Pollen development is an energy-consuming process that particularly occurs during meiosis. Low levels of adenosine triphosphate (ATP) may cause cell death, resulting in CMS . DNA sequence differences in ATP synthase genes have been revealed between the N- and S-cytoplasms in the cotton CMS system. However, very few data are available at the RNA level. In this study, we compared five ATP synthase genes in the H276A, H276B and fertile F1 (H276A/H268) lines using RNA editing, RNA blotting and quantitative real time-PCR (qRT-PCR) to explore their contribution to CMS. A molecular marker for identifying male sterile cytoplasm (MSC) was also developed.orf for the ATP synthase gene sequence in the three lines. Forty-one RNA editing sites were identified in the coding sequences. RNA editing showed that proteins had 32.43% higher hydrophobicity and that 39.02% of RNA editing sites had proline converted to leucine. Two new stop codons were detected in atp6 and atp9 by RNA editing. Real-time qRT-PCR data showed that the atp1, atp6, atp8, and atp9 genes had substantially lower expression levels in H276A compared with those in H276B. By contrast, the expression levels of all five genes were increased in F1 (H276A/H268). Moreover, a molecular marker based on a 6-bp deletion upstream of atp8 in H276A was developed to identify male sterile cytoplasm (MSC) in cotton.RNA blotting revealed the absence of any novel Our data substantially contributes to the understanding of the function of ATP synthase genes in cotton CMS. Therefore, we suggest that ATP synthase genes might be an indirect cause of cotton CMS. Further research is needed to investigate the relationship among ATP synthase genes in cotton CMS.The online version of this article (10.1186/s40659-019-0212-0) contains supplementary material, which is available to authorized users. The molecular mechanisms of CMS plants have been extensively studied for several decades . To date II rice  and pcf  petunia . The ATP petunia .Gossypium harknessii Brandegee (D2) and Gossypium trilobum (DC) Skovst (D8), respectively, into upland cotton  [In the cotton CMS system, CMS-D2 and CMS-D8 are derived from the introduction of the cytoplasm of um, AD1) , 7. Cottum, AD1) ; howeverorf77 has a similar sequence to atp9; however, orf77 has less RNA editing relative to atp9. The novel RNA editing of orf77 led to CMS by inhibiting the expression of atp9 [atp9 subunit [RNA editing is a post-transcriptional process that can synthesize different amino acid sequences from genomic sequences by conversion of cytidine (C) to uridine (U). In plant mitochondrial systems, RNA editing plays an important role in gene expression at the RNA level and is a critical process for generating functional proteins . It has  of atp9 . In rice subunit .cox1, cox2, and atp1 [atp1, atp9, ccmb, nad6, nad7c and rr18) have RFLPs between P30B and P30A and explored several male sterility cytoplasm markers. In the above studies, several significant RFLPs associated with ATP synthase genes have been revealed between the N- and S-cytoplasm in the cotton CMS system. However, little information is available at the RNA level. In this study, we compared five ATP synthase genes in H276A, H276B and the fertile F1 (H276A/H268) by RNA editing, RNA blotting and qRT-PCR to explore their contribution to CMS. In addition, a molecular marker for identifying MSC was developed.In previous studies of cotton CMS, investigations have focused on the mutation of mitochondrial genome sequences and exploration of the molecular markers of male sterility cytoplasm (MSC). RFLP polymorphisms were detected between CMS-D2 and normal AD1 cytoplasm with probes for and atp1 . Zhang eand atp1  reportedFor gene cloning, qRT-PCR and RNA blotting, three cotton lines were used: CMS (H276A), a maintainer (H276B) and a fertile F1 (H276A/H268). To identify the cytoplasmic characteristics, 37 varieties of cotton were employed including nine CMS lines  with abortive-type H276A cytoplasm; five CMS lines  with abortive-type Zhong16A [Genomic DNA was extracted from young leaves of each genotype by the CTAB method . Total RGossypium hirsutum L. mitochondrial genome (http://www.ncbi.nlm.nih.gov/nuccore/JX065074.1). For each sample, 1 μg total RNA was reverse transcribed into cDNA using TransScriptRII One-Step gDNA Removal and cDNA Synthesis SuperMix . Elimination of gDNA was verified with primers for cox2, which amplified a 1887 bp fragment of the gDNA, including a 1494-bp intron, but a 393-bp fragment was amplified for cDNA. For amplification of ATP synthase genes, 50 ng DNA and 0.5 μl cDNA were used as templates. The amplicons were cloned into the PMD-19T simple vector , then three gDNA positive clones were selected randomly and sequenced [All primers  with TransStartR Tip Green qPCR SuperMix . A housekeeping gene, 18s, served as the internal reference. The 18 s primer sequences were 5′-ACACTTCACCGGACCATTCAAT/5′-CCTGGAAGAACCCTTTGTGA. The qRT-PCR conditions followed the manufacturer’s procedure: 30 s at 95 °C followed by 42 cycles of heating at 95 °C for 5 s and annealing at 60 °C for 30 s, then heating to 95 °C with an increment of 0.5 °C for 5 s to generate the melt curve. The relative expression level was calculated by the 2−∆∆Ct method with three replicates [Relative quantification of five genes in the three cotton lines were conducted by real-time qRT-PCR analysis with a C1000 Touchplicates .The percentage of RNA editing efficiency was determined with at least 13 cDNA clones from each of the three materials  for each gene . For an Approximately 30 μg total RNA was denatured and separated on 1% denaturing formaldehyde agarose gels and transferred to a Hybond N+ nylon membrane . The blots were hybridized with a DIG-high labeled cDNA probe at 42 °C for 12 h, and subsequently the manufacturer’s instructions for the DIG-High prime DNA Labeling and Detection Starter Kit II  were followed.3 and colored with formaldehyde.The primer sequences of molecular marker were 5′-TACAGGAAGGACTCGCTTTCTCTTT/5′-AAGGCATAACCAGAAGAATTGTGAA. The PCR conditions for the molecular marker were 3 min at 95 °C followed by 42 cycles of heating at 95 °C for 30 s, annealing at 56 °C for 30 s and amplification at 72 °C for 30 s, and a final amplification at 72 °C for 5 min. To analyze the PCR product of the molecular marker, 10% native polyacrylamide gel electrophoresis was used; the 50 ml reagent mix included 12.5 ml 40% polyacrylamide, 40 μl tetramethylethylenediamine (TEMED), 5 ml 10 × tris boric acid (TBE), and 400 μl 10% ammonium persulfate (APS), with ddH2O added to 50 ml. The gel was stained by 10% AgNOData on gene expression among CMS, maintainer and fertile lines of cotton were statistically analyzed with one-way ANOVA using SPSS 18.0.atp1, atp4, atp6, atp8 and atp9) in H276A, H276B and a fertile F1 (H276A/H268) were obtained using homology cloning. Sequences analysis  at the 56th amino acid. However, a 6-bp deletion in the 5′ flanking region of atp8 and six base conversions in the untranslated regions (UTR) of atp1 were identified in H276A in comparison with H276B. In addition, the fertile F1 (H276A/H268) had sequences similar to CMS line H276A for all five genes.The sequences of five genes  were detected by sequence cloning . The editing frequency of the other partial editing site at the 247th base was significantly increased in H276A compared with that of H276B (50% vs. 27.2%).Twelve editing sites  were identified in atp6. A partial editing site at the 787th base caused a change from glutamine to a stop codon, and the frequency of editing was 63.6% and 45.5% in CMS and the maintainer line, respectively. A novel stop codon was created in the 789 bp coding region though the 816 bp coding region in the wild-type. At the other special editing site at the 294th base, the maintainer line had a significantly higher RNA editing efficiency compared with the CMS line (18.2% vs. 0%).A total of eleven partial editing sites were identified in atp9. However, a new stop codon with a similar editing frequency between H276A and H276B was detected. The partial editing site (the 243th base), which had a substantially decreased editing frequency in H276A, was a silent RNA editing site (F > F).Seven full and one partial editing site were found in atp4 (225th base), atp6 (331th base) and atp8 (77th base), all other RNA editing sites in the F1 had a relatively higher editing efficiency compared with H276A. An increased editing efficiency may be associated with the presence of the restorer gene.In addition, comparative analysis of the RNA editing frequency between CMS line H276A and the fertile F1(H276A/H268) indicated that, except for atp1 (0.70), atp6 (0.70), atp8 (0.59), and atp9 (0.61) compared with H276B (P < 0.05). By contrast, a significant increase in the expression levels of atp1 (1.25), atp4 (2.06), atp6 (1.64), atp8 (1.55) and atp9 (2.27) in the F1(H276A/H268) relative to the maintainer line H276B (P < 0.01) was observed  were detected using real-time qRT-PCR. Data were analyzed using SPSS software. The results showed a substantial reduction in the expression levels of ved Fig. .Fig. 3Exatp8 in CMS line H276A was developed. The product of special primers BQatp8-F and BQatp8-R was 123 bp and 129 bp in MSC and MFC, respectively, as shown in S.5. Nine cotton CMS lines with abortive type H276A cytoplasm, their maintainer lines and the hybrid F1 were used to detect the accuracy of the molecular marker. A 123 bp fragment was amplified from all CMS lines and their hybrid F1 compared to a 129 bp fragment from their maintainer lines  were increased dramatically. These results were consistent with atp8 and atp9 in kenaf [atp1, atp6, atp8 and atp9). However, the relationship between cotton CMS and ATP synthase genes needs further investigation.Pollen development is an energy-consuming process, especially during meiosis, and cell death might be caused by low levels of ATP leading to CMS . qRT-PCRin kenaf , which satp1, such as scar611 [Molecular markers have become an important and more convenient tool for identification of MSC in comparison to traditional breeding. In cotton breeding, a shortage of CMS prevented the utilization of cotton heterosis. To overcome this problem, cotton MSC molecular markers were explored based on different sequences. However, to date, all MSC molecules based on PCR were based on mutations around  scar611  and ssr1 scar611 .orf in ATP synthase gene sequences in three cotton lines and 41 RNA editing sites in coding sequences. The result of RNA editing in three materials indicated that no differences were associated with CMS. However, the results of qRT-PCR suggested that ATP synthase genes may be an indirect cause of cotton CMS. The relationship between ATP synthase genes and cotton CMS needs further study.We identified no novel Additional file 1. Primers used in this study.Additional file 2. Sequences analysis of atp1 in three materials.Additional file 3. Sequences analysis of atp4 in three materials.Additional file 4. Sequences analysis of atp6 in three materials.Additional file 5. Sequences analysis of atp8 in three materials.Additional file 6. Sequences analysis of atp9 in three materials.Additional file 7. Amplification of molecular marker specific to MSC.


Bladder cancer often recurs due to incomplete elimination of the cancer stem cells (CSCs). Therefore, new strategies targeting bladder CSCs are needed and the aim of this study was to investigate the effect of S100A4 on the proliferation capacity of MB49 bladder cancer stem cells (MCSCs).MCSCs were established and validated. The expression level of S100A4 in MCSCs and MB49 cells was evaluated using Western blotting and quantitative polymerase chain reaction (QPCR). S100A4 was overexpressed or knocked-down by transfection of pCMV6-XL5-S100A4 plasmid or RNA interference (RNAi) respectively. Proliferation capacity of MCSC was evaluated by cell proliferation assay and in vivo tumorigenicity study. Transcriptional activity of nuclear factor kappa B (NF-κB) was analyzed using luciferase reporter assay, and the level of interleukin (IL)-2 as well as tumor necrosis factor (TNF) was quantified by QPCR. Protein-protein interaction of S100A4 and inhibitor of nuclear factor kappa B NF-κB kinase (IKK) was analyzed by immunoprecipitation.S100A4 was significantly up-regulated in MCSCs, which positively associated with the proliferation capacity, as well as the level of NF-κB, IKK, IL-2 and TNF in MCSCs. Knock-down of S100A4 could reverse such effects. Using immunoprecipitation assay, an interaction between S100A4 and IKK could be observed.S100A4 is upregulated in MCSCs and possibly enhance the proliferation ability of MCSCs by way of activating the IKK/NF-κB signaling pathway, and S100A4 maybe a hopeful therapeutic target for MCSCs. Bladder cancer is a most common urological cancer in China, and the rest of the world . RadicalS100A4, also known as metastasin/FSP1/pEL98/mts-1, is a gene encoding a small calcium binding protein that interacts with other proteins to enhance apoptosis, cell motility, and tumorigenesis [S100A4 is overexpressed in most cancers, including breast cancer, gastric cancer, and non-small cell lung cancer (NSCLC) [S100A4 expression is correlated with patients’ outcome and cancer metastasis [igenesis . S100A4  (NSCLC) . In additastasis . These fS100A4 is up-regulated in MB49 bladder cancer stem cells (MCSCs). Additionally, overexpression of S100A4 enhances the proliferation capacity of MCSCs in vitro, and also upregulates inhibitor of nuclear factor kappa B NF-κB kinase (IKK) and activates the NF-κBsignaling pathway, whereas knockdown of S100A4 resulted in the opposite effects. The findings of this study suggest that S100A4 may promote the proliferation capacity and upregulate IKK in MCSCs by activating the NF-κB signaling pathway. Therefore, S100A4 may have the potential to be a therapeutic target in MCSCs.In the present study, we demonstrate that MCSCs were obtained from MB49 bladder cancer cells, which was a mouse cell line, using limited dilution and serum-free culture medium method described previously . The serThe validation of MCSCs was performed as previously reported . Cancer The MB49 cells and MCSCs were respectively harvested. Equal amount proteins were extracted from cells, and separated by 10% sodium dodecyl sulfate -polyacrylamide gel electrophoresis followed by transferring to polyvinylidene difluoride membranes . The membranes were blocked with 5% skim milk in PBS, and incubated overnight at 4 °C with the primary antibodies including anti-S100A4 , anti- IKK (Abcam) and anti-β-actin antibody (Abcam) followed by secondary antibodies (Abcam). Bands were visualized using Fluor Chem FC2 .t method. GAPDH was used as the internal control.Total RNA was extracted using the Arcturus PicoPure RNA isolation kit . RNA quality was tested using the Bioanalyzer RNA Pico Chip . Total RNA were transcribed reversely with Superscript III (Invitrogen), followed by synthesizing the first-strand cDNA which was amplified using a SYBR green PCR master mix  performed on a 7500 real-time PCR system . The cycling systems were denaturation (95 °C for 10 s), annealing and extension (60 °C for 60 s). The primers were designed using Primer Express version 2.0 , and are shown in Table S100A4 gene were inserted into a vector plasmid pCMV6-XL5 (OriGene Technologies). MCSC cells were transfected with pCMV6-XL5-S100A4 plasmid (pCMV-S100A4) performing in Lipofectamine™ 2000 (Invitrogen) following the manufacturer’s instructions, which were referred to as MCSCs/S100A4-vector. The expression of S100A4 was detected at 48 and 72 h after transfection on transcription and translation level respectively.Full length of S100A4 was knockdown by S100A4-siRNA transfection. Double-stranded siRNAs specific to S100A4 were bought from Shanghai GenePharma Co., Ltd. . The S100A4 siRNA sequences were 5’-TGTAACGAATTCTTTGAAG-3′, and 5’-ACGAATTCTTTGAAGGCTT-3′. The non-coding (NC) siRNAs sequence was 5’-UUCUCCGAACGUGUCACGUTT-3′. MCSCs were transfected with a final concentration of 20 nM of siRNA performing in Lipofectamine™ 2000, which were referred to as MCSCs/S100A4-siRNA or MCSCs/NC-siRNA cells, respectively.3 cells per well in a 96-well plate, followed by incubating for 72 h. After incubation, cell counting kit-8 reagent  was added to each well with 10 μl at a time periods of 24, 48, and 72 h. Incubated for 4 h, the absorbance value was read at 450 nm performing in EnSpire 2300 multilabel reader .Cells were plated at a density of 1 × 10All animal experiments were obeyed the Chinese animal protection laws and guidelines, and approved by the Ethics Committee of Southern Medical University .4) were injected into mice subcutaneously. Tumor xenograft formation was recorded at 10, 20, 30, and 45 days, calculated the tumor volume according to the formula d2 × D/2, where D and d were the longest and the shortest diameters, respectively. Then mice were sacrificed after CO2 anesthesia.Four-week-old immune deficient nude mice were purchased from Experimental Animals Center , and fed under specific, pathogen-free conditions. Cells . The cells were plated at a sub confluent density, followed by co-transfecting with 0.5 μg of NF-κB luciferase reporter plasmid or negative plasmid, and 0.02 μg of Renilla luciferase pRL-TK plasmid  performing in Lipofectamine 2000 reagent (Invitrogen). Cell lysates were prepared 24 h after transfection, and the reporter activity was measured using the Dual-luciferase reporter assay system (Promega).3VO4, 10 mM NaF, 2.5 mg/ml aprotinin and leupeptin, 1 mM β-glycerophosphate and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and 10 mM iodoacetate. After incubation, cellular debris and nuclei were removed by centrifugation. Cell lysates were incubated with specific antibody overnight, and then with Protein A-Sepharose  beads for another 4 h. The immunoprecipitates were washed in Tris-buffered saline four times and boiled in Laemmli buffer included of 0.02% blue bromophenol and 2% bmercaptoethanol.Cells were washed with ice-cold PBS, followed by lysing in Tris-buffered saline (pH 7.4), containing 150 mM NaCl, 50 mM Tris, 0.1% Nonidet P-40, 1 mM EDTA, 1 mM NaP < 0.05 was considered to indicate statistical significance.SPSS19.0 software was used for all statistical analyses. Numeric data were described as the mean value ± standard deviation. Comparisons were performed by Students t-test. A value of S100A4 mRNA expression level in MCSCs was also significantly increased as detected by QPCR , while suppressed by S100A4-siRNA transfection .MCSCs were transfected with S100A4 overexpression (Fig. S100A4 suppression (Fig. S100A4 overexpression, while was decreased by S100A4 suppression (Fig. The proliferation capacity of transfected MCSCs was evaluated by Cell Counting Kit-8 and tumorigenicity assay. The cell proliferation curve was increased by ion Fig. , while wion Fig. ., Tumor S100A4 enhanced the transcriptional activity of a NF-κB reporter gene (Fig. S100A4 (Fig. S100A4 increased the level of IKK (Fig. S100A4-overexpressing cells (Fig. S100A4-silenced MCSCs (Fig. S100A4 in MCSCs.Overexpression of ene Fig. , which w0A4 Fig. . WesternIKK Fig. . In addills Fig. , and dowSCs Fig. . Taken tWe hypothesized that S100A4 may regulate NF-κB signaling pathway through a direct interaction with IKK. To verify this hypothesis, interaction between S100A4 and IKK were analyzed using immunoprecipitation assays. As shown in Fig. S100A4 may have oncogenic effects in a multitude of tumor types [S100A4 has been reported to be an important regulator for modulating the cell cycle, proliferation, apoptosis, and migration in different kinds of cells through various mechanisms . In adulor types . HoweverS100A4 overexpression, while was inhibited by S100A4 suppression. Luciferase reporter assays manifested that the transcriptional activity of NF-κB was enhanced significantly by overexpression of S100A4, implying NF-κB may play an crucial part in the S100A4-induced proliferation capacity of MCSCs.In the present research, we found that cell proliferation was significantly prompted by NF-κB is the collective name of a family of transcription factors consisting of seven proteins, encoded by five genes: c-Rel, RelA, RelB, p100/p52 and p105/p50 . NF-κB hConsistent with those previous works, our research also demonstrated that overexpression of S100A4 upregulated the level of IKK, followed by raising the activation of NF-κB ultimately. In addition, overexpression of S100A4 upregulated some genes, IL-2 and TNF, which downstream of the NF-κB signaling pathway. IL-2 has been demonstrate to play an crucial role in tumor proliferation . TNF-α tThere are some limitations to this study that needed to be taken into account. The IKK complex is formed by three subunits: IKKα, IKKβ, and IKKγ , and it In summary, the present work showed that S100A4 is upregulated in MCSCs and possibly enhance the proliferation ability of MCSCs by way of activating the IKK/NF-κB signaling pathway. These results may offer a mechanisms for regulation of proliferation in MCSCs, and S100A4 maybe a hopeful therapeutic target for MCSCs.


As the process parameters lead to a variety of texture and preferential orientation, the influence of the sputtering conditions on the deposition of LiCoO2 thin films are considered. The electrochemical performance is examined as a function of composition of the sputter Ar/O2 gas mixture, gas flow rate, pressure, nature of substrate, substrate temperature, deposition rate, and annealing temperature. The state-of-the-art of lithium microbatteries fabricated by the rf-sputtering method is also reported.This review article presents the literature survey on radio frequency (RF)-magnetron sputtered LiCoO T. T2 emerporation c. Recentectively . Another (Korea) .2 gas mixture, gas flow rate, pressure, nature of the substrate, substrate temperature (Ts), deposition rate (Θdr), and annealing temperature (Ta) were considered. LCO material being highly anisotropic (lamellar structure), the thin-film deposition process must produce desirable crystalline texture that favors the lithium diffusion at the electrode–electrolyte interface. Typically, there are three structural configurations: (003), (101), and (104) as shown in + ions passed through the grain boundaries. In contrast, (104)- and (101)-oriented grains favor the Li+-ion transport. It has been shown that the mechanism responsible for the LCO thin film texture is the volumetric strain energy imposed by thermal mismatch between the film and the substrate during the annealing process ubstrate , Cu foilubstrate , or Al lubstrate , which a–1 and the deposition temperature in the range 50–300 °C, different degrees of (003), (101), and (104) orientations were determined. It was also mentioned that at high substrate temperature, due to the increase of the surface mobility of adatoms, the texture changed from (101)–(104) to (003). As shown in Θdr values using by the researchers. Nevertheless, it appeared that Θdr varies with both the working pressure  substrates, on the microstructure of sputtered LCO thin films. Film were deposited at the rate of 0.9–1.2 nm min−1 in the presence of 0.5 Pa Ar/O2 mixture gas (9:3 ratio) flowing at 12 sccm and post-annealed at 800 °C in O2 atmosphere for 30 min. For both substrates, a 300 nm-thick layer of Pt was deposited as the current collector.LiCoOates see , the mosrmed see . Lee et 2O3/Pt substrates displayed a rough surface with several cracks induced by thermal expansion of the substrate, while films deposited on Si/SiO2/Ti/Pt substrates consisted of small grains without cracks. Such films exhibited the specific capacity of 27 µAh cm−2 µm−1 at a current density of 50 µA cm−1 after 150 cycles with an average capacity decrease rate of 0.05% per cycle. Jeevan-Kumar et al. [2 target to compensate the volatility of Li. These films deposited on metallized Si substrate kept at Ts = 250 °C with subsequent annealing at 650 °C under an oxygen pressure of 5 Pa showed a well-oriented HT-LiCoO2 phase with predominant (104) planes. The high c/a ratio of 4.997 characterized the layered (R-3m) structure. Jeong et al. [2O3 layer (10 nm thick) deposited at the rate of 3.5 nm min−1 between the LCO film cathode and the LiPON film electrolyte, which decreases the interfacial resistance owing to the formation of a solid solution LiCo1−yAlyO2 at the interface during the heat treatment at 400 °C for 5 h. The same group identified the importance of substrate texturing and the formation of LCO film avoiding post-deposition treatment. The morphology of the LCO thin-film deposited on the Au/Ti/SiO2 stack was investigated as a function of the substrate texture using polished Si and textured Si  solution; −2 µm−1 [2 thin film cathode. A comparison between textured and annealed STS304 was established [2 mix gas of (4:1), the substrate having a strong (220) texture produces LCO films with a (003) preferred orientation, while the film deposited on annealed STS304 exhibited the (104) preferred orientation. As a result, the initial discharge capacity of the film deposited on the annealed stainless steel was higher than that of the film deposited on the textured substrate, but reverse behavior was observed for the LCO cathode cyclability. When Al foil is used as substrate, the degree of crystallization of the sputtered LCO films varies with the thickness of the Al coating deposited on the Al foil. The thinner the Al coating, the faster the crystallization process [LCO films deposited on Alr et al.  producedg et al.  interpos −2 µm−1 . Taking ablished . Using a process .Ts = 25 °C were found to be amorphous and subsequently developed a (003), (101), (110), or (104) out-of-plane texture upon annealing, depending on thickness [2 phase is obtained at Ts < 300 °C, while HT-LiCoO2 is prepared at Ts > 300 °C and in the Ar/O2 (3:2) atmosphere. LCO films were grown on Pt-coated Si wafers heated in the range 25–600 °C in an Ar/O2 (ratio 3:1) gas flow rate of 12 sccm. Films deposited at 250 °C and annealed at 600 °C showed strong (104) orientation. An electrochemical test carried out under a current density of 10 µA cm−2 in the potential range of 4.2–3.0 V displayed responses depending on preparation conditions. Specific discharge capacities of 50, 42.5, and 30 µAh cm−2 µm−1 were obtained after 10 cycles, for films 25 °C-deposited and annealed at 600 °C, 250 °C-deposited and annealed at 600 °C and 600 °C-deposited, respectively [Ts = 400 °C displayed the well-defined discharge voltage plateau of crystallized LCO at 3.9 V and delivered an initial specific capacity of 54.5 µAh cm−2 µm−1 [3N4/TiOx/Pt/LCO has been fabricated, in which Si3N4 is used as a barrier against the Li diffusion into silicon and TiOx is an adhesion layer with an optimum thickness of 25–45 nm [2 between LiCoO2 and Si can work as a solid-state electrolyte allowing transport of Li ions and trap Li ions when external voltage is removed thus increasing device retention. Recently, Hu et al. investigated the effect of the SiO2 thickness on the properties of the Pt/LiCoO2/SiO2/Si stacks demonstrating the Li trapping mechanism [In this section, we examined the relationship between the structure and morphology of LCO films and the experiments conditions, i.e., substrate temperature, deposition rate, sputter power, working pressure, substrate bias, and post-annealing process. The use of a heated substrate rather than a deposition at room temperature has been a debating issue. In most cases, the films deposited at hickness ,49. Stochickness  stated tectively . The strm−2 µm−1 . The mul25–45 nm . Note thechanism .−3 for increasing pressure from 0.15 and 1 Pa, respectively for 3-µm thick LCO films. At pressure of 10 Pa, the density strongly decreased to 3.5 g cm−3. SEM images displayed visible gaps and voids, giving evidence of an increase of the porosity. For 100-nm thick films, the decrease of the density is less dramatic, at 10 Pa a value of 4.2 g cm−3 was measured.The gravimetric density and the porosity of films are important parameters playing a major role for the transport of Li ions in the cathode material. Experimental results have evidenced the dependence of the density on the sputter pressure and film thickness ,79. The 2 in the mixed gas (≤25%) under working pressure of 2 Pa using a sputter power in the range 75–100 W. Higher sputter power produces a reduction of the compositional Li/Co ratio. Pan and Yang reported the effects of the radio-frequency sputtering powers (80–200 W) on the micro-structures and electrochemical properties of LCO thin film electrodes. LCO films, grown on Pt-wafer substrates kept at 55 °C and at high sputtering power of 200 W, had (101)-oriented with big grains of 150 nm in size and exhibited a specific capacity of 61 µAh cm−2 µm−1 when discharged at a constant current of 20 µA cm−2 between 4.2 and 3.0 V [−2 µm−1 taken at 30 µA cm−2 for the LCO films deposited on stainless-steel substrate at 150 W sputtering power in an Ar/O2 mixture of 9:1. Pracharova et al. [2/Ti/Au substrate, while the radio-frequency discharge power is an important parameter to control the stoichiometry. For a sputter power of 60 W , the films were stoichiometric but an unfavorable (003) orientation was observed by X-ray diffraction and Raman spectroscopy. Using a low power of 25 W, Rao et al. [xCoOy films with x< 1 and y> 2. Electrical conductivity measurements showed that the film Li0.8CoO2.7 is metallic, which can be beneficial for its role as a cathode material but contains Co3O4 impurity phase, which is detrimental.Due to the difference of the atomic weights between Li and Co, the Li/Co ratio is sensitive to the rf power . LCO filnd 3.0 V . Similarg powers –200 W ona et al.  stated to et al.  preparedPAr ≤ 1 Pa) and oxygen deficiency in the range 0.5–1.0 Pa. An annealing treatment in Ar/O2 (4.5:5) atmosphere of 10 Pa at temperatures between 100 and 600 °C for 3 h compensate for the oxygen deficiency.The effect of the sputter gas mixture on the LCO thin film composition has been widely demonstrated. For sputter gas consisting of 100% Ar, the Li/Co ratio was found to be 0.88  or 1:0 ±2 mixes of 1:10, 1:2, or 1:1, the Li/Co ratio was reported to be 0:8 ± 0:08 (O/Co = 2.7) [Ts = 300 °C, with background pressure of 0.7 Pa for the Ar/O2 mixture in 3:1 ratio. At a growth rate of 0.5 nm min−1 and after annealing at 600 °C for 3 h, the films were preferentially oriented with their (110) planes parallel to the substrate surface. However, an O/Co ratio of 2.02 was reported. The use of only argon in a conventional sputtering process may create cracks in LCO films. In thick films, distinct columnar structures appeared after annealing, which must be eliminated, because they result in lower film density and formation of cracks limiting the lithium diffusion. Mixing O2 with Ar during the deposition allows the fabrication of thick films with better morphology, good stoichiometry, and battery performance as a result of the presence of oxygen improving the film nucleation and growth. Park et al. [2 thin films prepared from a 4 inch target sputtered at 200 W under operating Ar/O2 gas ratio of 8:2 maintained at pressure in the range of 0.3–1.8 Pa. Films exhibiting the best performance were annealed at a temperature that does not exceed 400 °C. The higher value of specific capacity (ca. 38 µAh cm−2 µm−1) is obtained for film grown at pressure of 1.3 Pa. The matter transport through Ar plasma in LCO thin films sputtering was analyzed using the Monte Carlo method [For oxygen-rich Ar/Oo = 2.7) , 0.4 (O/o = 2.7) , or 1:15o = 2.7) , respecto = 2.7)  preparedk et al.  investigo method . Stable Vb) in the range 0–100 V has been investigated on the morphology and electrochemical performance of LCO thin films (350 nm thick) deposited on Si(100)/SiO2/Ti/Pt under Ar/O2 (45:5) gas flow at a working pressure of 0.5 Pa using a sputtering power of 200 W [Vb = 0 V, the XRD patterns showed tiny (003) and (006) peaks that disappeared with application of Vb in favor of the (101) Bragg line. The LCO films deposited at Vb = −50 V had an average grain size of 4.9 nm (density of 5.16 g cm−3), while at Vb = −70 V the presence of Co3O4 was detected. The charge–discharge tests carried out at a 1C rate in the voltage range of 3.0–4.3 V displayed a specific capacity of 60 µAh cm−2 µm−1. The cycling performance of a Li/LiPON/LCO/Pt solid-state microbattery is shown in Among the various ways of improvement of the growth process of crystalline LCO thin films, the use of a substrate bias has been proposed as the key parameter for decreasing the use of annealing treatment. The influence of the substrate bias on the LCO film crystallinity was recognized since 2005. The effect of substrate biases (of 200 W . At Vb =2 ratio 3:1) that produced 0.5-µm thick crystalline deposits without any trace of the cubic phase. Such films delivered a specific capacity of 50 µAh cm−2 µm−1 after 140 cycles at 10 µA cm−2 current density. Next, the same group of research recognized that the crystallinity is not only controlled by the annealing temperature and the bias voltage but also by the pressure of the gas mixture. LCO films deposited by RFS onto Al substrates and post-annealed treated at 500 °C in air for 2 h show various morphologies and structures as a function of the operating Ar/O2 gas pressure ranging from 0.55 to 3 Pa. Variation of gas mixture, gas pressure, substrate bias promoted the relative amount of LCO phase mixture, i.e., mixture of the layered HT-LCO and cubic LT-LCO.In 2011, Navone et al.  lowered 2 gas mixture of 3:4 delivering a high specific capacity of 67 µAh cm−2 µm−1 at C/5 rate but these studies were carried out in a liquid electrolyte only [2/Sn3N4/Ti/Au substrate that delivered an initial specific capacity of 49 µAh cm−2 µm−1 at 10 µA cm−2 current density and showed a capacity retention of 82% after 800 cycles [Optimized films were produced at 3 Pa in an Ar/Oyte only . Taking yte only  and, fin0 cycles .2 ambient at 650 °C leads to LCO films with the characteristic (104)-preferred plane [2 (ratio 3:1) gas flow rate of 12 sccm. Films deposited 250 °C and annealed at 600 °C showed strong (104) orientation. An electrochemical test carried out under a current density of 10 µA cm−2 in the potential range of 4.2–3.0 V displayed responses depending on preparation conditions. Specific discharge capacities of 50, 42.5, and 30 µAh cm−2 µm−1 were obtained after 10 cycles, for films 25 °C-deposited and annealed at 600 °C, 250 °C-deposited and annealed at 600 °C and 600 °C-deposited, respectively [2 ambient. The films were deposited at the rate of 0.08 nm s−1 at room temperature on NASICON-type electrolyte Si- and Ge-doped Li1.3Al0.3Ti1.7(PO4)3 (LATP) substrates in a working Ar atmosphere kept at 0.5 Pa [−2 µm−1 that decreased to 15 µAh cm−2 µm−1 after 50 cycles. Xie et al. [1+x+yAlxTi2−xSiyP3−yO12 (LATSP) at Ts < 180 °C. An initial discharge capacity of 210 mAh g−1 was delivered by as-deposited LCO thin films (0.5 µm thick) on LATSP in Ar/O2 (7:3) at a power of 100 W. A rapid-thermal annealing (RTA) process at 700 °C under flowing oxygen gas (for 20 min) was shown to be a successful method to obtain LCO thin films that consist of grains with (101) and (104) preferred orientations [Annealing is requested for the formation of well-crystallized films with preferential (104) or (101) orientation. Sufficient annealing temperature is also especially important in order to get the HT-LCO phase that avoids poor cyclability. However, heat treatment at temperature as low as 500 °C could be preferable to maintain a good adhesion on the substrate. The drawbacks of annealing at temperatures greater than 700 °C producing cracks and voids, i.e., micro-short paths, has been also mentioned ,89. The ed plane . Rapid tt 0.5 Pa . It appee et al.  show thantations .Qth in ampere-hour per mass (Ah·kg−1), or equivalently in mAh·g−1 is obtained from the Faraday law [Mw is the molecular mass of the “limiting” electrode material. With the transfer of n = 1e− per formula unit, the theoretical specific capacity of LiCoO2 (Mw = 97.87 g mol−1) is 273.8 mAh·g−1. Note that the reversible capacity, however, is limited to 140 mAh g−1 when LiCoO2 is cycled between 3 and 4.2 V, corresponding to extracting and inserting about 0.5 Li per LiCoO2.The relation between the gravimetric capacity, mQ, of the material, and the volumetric capacity of a film, fQ, is given by:mQ is expressed in mAh g−1, fQ is translated in µAh cm−2 µm−1 (or mC cm−2 µm−1), and d is the density of the material in g cm−3. With a density of 5.06 g cm−3, the theoretical volumetric capacity of LiCoO2 is 137.8 µAh cm−2 µm−1 if porosity is ignored. Decreasing the film thickness implies a large electrode area or a limited capacity. A thin electrode film exhibiting high intercalation rates is expected to have a short diffusion pathway L for the Li+ ions according to Fick’s law. In case the chemical reaction proceeds by a single-phase process, i.e., within a solid solution, the characteristic time τsp for Li+ ions to reach the surface of any active particle of dimension L is given by:DLi is the chemical diffusion coefficient of moving ions in the host framework. However, Wang et al. [−1 LiPF6 in ethylene-dietylene carbonate) or a LiPON solid film electrolyte. One of the first attempts show that, even annealed at 600 °C in air, the 0.2-µm thick LCO films deposited with a low power of 50 W on SnO2-coated glass under working pressure 0.7 Pa have only delivered a discharge capacity of 114 mC cm−2 µm−1 (in the voltage range 4.1–3.0 V) due to the mixed LiCoO2 + Li1.47Co3O4 crystalline phase [−2 µm−1 at a discharge rate of 10 µA cm−2 in the potential range 4.25–3.0 V for LCO film (1.3 µm thick) annealed at 500, 600, and 700 °C for 2 h, respectively. These films originally deposited on Pt/Ti(20 nm)/SiO2(600nm)/(100)Si substrate heated at 250 °C under P2O = 0.5–5.0 Pa  had a nanocrystalline structure with (104) out-of-plane orientation.The maximum capacity (in Ah) of any lithium cell is largely determined by the amount of active material in the positive electrode. The specific capacity aday law :(1)Qth=1g et al.  reportedne phase . Liao anne phase  obtained−2 [−1 µm−1 (170 mAh g−1), which correspond at x = 0.63 Li extracted when the cell is charged at 4.4 V. However, upon cycling to further voltage (>4.4 V), a two-phase reaction (CoO2 and LixCoO2 phases) associated with the variation of the c-lattice parameter of 3.2% induced an increase of cell resistance and capacity fades  measurements were carried out in the voltage range of 3.0–5.0 V at a current density of 10 µA cm−2 . Figure ty fades b. The fo2/LiNiO2 multilayer thin film cathode with that of LiCoO2 single-layer [2 gas mixture (4:1) at a flow rate of 150 sccm with a pressure maintained at 0.3 Pa, both electrodes deposited at Ts = 65 °C had the (003)-preferred orientation. The initial discharge capacity of the multi-layer electrode was ~53 µAh cm−2 µm−1 at a current density of 10 µA cm−2 in the potential range of 3.0–4.2 V, approximately 30% larger than the single LCO electrode. Via in situ measurements, Cho et al. [xCoO2 films electrochemically delithiated in the range 1.0 ≤ x ≤ 0.6. LCO films (500-nm thick) were deposited by reactive sputtering at a rate of 0.8 nm min−1 on c-plane oriented sapphire substrates coated with ~100 nm of SiO2 and an ~80 nm Al layer as current collector. They were randomly textured after an annealing process at 500 °C in air. During delithiation, the thermal conductivity decreases reversibly (from 5.4 to 3.7 W m−1 K−1) and the elastic modulus decreases as well from 325 to 225 GPa.Noh et al. compared the microstructure and electrochemical performance of sputtered LiCoOle-layer . Using ao et al.  investigDLi, were measured using various electrochemical methods, i.e., cyclic voltammetry (CV), galvanostatic intermittent titration technique (GITT), potentiostatic intermittent titration technique (PITT), and electrochemical impedance spectroscopy (EIS). In CV measurements, DLi is determined from the Randles–Sevcik relation, which describes the influence of the sweep rate on the redox peak current, while other methods evaluate the composition dependence of DLi. The PITT and GITT methods rely on solving Fick’s laws the variation of current (voltage) vs. time is measured after application of a potential (current) step to slightly modify the electrode composition of the electrode. In EIS measurements, the composition dependence of DLi is estimated from the Warburg impedance related with the straight line of 45° slope of the Nyquist diagram. The DLi values are considered to be more reliable when obtained from GITT and PITT methods [DLi are often due to a poor evaluation of the actual surface area of the electrode. It is recommended to use Brunauer–Emmett–Teller (BET) surface measurements.The Li-ion chemical diffusion coefficients,  methods . Howevera-axis orientation, which favors the intercalation rate and cycling efficiency. However, Bouwman et al. reported an intercalation rate hindered by a large charge-transfer resistance and phase boundary motion rather than the diffusion-limited behavior currently observed in LCO crystal [+ ion kinetics in three LCO thin films sputtered for different duration (td) of 30, 60, and 120 min on polished Al2O3 substrates covered with a 900 nm thick Au layer. A 0.31-μm thick LiCoO2 thin film (sputtered for td = 30 min) showed a high (003) orientation, while a 1.35-μm thick film (sputtered for td = 120 min) exhibited high (104) orientation. CV measurements showed that the peak current (Ip) follows a linear relationship with the square root of the scan rate (ν1/2) indicating a diffusion-controlled process. Thus, DLi can be calculated using the Randles–Sevcik relation Ip = f(ν1/2):R and F are the usual constants, T is the absolute temperature, A is the surface area of the electrode, and CLi is the concentration of Li in the electrode. The (104)-oriented film exhibited a larger DLi value of 7.7 × 10−12 cm2 s−1 than 6.4 × 10−13 cm2 s−1 for the (003) oriented thin film. Note that these values are “apparent” diffusion coefficients because the compositional dependence of DLi cannot be determined by the CV technique. However, the PITT method based on solving the Fick’s diffusion equation determines DLi as a function of x(Li) in LixCoO2 by recording the time dependence of the transient current (It) when a potential step is applied to the film:L is the thickness of the film. The compositional dependence of DLi calculated using Equation (5) is presented in 2 electrode according the phase diagram reported by Bouwman et al. [The influence of the diffusion plane orientation on electrochemical properties of thin film LCO electrodes has been pointed out by several authors ,79,97. S crystal ,79 repor+ ion diffusion coefficient if LCO films deposited on Pt-coated Si wafers. The 700 °C-annealed film showing good crystallinity with (104) preferred orientation exhibit a high discharge capacity of 61 and 56 µAh cm−2 µm−1 at a discharge rate of 10 and 50 µA cm−2, respectively, while the 500 °C-annealed film displays a discharge capacity of 35 µAh cm−2 µm−1 at a discharge rate of 50 µA cm−2 [+ ions (DLi) in LCO thin films appears as a two-step behavior with a decrease of almost two orders of magnitude around 3.9 V  and indicates a stable layered structure for Ta = 700 °C.Liao et al.  investig µA cm−2 . The difnd 3.9 V . Due to 2− and NO3− [−1 LiClO4 in propylene carbonate (PC), has been investigated by several electroanalytical techniques, i.e., electrochemical impedance measurement, in situ Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The formation of decomposition products, i.e., the organic surface layer, greatly depends on the crystal orientation and amount of the impurity (2 mol% Co3O4 cubic phase). The best electrochemical performance (229 mC cm−2 µm−1 in the voltage range 3.5–4.4 V) is attained for LCO films sputtered on Au substrate  with a (003) plane perpendicular to the substrate [+/Li [+/Li [6− anions of the Li salt [2 microcrystalline buffer layer (−20 nm thick) was deposited by rf-sputtering between the cathode (LiNi0.5Co0.2Mn0.3O2) and solid electrolyte (Li7Al0.1La3Zr2O12 pellet) in an all-solid-state lithium battery [Numerous studies have been devoted to the behavior of the solid-electrolyte/electrode interface (SEI layer). The sputtered LCO/LiPON interface was investigated step-by-step by photoelectron X-ray-induced spectroscopy. The SEI layer, ~10 Å thick, contains new nitrogen-containing species, i.e., NOand NO3− . The reaubstrate . In the te [+/Li . FinallyLi [+/Li . The ele Li salt . A dense battery . Due to xCoO2, irreversible changes occur upon the charge process at x ≈ 0.5, which limits the specific capacity to 140 mAh g−1 or 69 µAh cm−2 µm−1. Reportedly, one of the significant approaches to overcome this problem is lattice doping by either isovalent or aliovalent ions that results in the stable cycling at high voltages (4.5 V) [xCo1−xO2 thin films deposited on Au/Ti/SiO2/Si (100) substrates [−1 on a substrate held at Ts = 250 °C under Ar/O2 (9:1) mix gas at working pressure of 0.6 Pa using a RF power of 130 W. 1.2-µm thick LCO films exhibit a (108) preferential orientation. A slight increase in lattice parameters and c/a ratio (5.01 vs. 4.98 for pristine film) has been noticed for the LiCo0.98Zr0.02O2 films, confirming the introduction of Zr4+ ions (0.72 Å) in the host lattice. It is assumed that the presence of some Co2+ ions is due to a charge compensation effect. Electrochemical properties of doped-LCO films were investigated by CV and GCD. The narrow anodic/cathodic peak separation in cyclic voltammograms and the high Li+ ion diffusion coefficient (1.8 × 10−11 cm2 s−1) indicate an enhancement of kinetics of Li+ ions by Zr doping. The Li//LiCo0.98Zr0.02O2 cell with non-aqueous electrolyte exhibited an initial discharge capacity of 65 μAh cm−2 μm−1 at a 1C rate with a fading of 3.8% after 80 cycles in the potential range 3–4.2 V. The extension to a higher voltage was not explored in this work. Improved kinetics is also evidenced by the Nyquist plots shown in Rct) from 156 Ω (LiCoO2) to 60 Ω (LiCo0.98Zr0.02O2).Due to the rhombohedral/monoclinic phase transition in Li (4.5 V) ,105. Thebstrates . LCO fil2 mosaic target including Ti metal strips [−2 μm−1 (245 mC cm−2 μm−1) at a 1C rate that retained to ~54 μAh cm−2 μm−1 at a 4C rate for LiCo0.98Ti0.02O2 thin film cathodes. The larger ionic radius of Ti4+ (0.605 Å) than that of the Co3+ ion  provokes a slight elementary volume expansion of 0.6% and produces a pillaring effect that result in an enhancement of the Li+ ion diffusion. Another reason for such a good electrochemical performance with aliovalent doping is based on the increasing Co3+ ions concentration and the lower concentration of Jahn-Teller Co4+ ions that cause spontaneous deformation and disrupt the LCO lattice on the charge process [y(Ti) > 0.02, the ion exchange provokes more cation disordering and the appearance of Co3O4 spinel impurities [Ts = 250 °C using a 10% Li-enriched target, the Ti-doped LCO films exhibited a preponderant (104) orientation [Ti-doped LCO thin films were prepared using a sputter Li-enriched LiCoOl strips . The chr process . For a hpurities . When prentation .2 gas mixture in a ratio of 3:1 at a total flow of 20 sccm and a partial pressure of 2.7 pa. As an experimental fact, LCO films over 1-µm thick deposited at Ts ≤ 50 °C at a rate of 2 nm min−1 are 100% (003)-oriented grains, which demonstrates the lowest surface energy of the (003) plane. In contrast, 4-µm thick LCO films deposited at Ts = 70 °C at a rate of 1.3 nm min−1 exhibit 84% (101)- and 16% (104)-oriented grains (0% (003)); such a predominant texture was attributed to the large bulky strain energy in thick film [As a summary figure, the experimental results by Bates et al.  have cleick film . As show2 thin films with adequate texture and microstructure that allows it to be associated with a solid-state electrolyte thin films applied in rechargeable lithium microbatteries. For rf-sputtered LiCoO2 films, experimental results have shown the strong influence of the growth conditions on the preferential orientation, microstructure, and stoichiometry, which govern the electrochemical performance of cathode films in microbatteries. The (104) orientation facilitates larger Li-ion transport at the electrolyte/electrode interface and an increase in the capacity than that of the (003)-oriented thin film. Such preferred texture has been obtained using the annealing process as low as 500 °C, which is a favorable condition for the device fabrication. Based on the optimization of parameters, good quality LiCoO2 films, from which more than half lithium ions can be extracted, delivered a specific capacity higher than 70 µAh cm−2 µm−1.In this paper, we reported the successful use of the rf-magnetron sputtering technique for the synthesis of LiCoO2/graphite materials taking into account the issues with inhomogeneous current distributions [We focused attention on planar batteries. With this geometry, the areal energy density is limited. The thickness of the films is also limited to obtain good kinetics. 3D microbatteries are a promising design since it allows a scaling of the capacity by increasing the surface area of the 3D substrate, independent of film thickness. However, 3D TFBs have proven technologically very challenging to realize. The promise of a high capacity and stable microbattery has yet to be realized . Neverthibutions ,113,114,ibutions ,116.2 thin film cathode is almost mature and in the final form for actual applications. This class of power sources that belongs to the all-solid-state battery family including a lithium ion conductor (LIC) without undergoing liquid electrolyte leakage has the advantage of well-formed solid-electrolyte/electrode interfaces. Generally, LICs are safe with no risk of fire or explosion, thermally stable, and exhibit a wider electrochemical window than that of liquid electrolytes. However, the current density is quite low due to the poor ionic conductivity of the solid electrolyte such as LiPON. Development of a better electrolyte with the Li transport number close to unity such as perovskite-type oxides TiO3), garnet-type frameworks (Li5La3Ti2O12), or nitride-based glass ceramics are good candidates. Deposition of these materials by rf-sputtering should be the best technique in terms of film quality. Another prospective for the future is the development of thin films on flexible substrates for wearable electronics applications. The recent fabrication of a Li/LiBON/LCO microbattery demonstrates good electrochemical performance, i.e., capacity retention of 90% over 1000 cycles under heavy bending and twisting conditions.To date, lithium microbattery technology including LiCoO


Lateolabrax maculatus), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes  were analyzed by qRT-PCR in 10 tissues  of L. maculatus. Four algorithms, geNorm, NormFinder, BestKeeper, and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. The results showed the 18S rRNA was most stable in different tissues under normal conditions. During salinity stress, RPL7 was the most stable gene according to overall ranking and the best combination of reference genes was RPL7 and RNAPol II. In contrast, GAPDH was the least stable gene which was not suitable as reference genes. The study showed that different algorithms might generate inconsistent results. Therefore, the combination of several reference genes should be selected to accurately calibrate system errors. The present study was the first to select reference genes of L. maculatus by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in L. maculatus. The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.The aim of this study was to select the most suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) of spotted sea bass ( The fish were acclimatized at room temperature in seawater (30 ppt) with continuous aeration for a week prior to the experiment. Nine healthy fish were randomly divided into 3 groups as three biological replicates. The fish were treated with tricaine methanesulfonate  and immediately dissected. The intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen were collected. Ten tissues per fish were numbered and stored at −80 °C for RNA extraction.For the salinity challenge experiment, 60 spotted sea bass (100.00 ± 2.34 g) were acquired from Shuangying Aquatic Seed Company . The fish were acclimatized at a density of 5 individuals per tank (120 L). Water temperature, pH, dissolved oxygen and light-dark cycle were maintained at 21°C (±0.5 °C), 7.98∼8.04, 6.90∼8.54 mg/L and 14 L:10 D, respectively. After acclimation, the fish were randomly divided into 4 groups with different salinities . After rearing for 30 days, 9 fishes per group were randomly selected as three biological replicates and treated with MS 222 (200 mg/L). Gill tissues were immediately frozen in liquid nitrogen and then stored at −80 °C until further use.Two experimental sample sets were constructed. Set A: different tissues  and set B: different salinities . The total RNA was extracted from samples by using TRIzol reagent  according to the manufacturer’s instructions and digested with RNase-free DNase I  to remove genomic DNA contamination. Equal amounts of RNA from the same tissues of 3 individual fish under the same conditions were pooled as one sample to minimize the variation among individuals, and three such pools were obtained for each tissue and salinity treatment group. The concentration was determined by nucleic acid protein analyzer BD1000 , and the quality of RNA was assessed by gel electrophoresis. Samples with 260/280 absorbance ratios greater than 1.9 were used for cDNA synthesis. A 0.5-µg aliquot of total RNA from each sample was reverse transcribed by using the PrimeScript™ RT reagent Kit with gDNA Eraser  employing a RT Primer MIX (Random 6 mers and Oligo dT Primer) in a 20 µl reaction according to the manufacturer’s instructions. The synthesized cDNA was stored at −20 °C.18S rRNA) sequences from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) and 8 reference gene sequences from the transcriptomics database by the IlluminaHiseq 4,000 platform . The specificity of amplification was verified by melting curve and agarose gel electrophoresis, and the primer amplification efficiency was calculated as E(%) = (10(−1∕slopes) − 1) × 100 . Each well contained 10 µl of SYBR® Premix Ex Taq™, 0.4 µl of ROX Reference Dye, 6.8 µl of sterilized ddH2O, 0.4 µl of each primer (10 µmol L−1), and 2 µl of cDNA template. The reaction conditions were 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. After PCR amplification, a melting curve was obtained by the following process: 95 °C for 5 s, 60 °C for 1 min, followed by 95 °C at the rate of 0.11 °C per second to verify primer specificity. All RT-qPCR assays were carried out in three biological replicates with three technical replicates.All reference gene primers were designed by the Primer 5.0 software. Nine primer pairs were synthesized by the Beijing Genomics Institute (BGI) and tested via standard RT-PCR by using six serial five-fold dilutions of sample cDNA with SYBR1) × 100 . The qRTE)ΔCt, ΔCt = lowest Ct value—Ct value of sample. E is equal to 2 when PCR efficiencies approach 100%. The highest relative quantities for each gene are set to 1. Finally, all the reference genes were ranked by four programs. Then, all graphs were generated by using SPSS 19.0 and OriginPro 8.0.The expression stability of the 9 reference genes was evaluated by 4 different algorithms: geNorm , NormFinR2) were determined based on the standard curves. The primer efficiency (E) of the nine genes ranged from 94.16% to 106.67%, and the correlation coefficients (R2) ranged from 0.9966 to 0.9994 , while RNAPol II showed the lowest expression level (average Ct = 26.399). The expression trend of these candidate reference genes in the samples after salinity treatment was similar to that in different tissues, and the CV of all reference genes ranged between 1.078 to 3.830% and Ct values varied from 9.661 to 28.133. GAPDH displayed the highest transcription-level variation. The lowest transcription level variation was observed for HRPT, followed by RPL7 and RNAPol II. The minimum mean Ct value was 10.386, and the maximum mean Ct value was 26.517 for the highest and lowest expression levels for 18SrRNA and GAPDH  of the raw Ct values was calculated to evaluate transcription level variations. The CV of all reference genes ranged from 4.011 to 17.550%, and the Ct values varied from 9.402 to 34.015. nd GAPDH .To select optimal reference genes for accurate normalization under the same experimental conditions, four common algorithms were used to analyze expression stability and rank the reference genes.18S rRNA and ACTB have the least M value of 0.90, while GAPDH showed the highest value, indicating that 18S rRNA and ACTB were most stable in expression and that GAPDH was the least stable in expression. Furthermore, on the basis of geNorm analysis, the default limit of the stability value (M) is <1.5; thus, GAPDH, HRPT and B2M, with stability values (M) above 1.5, were not selected in gene expression. However, under salinity stress, the stability values (M) of all candidate reference genes were below 1.5. RNAPol II and TUBA were the most stable genes, with M values of 0.16, while GAPDH was the least stable gene, with an M value of 0.67. Thus, the geNorm analysis indicated that 18S rRNA and ACTB were the most stable reference genes among different tissues, and RNAPol II and TUBA were the most stable genes in samples under salinity treatments, whereas GAPDH was the least stable gene in both experimental sets  of sequential normalization factors to determine the optimal number of reference genes. The cutoff limit was set as 0.15 for pairwise variation, below which the addition of more genes is not necessary. For both experimental sets, in different tissues, all the pairwise variation (Vn∕Vn+1) was above 0.15. The inclusion of the sixth gene had approximately the same effect (V5∕V6 = 0.217) on the NF as the inclusion of the eighth gene (V7∕V8 = 0.204) had. Therefore, no stable combination was desirable for the selected reference genes in different tissues. However, under salinity stress, all pairwise variation (Vn/Vn+1) was below 0.15, and the V2∕V3 was well below 0.15. Thus, two genes (RNAPol II and TUBA) had the optimal number combination under salinity stress  was the most stable gene, followed by RNAPol II (0.760), while GAPDH (2.981) was the least stable gene (RPL7 (0.166) and RNAPol II (0.235) were the most stable genes, while GAPDH (0.590) was the least stable gene  and standard deviation (SD). The most stable reference gene was selected based on the size of the SD value. The SD values of the candidate reference genes were negatively correlated with the stability of the gene, indicating that the lowest SD value shows the highest stability. Similarly, the results for different tissues by BestKeeper were highly similar to those obtained by geNorm and NormFinder. For example, y stress .18S rRNA was ranked the 4th most stable reference gene by this method 0.25]. The gene with the lowest geometric mean shows the highest stability. As shown in 18S rRNA > ACTB > RNAPol II > EF1A > TUBA > RPL7 > HRPT > B2M > GAPDH. Under salinity stress, RPL7 > RNAPol II > HRPT > TUBA > B2M > 18S rRNA > EF1A > ACTB > GAPDH.Based on the rankings results from four algorithms, the overall ranking of reference genes was obtained. The geometric mean of each reference gene ranking was calculated for the overall final ranking. For example, GAPDH showed a significant difference in black rockfish(Sebastes schlegeli) during larvae developmental stages and tissue analysis   , ACTB anRPL7 was considered a suitable gene in salinity stress in the present study. This gene was also confirmed in other studies. For example, RPL7 was the most stable gene in the liver of zebrafish under bacterial expression. Dicentrarchus labrax), showing that RPL17 was a valid candidate references in seawater and following acclimation to fresh water. Similarly, L13a (RPL13a) are recommended for qPCR normalization according to BestKeeper and NormFinder ; ACTB (B); B2M (C); EF1A (D); GAPDH (E); HRPT (F); RNAPol II (G); RPL7 (H); TUBA (I), reveal single peaks, which obtained from three technical replicates of different cDNA samples.Click here for additional data file.10.7717/peerj.5631/supp-2Figure S2Trans2K DNA marker (A); RNAPol II (B); RPL7 (C); HRPT (D); GAPDH (E); TUBA (F); B2M (G); 18S rRNA (H); EF1A (I); ACTB (J).Click here for additional data file.10.7717/peerj.5631/supp-3Table S1Click here for additional data file.10.7717/peerj.5631/supp-4Table S2The sequence information of these 9 reference genes including B2M, GAPDH, TUBA, EF1A, HRPT, RNAPol II, RPL7, ACTB, 18SrRNA.Click here for additional data file.10.7717/peerj.5631/supp-5Table S3The raw data  of two experimental sample sets: different tissues, salinity stress. Each Ct value of the replicated sample indicates the average performance of three technical duplicates by qRT-PCR.Click here for additional data file.


Securiflustra securifrons. Securidine A was isolated using semi-preparative HPLC, and the structure was elucidated by spectroscopic methods. The isolated Securidine A was tested for cytotoxic, antibacterial, and anti-diabetic activities as well as for its potential for inhibition of biofilm formation. No significant biological activity was observed in the applied bioassays, thus expanded bioactivity profiling is required, in order to reveal any potential applications for Securidine A.A novel brominated alkaloid, Securidine A, was isolated from the cold water marine bryozoan  Bugula neritina, and it is proven to be an antineoplastic agent  colorimetric method. The growing cells were suspended in 100 µL of supplemented medium seeded at a density of 2000 cells/well in a 96-well microtiter plates . The cells were incubated at 37 °C for 24 h. After removal of old medium, the cells were treated with 50 µM and 100 µM of Securidine A (1). After 72 h incubation, 10 µL of Cell titer 96 ® Aqueous One Solution Reagent  was added to each well and the plates were incubated for 1 h. The absorbance readings at 485 nm was measured using DTX 880 multimode detector . Cells with RPMI-1640 medium were used as negative control and cells treated with Triton® X-100  as positive control. Growth inhibition was measured by optical density (OD) and was calculated as: Cell survival (%) = /(negative control – positive control) × 100Cell viability was tested against three adherent cancer cell lines, human melanoma A2058, human colon adenocarcinoma HT29, and human breast cancer MCF7 cell lines in vitro, using an MTS [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Test strains and culture media—The antibacterial activity of Securidine A (1) was determined by using a minimum inhibitory concentration (MIC) assay against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), and Streptococcus agalactiae (ATCC 12386). Antibacterial test was performed in sterile Muller Hinton broth  and brain heart infusion broth (Becton Dickinson). The bacterial cultures were obtained from the stock (−80 °C) and were grown on blood agar  at 37 °C for 24 h and working bacterial stock culture were maintained at 4 °C. An overnight culture of each strain was prepared and 2 mL of overnight culture was inoculated in 25 mL of growth media and incubated at 37 °C on the shaker at 180 rpm, until the culture reaches the turbidity according to 0.5 McFarland standard (1.0 × 108 colony forming unit (CFU)/mL). In this study, bacterial cultures were diluted in 1:100 and then 1:10 in growth media and the final concentration of bacterial cells in the wells were adjusted to 0.5–3.0 × 105 (CFU)/mL of S. aureus, E. coli, E. faecalis, and Streptococcus agalactiae and 3.0–7.0 × 104 CFU/mL of P. aeruginosa. Test concentrations (50 µM and 100 µM) were prepared from the stock solution of Securidine A (1) (10 mg/mL in 100% DMSO), and was dissolved in MilliQ water.TM Delta Surface, Thermo Fisher Scientific, Denmark). Subsequently, 50 µL of final concentration of bacterial cells were added and incubated at 37 °C for 24 h. After incubation, the activity was measured as absorbance at 600 nm in a plate reader  and WorkOut 2.0 software  was used for plate reading. Bacterial suspension with MilliQ water was used as positive control and growth medium with MilliQ water as negative control. Gentamycin  was used as the reference control of this assay. The inhibition was evaluated by the average of parallel OD value. The OD value ≤0.05 considered as active and ≥0.09 was considered inactive.From the test concentrations, 50 µL was transferred into a 96-well microtiter plate (NunclonReagents—(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , sodium chloride , dithiothreitol (Sigma-Aldrich), ethylene diamine tetra-acetic acid disodium salt dehydrate , bovine serum albumin (Sigma Aldrich), human recombinant protein tyrosine phosphatase 1B enzyme , protein tyrosine phosphatase inhibitor IV , 6,8-difluoro-4-methylumbelliferylphosphate .1) and was dissolved in the assay buffer.The test concentration (10 µM and 50 µM) was obtained from stock solution (10 mg/mL in 100% DMSO) of Securidine A (1) was measured in the presence of human recombinant PTP1B enzyme, flurogenic substrate DiFMUP, and the buffer solution in black polystyrene 96-well microtiter plate . The final volume of the mixture was 100 µL in each well. Each well contained 25 µL of buffer solution (pH 7.2) , 25 µL of Securidine A (1), and 50 µL of PTP1B enzyme (31.2 ng/mL). Then, the plate was incubated at 37 °C. After 30 min incubation, 25 µL of 10 µM fluorogenic substrate DiFMUP was added into wells and incubated again at 37 °C for 10 min. The inhibition of enzyme activity was measured as the intensity of fluorescence at 360 nm excitation and 465 nm emission wavelengths by using DTX 880 Multimode detector . The assay buffer was used as the positive control and protein tyrosine inhibitor IV (160 µM) was used as the negative control. The inhibition of enzyme activity was measured from the fluorescence and calculated with positive and negative control. The inhibition of enzyme activity was expressed in percentage and the activity lower than 30% was considered active. The inhibition of enzyme activity was calculated by (Average of triplicates – negative control)/(positive control – negative control) × 100.PTP1B activity of Securidine A (Test strains—The test strains Staphylococcus epidermidis (RP62A-42-77 ATCC-35984) and Staphylococcus haemolyticus  from the stock were grown on blood agar  at 37 °C for 24 h and these working bacterial stock were maintained at 4 °C.S. epidermidis was used to test the biofilm formation on the polystyrene 96-well microtiter plate . The overnight culture was grown in 5 mL of sterile tryptic soy broth  in a shaking incubator at 37 °C and diluted in 1:100 in TSB medium with 1% of glucose (Sigma-Aldrich). Securidine A (1) from the stock (10 mg/mL) was dissolved in the MilliQ water, to prepare 100 µM test concentration. From the test concentration, 50 µL was transferred into the wells, subsequently the diluted test strain 50 µL was added and incubated at 37 °C for overnight. S. epidermidis with sterile MilliQ water was used as positive, S. haemolyticus with sterile MilliQ water was used as negative control and medium, with sterile MilliQ water used as blank.The bacterial strain 3 TM multilabel counter at 600 nm and measuring the optical density (OD). If the inhibition of biofilm rate was 0.25 or lower the OD value was considered active.The biofilm formation in the well was determined by using crystal violet (Merck) staining. The bacterial suspension was removed carefully on the cell paper and the plate was gently washed with tap water two or three times. After the biofilm fixation was done at 65 °C for 1 h, the cells were stained with 70 µL 0.1% of crystal violet. After 10 min incubation, the crystal violet was removed carefully on the cell paper and the plate was washed with tap water two or three times. The plate was dried at 65 °C for 1 h and 70 µL 70% ethanol (VWR) was added into the wells. Thereafter, the microtiter plate was placed on a shaker for 5–10 min. The inhibition of biofilm was assessed by using in the Victor1) was isolated from the aqueous extract of the marine bryozoan Securiflustra securifrons. The structure was determined by interpretation of data from 1D and 2D NMR experiments and mass spectrometry analysis. In this study, Securidine A (1) did not show any significant biological activities in the applied bioassays. Further bioactivity profiling is required in order to identify any potential biological activities of the molecule.The new brominated tyrosine derivative Securidine A (


Of particular importance is that this ceramic composition exhibits excellent energy storage performance over a wide work temperature up to 150 °C, with strong fatigue endurance and fast discharge speed. All these merits demonstrate the studied ceramic system is a potential candidate for high-temperature capacitors as energy storage devices.Due to the enhanced demand for numerous electrical energy storage applications, including applications at elevated temperatures, dielectric capacitors with optimized energy storage properties have attracted extensive attention. In this study, a series of lead-free strontium bismuth titanate based relaxor ferroelectric ceramics have been successfully synthesized by high temperature solid-state reaction. The ultrahigh recoverable energy storage density of 4.2 J/cm Dielectric capacitors have been widely used as energy-storage components in various electronic and electrical applications ,2, with W and Wrec represent the stored energy density and recoverable energy density. The energy efficiency (η) is defined by the ratio between Wrec and W. It can be seen that high maximum polarization (Pmax), low remnant polarization (Pr), and high breakdown strength (BDS) relating to the various electric field will greatly benefit the recoverable energy density and energy efficiency. However, the Pmax or Pr for many dielectric materials are strongly temperature-dependent [3 and O3 (PLZT)-based systems [The energy density and efficiency of capacitors can be calculated by the following the Equations:ependent , leadingependent ,9. The o systems . However3, ST) has been extensively studied for energy storage applications in the past few years due to its high permittivity (~300), low dielectric loss (<1%) and relatively high BDS (~200 kV/cm) [3+ cation, Ca2+ cation or rare earth cations for increasing permittivity [3+ ions possess the lone-pair electronic configuration 6s2 being analogous to Pb2+ ions, which can be hybridized with O 2p orbitals, resulting the high saturation polarization. It was reported that bismuth-modified strontium titanate ((Sr0.7Bi0.2)TiO3 abbreviated to SBT) dielectric material exhibited strong relaxor-like behavior, showing smeared phase transition temperature and high polarization [3 at 217.6 kV/cm was reported for relaxor ceramic (Sr0.85Bi0.1)TiO3 [0.5Bi0.5)TiO3–0.45SBT multilayer ceramic capacitor showed a high energy density of 9.5 J/cm3 and energy efficiency of 92% at 720 kV/cm [3 was obtained in 0.9SBT–0.1BiFeO3 thin-film capacitor, the energy efficiency was relatively low (48%) [Strontium titanate (SrTiO0 kV/cm) . Many efittivity ,14,15, mittivity . Of part0.1)TiO3 . In addiow (48%) .3  endmembers have been attempted for dielectric solid solutions. For example, BaTiO3–Bi(Me′Me″)O3 ceramics transfer from typical ferroelectric to relaxor with increasing Bi(Me′Me″)O3 concentration, showing improved temperature stability, low dielectric loss, as well as improved energy efficiency [3–0.3Bi(Mg0.5Zr0.5)O3 was found to exhibit high stability over a wide temperature range from −20 °C to 430 °C with low permittivity variation (< ±15%) and relatively low loss (~2%) [0.5Zr0.5)O3-modified SBT relaxor ferroelectric solid solution (1-x)(Sr0.7Bi0.2)TiO3-xBi(Mg0.5Zr0.5)O3 ((1-x)SBT-xBMZ) was fabricated, and its energy storage properties were studied in detail. A high energy storage density of 4.2 J/cm3 at applied electric field of 380 kV/cm was obtained for the x = 0.06 sample. The energy efficiency at 200 kV/cm was maintained above 90% over the temperature range of 25 °C to 125 °C, exhibiting an excellent temperature stability.Based on the unique relaxor characteristic of Bi-based compound, in order to improve the temperature stability and energy storage density, numerous Bi(Me′Me″)Oficiency ,21,22,23ss (~2%) . Therefox)SBT-xBMZ ceramics with x = 4%, 6%, 8%, 10%, 15%  were fabricated by a conventional high-temperature solid state reaction method using reagent powders SrCO3 , TiO2 , Bi2O3 , MgO , and ZrO2 . These powders were baked at 150 °C for 12 h to remove the absorbed moisture and then stoichiometrically weighed with the addition of 1–2% excess MgO [P-E and breakdown strength measurements. The X-ray diffraction patterns were recorded on a diffractometer (GBC MMA XRD) using Cu Kα radiation with the ground pellets. The microstructure information of polished and thermally etched surfaces were collected using scanning electron microscopy (SEM) . Grain size distributions were calculated using Nano Measurer by measuring the selected area of SEM images. In order to measure the dielectric properties of the studied samples, silver electrodes were fired on both sides of polished samples. The dielectric data was collected on a LCR meter  from 100 Hz to 1 MHz over temperature range of −100 °C to 400 °C. For the electrical measurements, various electric fields were applied to the sample at 10 Hz and room temperature using a ferroelectric tester  to obtain P-E hysteresis loops. Thermal stability and fatigue endurance were measured at 200 kV/cm from 25–150 °C and 1 to 105 cycles, respectively. The charge–discharge rate was measured with charge–discharge system  based on the resistance–capacitance circuit with 2 kΩ load resistance.(1-cess MgO . The mixx)SBT-xBMZ ceramics with x = 4–15% are shown in r) of (Mg0.5Zr0.5)3+ (r (Mg0.5Zr0.5)3+ = 0.72 Å calculated from the average ionic radii of Mg2+ and Zr4+) than that of Ti4+ (r(Ti4+) = 0.605 Å) on the B-site. The lattice parameters are obtained from XRD patterns, the linearly increasing trend with BMZ concentration from x = 4% to 15% can be seen in 2Ti2O7 was observed for BMZ in the range of 6–8%, above which, the secondary phase was changed to Sr2Bi4Ti5O18 [3 for x = 0.04, slightly increasing to 5.68 g/cm3 for x = 0.15.The XRD patterns of (1−i4Ti5O18 . The denx)SBT-xBMZ ceramics not only show minimal porosity but also have fine grains with average grain size smaller than 1 μm, as shown in The SEM images of studied ceramics with thermally etched surface are shown in 3-based ceramics have been widely investigated to acquire deeper understanding, including the relationship of relative dielectric permittivity (εr) and the component Bi(Me′Me”)O3 endmember. Several previous studies show that the phase transition peaks became broader and smeared with increasing Bi(Me′Me”)O3 substitutions in BT-Bi(Me′Me”)O3 solid solutions, leading to the enhancement of thermal stability [εr) and loss (tanδ) for (1−x)SBT-xBMZ ceramics. The smeared phase transition and frequency dispersion behavior of permittivity and dielectric loss are observed near the dielectric maximum temperature (Tm), exhibiting a typical relaxor behavior. Generally, the diffuseness of phase transition can be evaluated by modified Curie–Weiss law 1/ε − 1/εm = (T − Tm)γ/C, where εm and Tm represent maximum permittivity and the corresponding temperature, respectively. The value γ = 2 represents a complete diffuse phase transition while γ = 1 represents a classical ferroelectric transition [γ values for (1-x)SBT-xBMZ ceramics are calculated using permittivity data at 1 MHz. γ values of (1-x)SBT-xBMZ ceramics, being above 1.5, indicating relaxor characteristics. With increasing BMZ, the maximum permittivity (εm) at 1 kHz for ceramics with x up to 10% are similar, but the Tm increases gradually from −68 °C to −27 °C, higher than the Tm of (Sr0.7Bi0.2)TiO3 ceramics which is around −100 °C [2Bi4Ti4O15) which was reported to possess a Curie temperature around 285 °C [The dielectric properties of BT-Bi(Me′Me”)Otability ,24,27,28ansition . In orde −100 °C . The lowd 285 °C .P–E loops at 200 kV/cm for (1−x)SBT-xBMZ ceramics with different BMZ content are compared and shown in P–E loops at 200 kV/cm, indicating high energy efficiencies above 92%. With increasing BMZ concentration, Pmax increases first then decreases, relevant to the dielectric permittivity, as listed in Pmax was found for 8BMZ, but its large Pr would reduce the recoverable energy density and decreases energy efficiency. The hysteresis increases obviously in 8BMZ, 10BMZ and 15BMZ samples, resulting in the decreased Wrec with increasing BMZ concentration, as shown in The P–E loops of 6BMZ ceramic are measured at 10 Hz with different applied electric fields as shown in P–E loops are slim with negligible hysteresis which are associated with the small value of Pr, contributing to the high energy efficiency. Pr and Pmax as a function of electric field for 6BMZ ceramic are shown in Pmax value enhances monotonically from 6.7 μC/cm2 to 30.3 μC/cm2, while the Pr remains at almost the same values. Thus, the ΔP (ΔP = Pmax − Pr) keeps increasing as function of applied field, which is beneficial for enlarging Wrec and η; the results are given in Wrec of 4.2 J/cm3 was obtained at 380 kV/cm. On the other hand, the energy efficiency exhibits remarkable stability over the applied field up to 320 kV/cm with values larger than 90%, above which the η slightly drops to 88%.The unipolar W as shown in Equation (1). The Weibull distribution has been used as a capable analysis method to explore the breakdown strength of ceramics, as given in the following equations:i is the serial number of sample. Ei, Pi and n are the breakdown strength of each test sample, the probability of breakdown and the summation samples, respectively. The ascending sort of breakdown strength for samples is shown as m) can be obtained by the slope of the fitted linear line of Xi and Yi, which is one of the important parameters that represents the reliability of experimental data. The Weibull characteristic breakdown strength (Eb) can be calculated by the intercept between the fitted linear line and Yi = 0. As shown in m value for 6BMZ ceramic is 17, leading to a high level of confidence. The calculated Eb of 6BMZ is 460 kV/cm which is much higher than other lead-free energy storage ceramics [3-Bi(Me′Me″)O3 ceramics have been widely studied, and the obtained energy densities have been found to be around 1–3 J/cm3 in previous research as shown in 3-Bi(Me′Me″)O3 ceramics are around or above room temperature, leading to a relatively high dielectric loss at room temperature. However, in this work, the Tm of 6BMZ is far below room temperature, resulting in a low dielectric loss at room temperature and above. The depressed tanδ is believed to be associated with high breakdown strength, and hence the high energy density as well as the high energy efficiency. The ultrahigh breakdown strength of 6BMZ ceramic leads to improved Wrec of 4.2 J/cm3, which is favorable for energy storage applications.High BDS is advantageous in dielectric materials for obtaining high ceramics ,34,35,36P–E loops for 6BMZ ceramic at various temperatures from ambient to 150 °C at 200 kV/cm are shown in P–E loops are slim, indicating the high temperature stability. The Pmax value was found to slightly decrease, which is related to the decreased permittivity at elevated temperature, while the Pr remains at a low value over a wide temperature range, indicating the 6BMZ ceramic capable of operating at high temperature. Wrec and η as functions of temperature, the decrease of Wrec is on the order of 14% due to reduced permittivity at elevated temperature. Of particular significance is that the variation of η is minimal up to 130 °C, associated with the low dielectric loss and temperature-insensitive Pr. The excellent thermal stability of η for 6BMZ ceramic make it very promising for energy storage applications working at elevated temperature.For high-temperature energy-storage applications, thermal stability plays a key role when working at elevated temperatures. For example, the operating temperature can be higher than 140 °C in hybrid electric vehicles, thus requiring some electronic components to be protected by cooling systems . DielectP–E loops for 6BMZ ceramic at 200 kV/cm under different cycle numbers. The energy storage performances of the 6BMZ ceramic are reproducible after 105 cycles, where the unipolar P–E loops maintain the same shape over the cycling, small difference can be seen in the inset figure of Wrec and η for fatigue cycling numbers are given in Wrec value remains almost the same value while the η slightly drops from 95% to 92% after 105 cycles, demonstrating that 6BMZ sample possesses excellent cycling reliability.To further investigate the energy storage properties for 6BMZ ceramic, the fatigue cycling endurance and a charge-discharge rate experiment were performed. Strong fatigue endurance is essential for energy storage capacitors in practical applications. RL, i(t) and Vol represent the total load resistor (2 kΩ), the discharge current through RL, and the sample volume, respectively. The calculated Wdis at an applied electric field of 200 kV/cm as a function of time for 6BMZ ceramic is given in τ0.9 is the time required to release 90% of total Wdis value. The τ0.9 for 6BMZ at room temperature is 0.62 μs, indicating the great potential of 6BMZ for pulse power capacitors.The fast discharge rate is critical for pulse-power applications. The discharged energy density can be calculated by x)SBT-xBMZ ceramics were fabricated by conventional solid state processing. The highest recoverable energy density of 4.2 J/cm3 and high energy efficiency of 88% were obtained at applied electric field of 380 kV/cm for 0.94(Sr0.7Bi0.2)TiO3-0.06Bi(Mg0.5Zr0.5)O3 ceramic. The 6BMZ ceramic shows a good temperature stability over a wide temperature range from room temperature to 150 °C. In addition, the 6BMZ ceramic also exhibits excellent cycling stability over 105 charging–discharging cycles. Together with its fast discharge rate, the τ0.9 is on the order of 0.62 μs, demonstrating that the 0.94(Sr0.7Bi0.2)TiO3-0.06Bi(Mg0.5Zr0.5)O3 ceramic shows promising potential as a lead-free dielectric for high-temperature, high-power energy storage applications.In summary, lead-free (1−


Dysregulated histone methyltransferase G9a may represent a potential cancer therapeutic target. The roles of G9a in tumorigenesis and therapeutics are not well understood in non-small cell lung cancer (NSCLC). Here we investigated the impact of G9a on tumor growth and signaling pathways in NSCLC.Immunohistochemistry analyzed G9a expression in NSCLC tissues. Both siRNA and selective inhibitor were used to target G9a. The impact of targeting G9a on key genes, signaling pathways and growth were investigated in NSCLC cells by RNA sequencing analysis, rescue experiments, and xenograft models.. Additionally, targeting G9a by siRNA-mediated knockdown or by a selective G9a inhibitor UNC0638 significantly inhibited tumor growth, and dramatically suppressed Wnt signaling pathway in vitro and in vivo. Furthermore, we showed that treatment with UNC0638 restores the expression of APC2 expression in these cells through promoter demethylation. Restoring HP1α and silencing APC2 respectively attenuated the inhibitory effects on cell proliferation and Wnt signaling pathway in cancer cells in which G9a was silenced or suppressed.Overexpression of G9a (≥ 5% of cancer cells showing positive staining) was found in 43.2% of 213 NSCLC tissues. Multiple tumor-associated genes including HP1α, APC2 are differentially expressed; and signaling pathways involved in cellular growth, adhesion, angiogenesis, hypoxia, apoptosis, and canonical Wnt signaling pathways are significantly altered in A549, H1299, and H1975 cells upon G9a knockdownThese findings demonstrate that overexpressed G9a represents a promising therapeutic target, and targeting G9a potentially suppresses growth and Wnt signaling pathway partially through down-regulating HP1α and epigenetically restoring these tumor suppressors such as APC2 that are silenced in NSCLC.The online version of this article (10.1186/s12943-018-0896-8) contains supplementary material, which is available to authorized users. Lung cancer is the leading cause of cancer-related death worldwide . About 8Recent studies have uncovered an important role for epigenetic changes in tumor progression and treatment resistance , 4. G9a,G9a is shown to directly bind DNA methyltransferase 1 (DNMT1), and G9a and DNMT1 colocalize with H3K9me2 on heterochromatic regions of chromosome. The complex of DNMT1 and G9a enhances DNA and histone methylation for stable repression of gene expression  and p53 Wnt/beta-catenin (β-catenin) signaling pathway promotes cell proliferation, maintains stem cell multipotency, and stimulates EMT in cancer , 16. InhIn this study, we first examined aberrant G9a expression and deciphered its transcriptional regulatory network and highlighted its complex role in gene expression, and we found that APC2 was dramatically upregulated upon G9a knockdown, while heterochromatin protein 1 alpha (HP1α), which binds euchromatic loci during the process of gene silencing in cooperation with DNMT1 , was sigThis study was reviewed and approved by the Institutional Review Board  of City of Hope National Medical Center. A total of 250 patients with lung adenocarcinoma and squamous carcinoma who underwent surgical resection for curative intent between 2002 and 2014 without preoperative chemotherapy or radiation therapy were included. Tissue microarrays were created using cancer and matched normal tissues.http://www.cbioportal.org/). For data analysis, normalized RNA-Seq data  was used as gene expression value and the median was used to classify samples into high and low expression groups. RNA-Seq gene expression data of LUAD and LUSC samples was also downloaded from TCGA data portal to investigate the correlation between the level of G9a mRNA with these selected gene mRNA in the same tissue.G9a mRNA expression and survival data for 517/494 lung adenocarcinoma (LUAD)/lung squamous carcinoma (LUSC) and 59/46 normal lung tissues was accessed from the Cancer Genome Atlas (TCGA) public data portal and extracted from expression dataset from Cancer Bioportal . UNC0638, a specific inhibitor for G9a [50) of UNC0638 to each cell was also calculated accordingly.Human lung cancer cell lines: A549 , H1299 , and H1975  cells were purchased from American Type Culture Collection (ATCC). All cells were cultured in DMEM or RPMI medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. For proliferation assessment, cells were seeded in 6-well plates in 3 replicates at densities of 2.0 × 10 for G9a , was pur5/well cell in a 6-well plate with Lipofectamine RNAiMAX (Invitrogen) to silence G9a expression using the protocol described previously [Two independent G9a siRNAs: Cat No.10620318 (5′-GGCAUCUCAGGAGGAUGCCAAUGAA-3′) and Cat No. 10620319 (5′-UUCAUUGGCAUCCUCCUGAGAUGCC-3′) purchased from Thermo Fisher Scientific Corporation  were applied to silence G9a expression. One APC2 siRNA: Cat No.121574 (5′-CCUACAGGGAAAACUGGAGTT-3′) and HP1α siRNA ID #: s23884 (5′-GCAGAGCAAUGAUAUCGCUtt-3′) were purchased from Thermo Fisher Scientific Corporation to silence APC2 and HP1α expression. A total of 45 pMol siRNA was used for 1 × 10eviously . At 72 heviously . Both geeviously , 30. Qua4 cells per well on 24-well plates. At 24 h post-transfection, cells were further transfected with 125 ng of T-cell factor (TCF)/LEF-1 reporter plasmid plus 5 ng Renilla luciferase reporters using Lipofectamine™-2000. Forty hours after reporter plasmid transfection, cells were treated with or without 100 ng/ml Wnt3a for another 8 to 12 h, firefly and Renilla luciferase activities were determined and calculated as described previously [NSCLC cells were first transfected with either control or G9a specific siRNA at a concentration of 5.0 × 10eviously . All expeviously , and it (IHC) was performed using anti-G9a antibody from GeneTex as described previously [The antibodies against Actin, APC2, DKK1, EpCAM, G9a, H3K9-Me2, HP1α, and WIF1, p53, c-Myc were purchased from Cell Signaling Technology, Abcam, Santa Cruz Biotechnology or GeneTex respectively. Immunohistochemistry eviously . Expresseviously . G9a IHCeviously . After ceviously .6 tumor cells (H1299) in 0.1 ml RPMI 1640 was mixed with 0.1 ml BD Matrigel™ (BD Science) and injected into the subcutaneous dorsa of mice at the proximal midline. When the tumor volume was 90–110 mm3, mice were randomized. Mice treatment with UNC0638 was performed by continuous administration of 100 μl of 5 and 10 mg/ml of UNC0638 intraperitoneal (i.p.) injection via mini-osmotic pump  as described previously [All animal protocols were performed in the animal facility at City of Hope National Medical Center accordance with federal, local, and institutional guidelines. NOD/SCID/IL2Rgamma null mice (NSG) mice  were used for xenograft experiment. A suspension of 5 × 10eviously . These peviously .Genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen). A total of 1.5 μg of genomic DNA were modified using sodium bisulfite to deaminate selectively unmethylated cytosine residues to uracil, while 5-methyl cytosine residues were not modified. The bisulfite modification was performed using the EZ DNA Methylation Kit™ , and 40 ng of modified DNA was used per PCR amplification. A forward (5′-GGGTYGTTATTGGTTGTTGTTATGG-3′) and a reverse (5′-AAACRCCTAAATCTAAAACCTCCTC-3′) primers specific for the bisulfite-converted DNA were used to amplify a highly methylated CpG island  in the APC2 gene promoter region . And thet-test or Pearson χ2 test. All data shown are mean ± s.d. Statistical significance was set at P < 0.05. Pearson’s correlation coefficient was used to measure correlation of gene coexpression.All experiments were performed in duplicates or triplicates and repeated at least two times in each experiment. Two group comparisons were analyzed for variation and significance using a Student’s N = 517; Fig. N = 105). Importantly, statistical analysis revealed that in comparison to corresponding normal lung tissues, G9a mRNA was significantly higher in NSCLC samples .In the present study, we first mined lung cancer gene expression data from TCGA to examine the level of G9a mRNA in NSCLC tissues. As shown in Fig. To further investigate the G9a protein in NSCLC, we examined G9a protein in about 213 cancer tissue samples by IHC analysis. The results show that G9a protein in normal lung tissues was generally absent or much lower compared to NSCLC tissues. Representative G9a immunostaining images of normal lung tissue Fig.  and NSCLP < 0.05). The majority of gene sets were down-regulated, and important sets in two lung cancer cells were these involved in cell proliferation, migration, and regulation of apoptosis, angiogenesis, response to hypoxia, canonical Wnt, and toll-like receptor signaling; while adhesion to matrix, response to DNA damage, regulation of epithelial cell differentiation, and histone H3-K9 methylation, were significantly enriched in both A549 and H1299 cells  analysis was utilized to characterize global gene expression change upon knockdown of G9a in A549 and H1299 NSCLC cells. Unsupervised hierarchical clustering analysis of RNA-Seq data depicts the changes in global gene expression profile in these lung cancer cells upon knockdown of G9a Fig. . G9a knolls Fig. , indicatP < 0.05). In addition, we found that knockdown of G9a induced cell cycle arrest of A549 in the G1/S phase . To further test if a small molecular inhibitor of G9a could suppress NSCLC proliferation, we treated three lung cancer cells with a selective G9a inhibitor UNC0638 [50 of UNC0638 was about 5.0, 2.5, and 3.5 μM for A549, H1299, and H1975 respectively. Overall, these data indicated that targeting G9a by siRNA or small molecule inhibitor significantly suppressed in vitro proliferation of NSCLC cell lines.Interestingly, RNA-Seq analysis showed that knockdown of G9a downregulated HP1α in these NSCLC cells. Study demonstrated that overexpression of HP1α correlated with enhanced cell proliferation and poor clinical outcomes of breast cancer . Therefo UNC0638 . UNC0638P < 0.05), H1299 cells  and H1975 cells . In agreement with the TOPFlash-Luc assay, double-label fluorescent immunohistochemical analysis showed that accumulation of nuclear β-catenin was relatively lower in cells without Wnt3a stimulation (data not shown), however, the accumulation was dramatically elevated in cells upon Wnt3a stimulation , increase in TCF transcriptional activity indicates upregulation of β-catenin. We first measured TOPFlash-Luciferase  reporter activity , and exaion Fig. -f. Knockion Fig. , H1299  analysis showed that APC2, DKK1, and WIF1 gene were significantly upregulated. Western blot analysis confirmed that UNC0638 treatment dramatically reduced the level of H3K9Me2, increased the levels of APC2, DKK1, and WIF1 proteins, and resulted in decreased β-catenin in these cancer cells  and H1299 cells  and WIF1  in 46 of lung cancer tissues, and a slight reverse but insignificant correlation between APC2 and G9a was observed in lung cancer tissues (data not shown).By RNA-Seq, we found that APC2, which forms a destructive complex capable of binding β-catenin to function as an important inhibitor of Wnt signaling pathway , was dra5alls Fig. . ConsideUsing RNA-Seq analysis, we found that besides APC2 other genes including ICAM5, JPH3, MGMT, MMP2, and RASSF1 whose expression are epigenetically silenced, were also upregulated with G9a inhibition . The average tumor size in the mice treated with UNC0638 on day 28 after the implantation was significantly lower than the tumor weight in the mice treated with PBS Additional file 2:Figure S1. Inhibition of G9a induces cell cycle arrest. a A549 in G1 phase b H1299 in G2 phase. The statistical analyses of G1 and sub-G1 are shown . Figure S2. Impact of knockdown of G9a on the levels of p53 and c-Myc proteins in A549 (p53 wild-type), H1299 (p53 null), and H1975 (p53 mutated) cancer cells. Figure S3. Impact of knockdown of HP1α on NSCLC cells proliferation. a Western blot of HP1α in three cell lines. b Cell proliferation after 72 h posttransfection of HP1α siRNA. , b WIF1 mRNA  in 46 of LUSC tissues. X-axis is for the normalized G9a (EMHT2) mRNA level, Y-axis is for the normalized DKK1/WIF1 mRNA level. Figure S5. Unsupervised hierarchical clustering analysis of selected upregulated genes upon G9a knockdown. These genes include  ICAM5, JPH3, MGMT, MMP2, RASSF1α etc. that are frequently silenced by epigenetic mechanisms in lung cancers. The two top rows represent two independent siRNA repeats, Ctrl1/2 is for control siRNA and G9a-KD1/2 is for G9A siRNA1/2. Figure S6. UNC0638 treatment decreased HP1α in H1299 cells. Cells were treated with defined UNC0638 for 72 hours and then protein was extracted for Western blot analysis of HP1 α, G9a, H3K9-Me2. (PDF 438 kb)