo Medical University for 2 weeks before use. The mice were maintained at 22 °C–24 °C and 50–60% relative humidity with a 12-h light–dark cycle. After approval by the Tokyo Medical University Animal Committee (S-23041, S-24018), all animal experiments were performed in accordance with the guidelines of the National Institute of Health.
|
other
| 99.8 |
Testes were excised from 10- and 2-week-old mice (n = 10 each), minced with scissors in cold Hanks’ balanced salt solution (HBSS), and passed through a stainless steel mesh. TGCs were harvested by centrifugation at 400 G for 15 min, washed three times in cold Hanks’ balanced salt solution, and then adjusted to a concentration of 1 × 107 TGC/200 μl/mouse after determining cell viability by trypan blue dye exclusion. The TGC suspension contained more than 99% germ cells at various stages of spermatogenesis; the remaining <1% consisted of Sertoli and interstitial cells21. The ratio of prepared cells was determined by visual microscopic inspection using a hemocytometer. In addition, prepared cells have been confirmed by immunohistochemical method and real-time RT-PCR (Figure S1).
|
study
| 100.0 |
At 10 weeks of age, male mice were subcutaneously injected with 1 × 107 TGC/mouse once on day 0 and again on day 14 (i.e., at a 2-week interval) for induction of active EAO. Male mice (aging 10 weeks) injected with HBSS alone were used as controls. At 120 days after the first immunization, the mice were deeply anesthetized with pentobarbital (65 mg/kg body weight) and their testes were removed. Histopathological samples were taken from the right testes, and immunohistochemical samples were collected from the left testes of the mice (n = 10 for both the TGC-immunized mice and control mice). Blood samples were collected from the mice by cardiac puncture (n = 10 for each group).
|
study
| 100.0 |
Isolated right testes obtained from EAO-affected mice and control mice were fixed with Bouin’s solution and then embedded in plastic (Technovit 7100; Kulzer & Co., Wehrheim, Germany) without cutting the organs to prevent artificial damage to the testicular tissue. Sections (3–4-μm thick) were obtained at 15–20-μm intervals, stained with Gill No. 3 hematoxylin and 2% eosin Y (HE stained), and observed with a light microscope (BX51; Olympus, Shinjuku, Japan).
|
study
| 99.94 |
Isolated left testes obtained from EAO-affected mice and control mice were placed in OCT compound (Miles Laboratories, IL, USA), frozen in liquid nitrogen, and stored at −80 °C until use. Five-micrometer-thick sections were cut with a cryostat (CM1900; Leica, Wetzlar, Germany) and then fixed in ethanol for 10 min at −20 °C. The sections were then rinsed in phosphate-buffered saline (PBS) and incubated with Block Ace (Yukijirushi, Hokkaido, Japan) for 20 min at room temperature to inactivate endogenous peroxidase activity. After rinsing in PBS, the sections were incubated with a rat anti-mouse B220 (clone: RA3-6B2, ×200; BD Biosciences) monoclonal antibody, followed by incubation with rabbit anti-rat IgG (Vector Labs, CA, USA) at room temperature. Bound antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZyMax, South San Francisco, CA) at room temperature. Immunoreactive cells were visualized using a Vectastain ABC Kit (Vector Labs, CA, USA) with 3,3′-diaminobenzidine (DAB) as the chromogen. Bound HRP was detected using 0.05% DAB and 0.01% H2O2. Sections processed with rabbit serum instead of the primary antibodies were used as negative controls. The stained sections were counterstained with methylgreen (Vector Laboratories, CA, USA).
|
study
| 100.0 |
ELlSA was performed using the method described by Hirai et al.32. For preparation of antigens, TGC obtained from 10-week-old normal mice (n = 3) was homogenized in carbonate–bicarbonate buffer (Sigma-Aldrich, MO, USA). Antigen concentrations were determined using the Bradford method with bovine serum albumin as the standard. Antigen was then added to each well of a microtiter plate (Nunc 96-well plate; Thermo Fisher Scientific, Kanagawa, Japan) and incubated at 37 °C for 30 min. After removing the coating solution, the wells were washed three times with PBS-Tween 20; Then, 200 μl of a rabbit anti-mouse monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (housekeeping gene; Bethyl Laboratories, Inc., TX, USA; 1/200 and 1/1000 dilution) or experimental mouse serum samples serially diluted with 1% goat serum in PBS (each sample run in duplicate) were added to microELISA wells and incubated for 2-h at room temperature. The wells were then washed five times with PBS-Tween 20 and incubated for 2-h at RT with 50 μl of HRP-conjugated goat anti-rabbit IgG (Cappel, PA, USA; 1:1,000), anti-GAPDH antibody, or HRP-conjugated goat anti-mouse IgG (Cappel; 1:1,000). A solution of one Alkaline Phosphatase Yellow (pNPP) tablet and one Tris Buffer tablet (Sigma-Aldrich) dissolved in 5 ml of water was used as a soluble substrate to detect alkaline phosphatase activity. After the wells were again washed five times with PBS-Tween 20, 200 μl of freshly prepared pNPP solution was added to each well. Thirty minutes later, the absorbance at 492 nm was determined using a microtiter plate reader (MPR-A4, Toyo Soda, Tokyo, Japan).
|
study
| 100.0 |
Testes from normal 10-week-old mice (n = 3) were lysed by sonication in isoelectric focusing rehydration buffer (7 M urea; 2 M thiourea; 4% CHAPS; 100 mM DTT; 0.2% Bio-Lyte, pH 5–8; 0.01% bromophenol blue; and protease inhibitor). Insoluble material was pelleted (12,000 g, 15 min and 100,000 g, 60 min) and the resulting supernatant was desalted using a 2D Cleanup Kit (GE Healthcare UK Ltd, Buckinghamshire, England). One hundred microgram of protein in a total of 200 μl of rehydration buffer was applied to 24-cm pI gradient strips (pI4–7 and pI6–9; GE Healthcare UK, Buckinghamshire, England) for overnight rehydration. First-dimension isoelectric focusing was preformed on a GE Multiphor II System (GE Healthcare UK). After focusing, strips were equilibrated with equilibration buffer (6 M urea; 50 mM Tris-HCl, pH 8.0; 2% SDS; 20% glycerol; and 2% w/v DTT) for 15 min. The strips were further equilibrated with equilibration buffer II (6 M urea; 50 mM Tris-HCl, pH 8.0; 2% SDS; 20% glycerol; and 2.5% w/v iodoacetamide) for 15 min and then directly applied to a 7.5% isocratic SDS-polyacrylamide gel for the second dimension separation. The resulting gel was then transferred to a membrane for Western blot or stained using a silver stain kit (Wako Pure Chemical Industries, Osaka, Japan).
|
study
| 100.0 |
In mice immunized with isolated TGC alone, only EAO is inducible without epididymo-vasitis21. This EAO model has been found to involve Th1-cell-dependent autoimmunity; however, B-cell lineages were also related to the development of EAO in the present study. Several IgG immune deposits are present in EAO lesions. Various immunoglobulins (IgG, IgM, and IgA) against TGC were detected in TGC-induced EAO mice sera in the present study. TGC-induced EAO is unique as serum autoantibodies are only produced against the acrosomes of ES and round spermatids252632. Previously, we had observed that there were some molecular differences between TGC and ES antigens that reacted with autoantibodies using Western blot32. Considering that epididymitis does not occur in TGC-induced EAO, the AI analysis was performed using TGC rather than ES.
|
study
| 100.0 |
After gel electrophoresis, proteins were transferred onto a nitrocellulose membrane. Membranes were blocked in 4% skimmed milk in TBST for 1 h, followed by serial incubation with EAO and normal serum samples (1:50) 4 °C overnight, a biotinylated goat anti-mouse whole IgG antibody (Amersham Biosciences, Freiburg, Germany; 1:10000), and finally, a streptavidin–HRP conjugate for 30 min (Amersham Biosciences). Bound antibodies were visualized using the ECL Plus Detection Reagent (Amersham Biosciences). Spots corresponding to the Western blot membrane were cut from the silver stained 2D gel for identification by MS. Peptidase protein digestion was performed as follows: A piece of a gel spot stained with silver was placed in a sampling tube, dried with a vacuum concentrator, rehydrated with a trypsin solution (10 μm/ml in 50 mM ammonium bicarbonate) and then placed in a small volume of 50 mM ammonium bicarbonate. After the incubation at 37 °C under vigorous shaking for 12–16 h, the supernatant solution was removed and stored for further use. The gel piece was successively extracted once with 50 μl and again with 25 μl of 5% trifluoroacetic acid/50% acetonitrile with shaking for 30 min each time. The supernatant and extracts were mixed and concentrated to a volume of 5–10 μl.
|
study
| 99.94 |
Testes, epididymis, submaxillary gland, spleen, heart, kidney, muscle, small intestine, liver, brain, lung, and pancreas from normal control A/J mice were evaluated at 8 weeks of age (n = 4) for expression analysis in the various organs. Testes from normal A/J mice were evaluated at 2 weeks of age (n = 4) for analysis of expression that was related to spermatogenesis. TGC and ES were excised from 10-week-old mice (n = 4), minced with scissors in cold HBSS, and passed through a stainless steel mesh. TGC and ES were harvested by centrifugation at 400 g for 15 min and washed three times in cold HBSS.
|
study
| 100.0 |
For analysis, total RNA was isolated from the entire above organs and cells using the TRIzol RNA Extraction Kit (Invitrogen, CA, USA), according to the manufacturer’s instructions, and the RNA pellets were dissolved in 10 ml of RNase-free distilled water. Total RNA was measured at 260/280 nm using a UV spectrophotometer and was stored at −80 °C prior to use. cDNAs were prepared from 10 μg of total RNA in a 100 μl reaction mixture using random primers according to a standard protocol (high capacity cDNA archive kit; PE Applied Biosystems, Foster City, CA, USA). The PCR reactions were conducted using an iCycler thermal cycler (Bio-Rad, Hercules, CA, USA), and the mixtures were stored at −80 °C until analysis. Real-time RT-PCR was performed using 3 ng of cDNA and the validated SYBR Green gene expression assay in combination with the SYBR Premix Ex Taq II (TaKaRa, Bio Inc., Ohtsu, Japan) for measuring Tubb2c, Atp6v1a, Pdhb, Hsc70t, Fbp1, Lrrc34, Tyrp1, Gapdhs, Pdha2, Dazap1, Fh1, and GAPDH. All of the primers used in this analysis are listed in Table 4. Quantitative real-time PCR was performed in duplicate in a thermal cycler dice real time system TP800 (TaKaRa). The data were analyzed using thermal cycler dice real time system software (TaKaRa), and the comparative Ct method (2∆∆Ct) was used to quantify gene expression levels. Real-time RT-PCR data were standardized to GAPDH, which was used as an internal control. To confirm the specific amplification of the target genes, each gene product was further separated on a 1.5% agarose gel to detect any single bands at the theoretical product sizes, and the dissociation curves were analyzed to detect any single peaks.
|
study
| 100.0 |
Plasmid vectors (pCMV6) of TGC-specific AIs and Native were purchased from Origene (Table S2). All proteins were expressed in a suspension-adapted HEK293 cell line (ExPi293F, Thermo Fisher). Cells, growing in deep 6-well blocks, were transfected in triplicate at a density of 1.0 × 107/ml using ExpiFectamine™ 293 according to the manufacturer’s instructions. Following transfection, the cells were grown at 37 °C by shaking in 5% CO2, for 48 hours. The conditioned media, containing secreted protein, were harvested by centrifugation at 1000 rpm for 5 minutes. The clarified media from each of the three replicates for each expression vector were pooled and stored at −20 °C. Each transfection was performed in duplicate, on separate days, and the conditioned media was analyzed separately by Western blot analysis.
|
study
| 100.0 |
The expression of TGC-specific AI proteins by HEK293 and testicular germ cells were evaluated by Western blot analysis for detection of the presence of TGC-specific AIs antibody in EAO serum. The concentration of protein was measured with a Protein Quantification Kit-Rapit (Dojindo Molecular Technologies, Kumamoto, Japan). Samples were mixed with LDS Sample Buffer (Thermo Scientific, Rockford, IL, USA) and an equal amount of protein per lane was run on a 4–12% SDS-PAGE and transferred by iBlot (Thermo Scientific, Rockford, IL, USA). Blots were incubated with anti-FLAG antibody (Sigma-Aldrich, MO, USA) at a dilution of 1:1000, EAO serum at a dilution of 1:50, control serum (10 week mice) at a dilution of 1:50 at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG second antibody (GE Healthcare, Little Chalfont, UK) at a dilution of 1:10000 for 2 h at room temperature. The proteins were visualized by chemiluminescence using an ECL Prime western blotting detection kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. Detection was performed with a ImageQuant 350 (GE Healthcare, Little Chalfont, UK).
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study
| 100.0 |
The functionality for a range of complex metal oxides is controlled by the interplay between lattice, spin, charge, and orbital degrees of freedom. Experimentally, it has been demonstrated that the oxygen content is one of the key parameters in controlling this interplay and thus significantly influence the physical properties; for example, Mn-O-Mn chains in manganite perovskite oxides determine the ferromagnetism and magnetotransport properties12. In semiconducting oxides, oxygen vacancies usually serve as dopants (since the energy level is above the middle of the bandgap) and impact the electrical behavior. Because the oxygen content in complex metal oxides allows the manipulation of desired physical properties345678, it is a crucial aspect of consideration in the design, synthesis, and application of functional oxide thin films. In perovskite oxides with ABO3 structure, oxygen content-driven A-site and B-site vacancies have been reported to significantly alter the physical properties of thin films. There have been tremendous efforts to understand the effect of changes in oxygen content on the properties of perovskite thin films that are not composed of cations with multiple valance states. While oxygen vacancies have been recently been reported to act as desired defects91011121314151617, the oxygen content effect in perovskite compounds with multiple valance states at the B site can provide a deeper understanding of the underlying mechanics of the functional properties observed in these materials18.
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study
| 89.0 |
During thin film growth, oxygen content in oxide thin films varies depending on the growth conditions or post-treatments. Three main approaches have been commonly used to accommodate oxygen vacancies: (I) by generating corresponding vacancies in cation sites; (II) by altering the valence state of cations without cation non-stoichiometry; and (III) by incorporating both cation vacancies and change of valence state. Generation of vacancies in cation sites or forming cation-anion vacancy pairs usually occurs in metal oxides without multiple valence states such as ZnO, SrTiO3, BaTiO3, etc19. For example, electronic conduction and superconductivity in SrTiO3 as well as ferroelectricity in BaTiO3 are influenced by oxygen vacancies induced cation stoichiometry202122. It was reported that the cation stoichiometry is strongly affected by the oxygen pressure during synthesis. In both SrTiO3 and BaTiO3, lower oxygen pressure during film deposition results in larger out-of-plane lattice parameter23.
|
study
| 100.0 |
Altering the valence state of cations without cation non-stoichiometry usually occurs in compounds with multiple valance states, where oxygen vacancies are often charge-compensated by the change of cation valence state24. Perovskite oxides with transition metals such as V, Co, Fe, and Mn belong to this category. SrFeO3−δ is an example of this type. The existence of multiple valence states of Fe allows for various stable states of oxygen occupancy in the lattice. Much of the recent attention in SFO thin films stems from the strong dependence of the crystal structure, magnetic and electrical properties of this material on the oxygen content (0 ≤ δ ≤ 0.5). SrFeO2.5 can exhibit semiconducting behavior and brownmillerite structure, while cubic perovskite SrFeO3 has been shown to exhibit metallic behavior with existence of helical antiferromagnetic-ordered spin structure252628.
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study
| 100.0 |
The control of vacancy state behavior in compounds with multiple valance states represents great promise for the malleability of multifunctional oxide thin films, but more investigation is still required to establish and understand the underlying mechanisms. Here, we report on the effect of various processing parameters on the oxygen content of SFO thin films and correlate oxygen content to the transport and dielectric properties. Studying the effect of oxygen vacancies in compounds with multiple valance states such as SFO provides an avenue to better understand the relationship between lattice, charge and structure. We propose that the metastable oxygen deficient states of SFO thin films offer an opportunity to accomplish highly tunable electronic properties applicable to a wide range of technological applications that leverage the variability in structural, magnetic and electrical properties of SFO.
|
study
| 100.0 |
SFO thin films with a thickness of 75 nm were deposited by PLD on Nb-doped (0.7% wt) STO (001) substrates (Nb:STO) at 800 °C in three different post-growth annealing conditions. O2 and vacuum anneals were performed by holding the films at 600 °C for 1 hour in O2 of 500 Torr and <10−6 Torr, respectively. The films were then cooled down to room temperature at 5 °C/min at the same oxygen pressure. SFO with no anneal was also investigated, in which the heater was promptly shut off after deposition and films cooled freely in the growth pressure (250 mTorr O2) environment.
|
study
| 100.0 |
Similar to well-studied transition metal oxide systems such as SrCoO3−δ29, bulk SFO exhibits two topotactic phases: the cubic perovskite SrFeO3 and the brownmillerite SrFeO2.53031. Perovskite SrFeO3 has a bulk lattice parameter of 3.851 Å. The oxygen-deficient brownmillerite SrFeO2.5 structure has unit cell parameters of a = 5.672 Å, b = 15.59 Å, and c = 5.527 Å. This structure can be reduced to pseudotetragonal with unit cell parameters of = 4.011 Å, = 3.898 Å, and = 3.908 Å3132. Figure 1 shows the X-ray diffraction (XRD) patterns of the films treated with different conditions. The out-of-plane lattice parameters of SFO thin films are shown to increase with decreasing oxygen pressure during anneal, as evidenced by the progressively smaller 2θ values of the SFO film peak when comparing O2 anneal, no anneal and vacuum anneal samples, respectively. A broad XRD 2θ scan indicates that films are c-axis oriented with no detectable mixed phases present (not shown here). The out-of-plane SFO film lattice parameter is ~3.83 Å for the O2 annealed film, ~3.85 Å for the film with no anneal, and ~3.97 Å for the vacuum annealed film. Reciprocal space mapping (RSM) scans around the (103) peak of the SFO films are compared in Fig. 1b–d. In all three films, it can be seen that the in-plane lattice parameter of SFO remains strained to the Nb:STO substrate, indicating a = b ~3.905 Å. The out-of-plane lattice parameters progressively increase from 3.83 Å to 3.97 Å with reducing oxygen content in annealing. This corresponds to a relative unit cell volume increase of approximately 3.6% when comparing anneal in vacuum vs. oxygen. Oxygen vacancies donate electrons to the empty 3d-orbitals of Fe, reducing the ion from Fe4+ to Fe3+ and increasing the ionic radius of the Fe ion14. Hence, the lattice parameter is directly related with the oxygen content. These results agree with SFO materials reported by Yamada et al.32.
|
study
| 100.0 |
The oxygen content and associated structural changes in SFO films significantly modify the electrical properties. The room temperature dielectric and leakage current properties of SFO films with various anneal treatments are shown in Fig. 2. A metal-insulator-metal (MIM) structure was employed with Au top circular electrodes of area approximately 0.3 mm2 sputtered on the SFO/Nb:STO samples. In samples with O2 anneal and no anneal, the dielectric constant is higher than that of the vacuum annealed samples, but the loss is also significantly greater, with dissipation factor, D, of 0.547 and 0.409 for SFO at 100 kHz without anneal and O2 annealed, respectively (see Fig. 2a,c and e). The dissipation factor of SFO annealed in vacuum is a magnitude of order lower, with a value of 0.064 at 100 kHz. The lower loss is due to the increased formation of the semiconducting SrFeO2.5 brownmillerite phase in the vacuum annealed SFO, which can also be seen in the trend of leakage current behavior in Fig. 2b,d and f. The phase transition between the semiconducting SrFeO2.5 phase and metallic SrFeO3 phase has been used to design filament type resistive switching devices31. The formation of conducting filaments in SFO is possibly related to the migration of oxygen vacancies within SrFeO3−δ., under application of electric field, which allows metallic SrFeO3 channels to flow current within the semiconducting matrix. As a result, the hysteretic behavior related to this filament formation is directly related to the density and mobility of oxygen vacancies within the SFO bulk. The asymmetry of leakage behavior between positive and negative applied bias is a product of the difference between conduction activity at the Au/SFO and Nb:STO/SFO interfaces3334. By applying fields in both directions in the MIM structure, the Schottky diode behavior allows for investigation in the dominant I-V characteristics at either of the metal-semiconductor contact interfaces35.
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study
| 100.0 |
To understand the oxygen vacancies’ effect on electrical properties, the current conduction mechanisms with applied field in the range of 0–66.6 MV/m were analyzed by fitting to both bulk and interface-mediated emission models. Mathematical fitting functions were applied to the I-V curves in regions of applied field above which the majority of hysteresis was observed, since fitting in these regions must also account for the high density of oxygen migration. One possible mechanism of conduction that considers the injection of charge carriers from a metal into nearby oxygen vacancy sites as traps, with an associated barrier height that governs bulk conduction in a solid, is the trap-assisted tunneling current model:33
|
study
| 100.0 |
where A is a constant, e is the electronic charge, is the corresponding tunneling barrier height, is the effective electron mass, E is the electric field magnitude and h is the Planck constant. Based on this model, linear fitting was performed on the SFO thin films by plotting ln(J) vs. 1/E. A good linear fit was obtained for no anneal and O2 annealed SFO samples, suggesting a trap-assisted tunneling conduction mechanism, as seen by the fitting data in the insets of Fig. 2b and d, respectively. The fitting shows that is 132 meV and 136 meV for the SFO samples without annealing and annealed in O2, respectively. However, the trap-assisted tunneling model did not yield a good linear trend for the SFO film annealed in vacuum. On the other hand, the Schottky emission model is used to explain temperature-dependent leakage mechanisms that are dominated by injection of charge carriers at the metal-insulator interface, and is given by the following:
|
study
| 100.0 |
where J is the field-dependent current density, T is the absolute temperature, is the Schottky barrier height, is the optical dielectric constant, is the permittivity of free space, and k is the Boltzmann constant. is known as the Richardson constant, represented as
|
other
| 99.8 |
This model also takes into account mediation of leakage current by defects such as oxygen vacancies, which makes it a useful tool to analyze the behavior in SFO thin films34363738. Fitting of the data yields a good linear fit of ln(J/T2) vs. for the vacuum annealed SFO thin film, as can be seen in the inset of Fig. 2f. An optical dielectric constant of 3.74 was extracted from the slope of the linear fitting curve. There are limited data on the optical dielectric constant of SFO, but similar studies on SrCoOx thin films suggest that this value is within reason39. Although the results suggest that the leakage current behavior in the vacuum annealed SFO thin film is well-explained by the Schottky equation, the SFO samples without annealing and annealed in O2 do not yield a linear trend of the ln(J/T2) vs. data. Dielectric properties for all SFO films at 100 kHz are compared in Table 1. We can use these data to infer that as the oxygen content in SFO varies, not only the structure but also the nature of the conduction mechanisms in the film undergoes a notable change. Further understanding of the conduction mechanisms is critical to improving and tailoring SFO thin films for specific applications.
|
study
| 100.0 |
There are several methods used to extract the Schottky barrier height, , in the Schottky thermionic emission model, but one of the most widely used is to compare the conduction behavior against variation in temperature3840. Investigation of the electronic properties of SFO thin film annealed in vacuum was therefore conducted in the temperature range of 120 K–300 K. Results for the temperature dependence of permittivity, dielectric loss and leakage current are shown in Fig. 3.
|
study
| 100.0 |
The dielectric constant and loss of the vacuum annealed SFO sample were observed to decrease with decreasing temperature while maintaining a similar frequency-dependent profile, as shown in Fig. 3a. The temperature dependence of the leakage current at the Au/SFO interface, shown in Fig. 3b, follows an exponential behavior with increasing temperature, which indicates a good fit to the thermionic emission model. An effective thermal barrier height, , is extracted from the Schottky equation by using the relationship between ln(J/T2) vs. 1/T at a given E, which is presented in Fig. 3c3338. By then extrapolating from the linear trend of vs. to zero electric field, the intrinsic thermal barrier height, , was calculated with a value of 225 meV for the Au/SFO interface.
|
study
| 100.0 |
Dielectric property data measured at 100 kHz is summarized in Fig. 4. As temperature is varied, the dielectric response depends on associated changes in volume and polarizability in the dielectric41. The linear increasing trend can be attributed to the increasing polarizability of the SFO thin film with temperature, as shown in Fig. 4a and 4b. Indeed, the temperature dependence of the tunability of the SFO thin film, as shown in Fig. 4c, follows an exponential trend in the investigated temperature range. The inset of Fig. 4c shows tunability curves at selected temperatures, where the maximum tunability was calculated at 66.6 MV/m. This behavior is suggestive of the suppression of polarizability in the SFO thin film with decreasing temperature, and indicates that the temperature dependence of polarizability is a dominant effect in governing the response observed in the vacuum annealed SFO thin film42. Similar dielectric response behavior has also been seen in LaFeO3 in studies reported by Gaikwad et al.43.
|
study
| 100.0 |
In summary, the effect of oxygen vacancies on structural and electrical properties in epitaxial thin films of SrFeO3−δ (SFO) is studied, where SFO is a compound with multiple valance states at the B site. Various annealing treatments are used to produce different oxygen contents in the films, which results in significant structural changes in the fully strained SFO films. The out-of-plane lattice parameter and tetragonality increase with decreasing oxygen concentration, indicating the crystal structure is closely related to the oxygen content. Importantly, variation of the oxygen content in the films significantly affects the dielectric properties, leakage conduction mechanisms, and the resistive hysteresis of the materials. Leakage current mechanisms are found to shift from dominantly bulk-mediated trap-assisted tunneling to interface-mediated Schottky thermionic emission depending on oxygen vacancy concentration. Temperature dependence of vacuum annealed SFO thin film is investigated in the temperature range of 120 K–300 K, and results suggested a suppression of polarizability of the SFO thin film with decreasing temperature, as indicated by a reduction in the dielectric constant and tunability of the SFO film. These results establish the relationship between oxygen content and structural and functional properties for a range of multivalent transition metal oxides.
|
study
| 100.0 |
SFO thin films were grown by PLD using a KrF excimer laser (Lambda Physik LPX 300, λ = 248 nm, 2 Hz). The laser beam, defined by the image beam method44, was focused onto the target with an energy density of 1.87 J/cm2 . Prior to the deposition, the chamber was pumped down to a base pressure of 1 × 10−6 Torr. A substrate temperature of 800 °C and an oxygen pressure of 250 mTorr were maintained during all depositions. After deposition, various anneal treatments were used to modify the oxygen content in the SFO thin films. An O2 anneal was used with an oxygen pressure of 500 Torr and the films were held at 600 °C for 1 hour to allow more oxygen to enter the thin film. The films were then cooled down to room temperature at 5 °C/min. A vacuum anneal was performed by maintaining films in a pressure environment of < 10−6 Torr, held at 600 °C followed by cooling to room temperature at 5 °C/min. Samples were also grown with no anneal, in which the heater was promptly shut off after deposition and films cooled freely in the growth pressure (250 mTorr O2) environment.
|
study
| 100.0 |
X-ray diffraction (Panalytical X’Pert PRO MRD) 2θ-ω and reciprocal space mapping (RSM) scans were employed to obtain information on the orientation, lattice parameters and epitaxial quality of the thin films. Dielectric measurements (E4980A Precision LCR meter) were performed in the temperature range of 120 K–300 K by using a physical property measurement system to produce the cryogenic environment. An MIM (Au/SFO/Nb:STO) structure was used for dielectric measurements, with positive bias applied indicating the high potential applied to the Nb:STO substrate and low potential at the Au electrode.
|
study
| 100.0 |
Olive tree (Olea europaea L.) is one of the emblematic plants of the Mediterranean landscape, where it features an important role either cultural and economic. Therefore, the prevention of plant diseases and the maintenance of fruit production are of great concern.
|
other
| 99.94 |
The olive grove agroecosystem is an agricultural system designed and managed by man where the soil is periodically modified by agronomic practices, and the plants undergo chemical pesticide treatments to control outbreaks of pests, among which one of the most important is the olive fly [1–4], Bactrocera (Daculus) oleae (Gmelin, 1790) (Diptera: Tephritidae). Agronomic practices may have a negative impact on soil properties and erosion , while the consequence of the intensive use of pesticides often generates environmental modifications, which extensively impact on biodiversity [6–9]. Conventional agricultural practices affect the soil’s fauna diversity and activity by tillage and removing of the herbaceous vegetation cover [7, 10], the same is likely to happen as an effect of soil cultivation in olive groves. The impact of these management practices may cause a decrease in the number of epigean insects, as well as changes in their abundance [11, 12], leading to a detrimental effect even to beneficial insects (e.g. pest predators).
|
study
| 99.9 |
In recent times, the need for low-input agronomic techniques has encouraged the maintenance of agroecosystems entirely covered with spontaneous herbaceous vegetation (i.e. cover-cropping) [13, 14]. Monitoring the effects of agricultural practices by means of organisms sensitive to such kind of impact is a useful way for approaching the complexity of a comprehensive biodiversity study even over a small agroecosystem [15–18].
|
other
| 99.44 |
Although functional diversity (i.e., ‘the value and range of those features in species and organisms which influence how an ecosystem works’ ) is a key biodiversity parameter, studies focused on functional biodiversity in olive agroecosystems and data about the impact of cover crop on biodiversity in agricultural ecosystems are scarce , while quantification methods are still underdeveloped [20–22]. Notably, the classification of arthropod species strictly characterising the olive grove agroecosystem is still missing.
|
study
| 99.9 |
Carabids are epigean beetles living within the soil’s top horizons where they occupy several ecological niches and are a crucial component of predator diversity either in natural and in agricultural ecosystems [26, 27]. They respond to ecosystem alterations either at fine and at broad environmental scale [28, 29]. Different species show different feeding strategies, such as zoophagy (predatory), phytophagy (seed-eating) and polyphagy (zoophytophagous). Zoophagous types have a special role in containing phytophagous insects of agrarian significance, especially if the latter spend part of their biological cycle in the soil or within the herbaceous layer [11, 23, 30, 31]. Carabid assemblages are distributed in the environment according to definite species’ habitat preferences, which is also affected by landscape managements [11, 23, 32–34].
|
review
| 88.0 |
Even if the role of carabid beetles as indicators is controversial (see e.g. [35, 36]), it has been found that at least in the agricultural landscape they respond to the human artificialization of the environment [10, 16, 23], so that they can be used as model organisms exemplifying the insects living on the ground [18, 37, 38]. In particular, the main environmental factors affecting the species distribution are: the soil structure [23, 39], the type of soil management [40, 41] and the use of pesticides [9, 42]. This paper aims to study carabid beetles distribution in olive groves, for disentangling the possible consequences of different types of soil management. Given that there is a lack of knowledge about the relationships between agroecosystem and carabid communities, the latter need to be assessed in the Mediterranean agricultural landscape, thus the first question is: i) is species distribution affected by increased frequency of soil cultivation?; then ii) what are the main species characterizing olive plantations?; and iii) which species traits are positively/negatively selected by soil cultivation?
|
study
| 99.94 |
The study area was in Calabria, one of the southernmost regions of Italy, and in a central position inside the Mediterranean basin. The olive plantations were located in the municipality of Rende (UTM 33S 606004.62 E; 4358146.54 N), Terranova da Sibari (UTM 33S 618990.68 E; 4392167.20 N) and of Mirto (UTM 33S 651740.77 E; 4386733.68 N). All the three study areas belong to the Cosenza Province, Italy (see Fig 1 and Table 1).
|
study
| 98.4 |
The code is composed by the first three letters of the municipality (i.e. direct intuitive geographically based classification), sequentially numbered, and followed by the management code. Management: tilled (ti) = periodically undergoing mechanical soil tillage; half-cropland (hc) = characterized by cover crop of spontaneous herbaceous vegetation only under tree rows, being the rest of the soil tilled; cover cropped (cc) = entirely covered with spontaneous herbaceous vegetation mowed in spring. Forest climax: the forest vegetation potentially growing in natural conditions (i.e. absence of agricultural landscape).
|
other
| 99.94 |
We compared ground beetle communities sampled from olive plantations, characterized by three types of soil management (i.e. the treatment) (Table 1). In the following the site code comes from the territorial location name of olive plantations (i.e. REN from Rende, TER from Terranova di Sibari and MIR from Mirto), so that a direct geographically based classification is intuitively given. Sample sites were (i) those periodically undergoing mechanical soil tillage (which, in the following, are called tilled sites with the code REN1-REN4; (ii) those characterized by cover crop of spontaneous herbaceous vegetation only under tree rows, with the rest of the soil tilled (half-cropland sites TER1-TER6); and (iii) those counted as no-treatment sites because entirely covered with spontaneous herbaceous vegetation, even if mowed in spring (cover-cropped sites REN5, REN6, REN7, TER7, MIR1, MIR2 and MIR4).
|
study
| 100.0 |
The pristine ecological landscape has been changed over the centuries by the spreading of agricultural practices, which lead to the present agricultural landscape. The natural vegetation substituted by olive plantations (i.e., the potential vegetation; see Table 1) belongs in the evergreen (mainly Quercus ilex) Mediterranean forest (TER sites), the sub-Mediterranean (Quercus virgiliana) deciduous forest (REN sites) and the lowland mixed forest (Quercus robur, Fraxinus oxycarpa; MIR sites). The soil of all sampled sites was a mixture of sand and clay, while only MIR sites grow on finer, silt-rich alluvium.
|
other
| 99.9 |
Among the most frequently used morpho-functional traits [43, 43–46] we analysed body size, food choice and wing status (i.e. three morpho-functional traits), a biogeographical trait, i.e. the type of distribution range (chorotype: see and habitat association and reproduction rhythm (i.e. two eco-physiological characteristics). The body size of each species has been measured on 20 individuals of both sexes, while other traits have been extracted from a database available from the Department of Biologia Ecologia et Scientiae Terrarum in the University of Calabria, as well as from recent literature (e.g., ). The species’ chorotypes were identified according to , after which they were grouped according to their range size (see also: ).
|
study
| 100.0 |
Ground beetles were collected by using pitfall traps, which were emptied every 20 days, from April to December in 2005, in order to obtain a “year’s sample”. In this way the different seasonal period of activity of each species was intercepted by the traps, so avoiding the possible bias affecting the samples (as in [28, 51]; see also ). This is a standardized sampling method successfully applied since many years by many authors (see and literature therein).
|
study
| 99.9 |
The traps were plastic vessels with an upper diameter of 9.2 cm, a depth of 11 cm and a small opening at 4 cm below the border, to avoid rainwater overflowing, filled with 200ml of a conservative mixture of wine vinegar and 5% ascorbic acid. In all sites, three traps were set up in a line at a distance of 15 m from each other. In total, 51 traps were utilized.
|
other
| 99.75 |
Pitfall traps intercept the activity of carabids, and the activity mirrors the way by which the species disperse and eventually cluster toghether, so that it is possible to describe the species groupings characterizing different habitats on the basis of their activity pattern. This was acknowledged in the traditional literature on carabid synecology (see the review in ), and by several authors working on carabid assemblages with an "abundance approach", as e.g. [28, 53–57].
|
review
| 99.9 |
Data have been analysed in R environment . The variation of the species richness (i.e., number of species) and the activity (i.e. aAD) have been evaluated for the sampled sites by means of bootstrapping (“rich” function in ) to compare the actual number of sampled species (i.e. that species whose activity was intercepted by the traps) with the expected one (i.e. the bootstrapped one) to evaluate the eventual amount of lacking data.
|
study
| 99.94 |
On the basis of quantitative data (i.e., aAD), species have been classified by means of correlation coefficient and average linkage clustering algorithm, while sample sites have been classified by means of Bray-Curtis coefficient and minimum variance clustering algorithm. Principal Component Analysis (PCA) has been used to identify the main factors influencing the distribution of species over different habitats. PCA depicts components as orthogonal axes, and the relative position of sites on these axes was chosen as a tracer for each component (see ).
|
study
| 100.0 |
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