There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions.We identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability.Replication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.The online version of this article (doi:10.1186/s12858-016-0074-9) contains supplementary material, which is available to authorized users. The basic repeating structural unit of eukaryotic chromatin is the nucleosome core particle (NCP), which is a structured package of 147 bps of DNA and a histone octamer consisting of 2 copies of each core histone: H2A, H2B, H3, and H4. There are also linker histones, H1, flanking NCPs and sealing the structure with about 20 bps of additional DNA. The involvement of H1 is crucial for the formation and stabilization of chromatin structures and for the regulation of gene expression [There are 11 variants of linker histone H1 in mammalian cells: somatic replication-dependent variants (H1.1 to H1.5), somatic replication-independent variants , and germ cell specific variants  . The repStudies on core histones indicate that replication-dependent and replication-independent histone variants can be involved in distinct protein complexes and assembly pathways. For example, histone H3.1 is assembled into chromatin in coordination with DNA replication during S phage, while another variant, H3.3, is exchanged throughout the cell cycle. The dynamics of H3.1 and H3.3 are mediated by distinct protein complexes that contain different chaperones for each of the H3 variants. While H3.1 and H3.3 complexes have some shared histone associated proteins, such as NASP, ASF1A, ASF1B, HAT1, and importin 4, there are also exclusive histone binding partners in each complex. All three subunits of histone chaperone CAF-1 are only found in the H3.1 complex, while another histone chaperone, HIRA, is only found in the H3.3 complex , 8. CAF-Currently it is not known whether the dynamics of replication-dependent and -independent variants of H1 are also regulated by distinct chaperones in a manner similar to H3.1 and H3.3 complexes. Previous studies proposed several linker histone chaperone candidates: Nucleosome Assembly Protein 1(NAP1) , NuclearTo purify H1 variants and their associated proteins for identification, we generated tetracycline-inducible U2OS cell lines over-expressing 6 × His-tagged H1 variants H1.1, H1.2 or H1x by transfecting U2OS cells engineered for tetracycline-inducible expression with pT-Rex-DEST31 plasmids carrying the corresponding H1 variant sequences Fig. . CellulaTo analyze proteins associating with the soluble pool of the H1 variants, we purified H1.1, H1.2 and H1x from U2OS whole cell extracts by sequential chromatography with an anion exchange column (Mono Q) and an affinity column (nickel column). The specific order of the chromatography was used to optimize the concentration of proteins prior to mass spec analysis. Interestingly, H1.1 eluted in two separate peaks on the Mono Q column, while H1.2 and H1x both eluted in only one peak Fig. –c. Each Peak samples eluting from the nickel chelate column containing each histone variant, as well as the comparable negative control fraction, were analyzed in duplicate by mass spectrometry. A complete list of the proteins identified, as well as the number of unique peptides observed in each analysis, is listed in Additional file The protein list contains a large number of ribosomal proteins. Several recent proteomic studies have also found that linker histones bind to ribosomes and this interaction has been implicated in transcriptional repression –17. ReceTable The group 1 proteins included two proteins that had previously been identified as histone chaperones, nucleolin and nucleophosmin (NPM1) . NucleolGroup 1 also included SRP68 and SRP72. Components of the signal recognition particle, SRP68 and SRP72 have also been shown to form a heterodimeric complex distinct from the signal recognition particle. In addition to its role in translation, the SRP68/72 complex was also found to bind histone H4 molecules that contain arginine 3 methylation .Bax in order to repress p53-induced transcription [YBX1 and YBX3 also co-purified with all three linker histones. Y box-binding proteins bind to Y box consensus promoter elements and are involved in mRNA processing . YBX1 wacription . Our dat6-methyladenosine modification of mRNA. VIR (virilizer homolog), WTAP (Wilms Tumor Associated Protein), ZC3H13 and Hakai are all components of the WTAP complex that serves to target the METTL3 and METTL14 methyltransferases to their substrate [6-methyladenosine [Five of the group 1 proteins are linked to Nubstrate –30. In adenosine –34.The remaining group 1 proteins include a cyclin/cdk complex; CCNL1 and CDK11b. Linker histones are highly phosphorylated and often used as non-specific substrates in kinase assays. In fact, CDK11b has been shown to be able to phosphorylate histone H1 in vitro . The obsThe group 2 proteins bind specifically to the replication-dependent H1 variants H1.1 and H1.2 but don’t form a complex with the replication-independent variant H1x. The group 2 proteins include 4 subunits of the PAF1 complex, PAF1, CTR9, CDC73 and LEO1 . The speCHD8 has previously been shown to function in transcriptional repression of p53 and β-catenin target genes through the recruitment of histone H1 , 39. TheThe nuclear pore complex protein Tpr was also found to be replication-coupled H1 variant specific. Tpr (translocated promoter region) is a component of the nuclear pore complex (NPC), forming fibrous structures that extend into the nuclear interior . Tpr is Proteins that specifically co-purified with histone H1.1 (group 3) included several known histone chaperones. NapP1L1 and SET were recently shown to bind histone H1.0 in vitro , 46. NAPGroup 3 contained several proteins involved in RNA metabolism. These included three poly A binding proteins, PABP1, PABP3 and PABP4, as well as the RNaseP subunit POP1 , 48. In H1.2 specific binding partners (group 4) included DNA damage-binding protein 1 (DDB1) and protein VPRBP . DDB1 and VPRBP are both members of an E3 ubiquitin-protein ligase complex, named CUL4A-RBX1-DDB1-DCAF1/VPRBP complex, which is responsible for methylation-dependent ubiquitylation . VPRBP aGroup 4 contained two RNA helicases, DDX21 and DDX50 and the nucleolar RNA binding protein EBP2. Group 4 also contained the transcriptional regulators TAF5 and TIF1B, as well as CHD7. While CHD7 has not been shown to be associated with linker histones, these results suggest that it may function in a manner analogous to CHD8.Only two proteins were found to be exclusive binding partners of H1x (group 6). These were ubiquitin carboxyl-terminal hydrolase 15 (USP15) and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). USP15 is a deubiquitinating enzyme that can bind to ubiquitinated H2A/H2B dimers, and removes ubiquitin from ubiquitinated H2B that are not in nucleosomes . IGFBP3 Group 6 is an eclectic collection of proteins that co-purified with both H1.1 and H1x. This group includes the RNA binding proteins SRSF3 and SRSF7, the zinc finger protein ZNF16 and the anti-microbial protein dermcidin.The proteomics data clearly demonstrate that linker histones participate in a complex set of interactions that display varying degrees of specificity for the H1 variants. To explore in more detail the distinction between replication-dependent (H1.1 and H1.2) and replication-independent (H1x) H1 variants, we examined whether the specificity observed for replication-dependent H1.1 and H1.2 displayed by Tpr was a reflection of a specific in vivo connection between Tpr and replication-dependent linker histones. Unlike the PAF-1 complex and CHD8, which were also identified in the H1.1 and H1.2 complexes but not in the H1x complex Table . In all r = 0.83 and 0.89, respectively) while there was no correlation between the level of Tpr and the level of H1x (r = 0.17). Consistent with the immunofluorescence data, we also observed reduced H1.1 and H1.2 protein levels in Tpr siRNA treated cells on western blots; however H1x protein levels were not affected  was not found to associate with any of the histone H1 variants in the present study and has not been identified in the recent proteomic analyses of in vivo linker histone complexes , 26, 37.In comparing the proteins that co-purified with each of the H1 variants, we found that the three H1 variants had shared binding partners that were identified in earlier studies, such as nucleophosmin, nucleolin and FACT complex subunits SPT16 and SSRP1, nucleolin, nucleophosmin (NPM1) and Y box-binding proteins YB1, YB2 and YB3. These H1-associating proteins may contribute to the common functions of H1 variants. Importantly, we identified shared components found in the replication-dependent H1 variant complexes that were not associated replication-independent H1 variants. These factors include the PAF1 complex, CHD8 and nucleoprotein Tpr. The recent study from Kim and colleagues demonstrated that the PAF1 complex is not associated with histone H1.0 consistent with the specificity of this complex for replication-dependent linker histones . These eThe interactions of the PAF-1 complex and CHD8 with linker histones have demonstrated functions in transcriptional regulation –39. HoweWhile Tpr is required for the stability of histones H1.1 and H1.2, the functional significance of their interaction is not clear. As Tpr is a component of the nuclear pore complex, the most straightforward model is that Tpr is specifically involved in the nuclear import of replication-dependent linker histones. This model predicts that if the levels of H1.1 and H1.2 were restored in cells lacking Tpr that the histones would accumulate in the cytoplasm. However, when the Tpr siRNA knockdown was performed in the presence of the proteasome inhibitor MG132, we observed no cytoplasmic accumulation of H1.1 and H1.2.A second possibility is that Tpr can function as a histone chaperone that participates in the targeting of replication-dependent linker histones to sites of chromatin assembly. Indeed, Tpr contains a region nears the COOH-terminus that is highly enriched in aspartic acid and glutamic acid as is observed in many histone chaperones. In addition, a recent proteomic analysis of nascent chromatin structure identified Tpr as a chromatin-associated protein .Another potential function for the interaction of Tpr with histones H1.1 and H1.2 may be related to involvement of Tpr in the formation of heterochromatin exclusion zones (HEZs) around nuclear pores. While heterochromatin localizes near the nuclear envelope, regions near the nuclear pores are devoid of heterochromatin through the formation of Tpr-dependent HEZs . The intWe have isolated and analyzed the soluble forms of the replication-dependent linker histones H1.1 and H1.2 and the replication-independent linker histone H1x. These linker histones interact with both a common group of proteins and with variant-specific interacting proteins. One protein that is specifically associated with H1.1 and H1.2 is nuclear pore complex protein Tpr. Tpr was not required for the nuclear import of histones H1.1 and H1.2. However, knockdown of Tpr expression resulted in a specific decrease in protein stability of the replication-dependent linker histones.6 from Roche was used for immunofluorescence microscopy; anti-Tpr from Abcam. Control siRNA (sc-37007) and siRNA targeting Tpr (sc-45343) was purchased from Santa Cruz Biotech.Sources of antibody employed are as follows: anti-His.M8 from Thermo Fisher Scientific was used for western blots; anti-His2-terminus. The resulting constructs were transferred into DH10B E. coli strain for production.The human Ultimate ORF of histone H1.1 (IOH35288), H1.2 (IOH5275), and H1x (IOH3417) (Thermo Fisher Scientific) were inserted into mammalian expression vector pT-Rex-DEST31 (Thermo Fisher Scientific), which adds a 6 × His-tag at the NHv/v) fetal bovine serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1 % L-glutamate (Sigma) at 37 °C in 5 % CO2 supply. The U2OS cells were transfected with pT-Rex-DEST31 plasmids carrying H1.1, H1.2 or H1x using X-tremeGENE HP DNA transfection reagent (Roche) following the manufacturer’s instruction. Transfected clones were selected against 200 mg/ml G418 and 10 mg/ml blasticidin . Stable clones were induced by 1 μg/ml doxycycline (Sigma) for 48 h. The expression of 6 × His-tagged H1 variants was confirmed by western blot.Tetracycline-inducible human osteosarcoma cell line U2OS (kind gift from Dr. Dan Schoenberg) was grown in McCoy’s 5A media supplemented with 10 %  for 5 min on ice. The cells were then broken with 7 gentle strokes in a type B Dounce homogenizer. Nuclei were pelleted by centrifuging at 120 × g for 10 min. The pelleted nuclei were washed with 1 ml MNase digestion buffer , resuspended in 1 ml MNase digestion buffer, and digested with 0.2 U/ml (Sigma units) of MNase at 37 °C for various lengths of time. Digestions were stopped with 10 mM EDTA followed by incubation with 0.1 mg/ml proteinase K at 37 °C for 5 min. The resulting DNA samples were isolated using phenol extraction and ethanol precipitation, and resolved on 1 % agarose gel [1 × 10rose gel .2+. The column was then washed extensively with NP40 lysis buffer containing 50 mM imidazole. Bound proteins were eluted with NP40 lysis buffer containing 500 mM imidazole. Fractions were collected and resolved on western blots.U2OS cells with 6 × His-tagged H1 inserts were induced by 1 μg/ml doxycycline (Sigma). After 48 h, cells were harvested, washed with PBS, and lysed with NP40 lysis buffer  containing EDTA-free protease inhibitor cocktail (Roche) and 0.5 mM phenyl methyl sulfonate fluoride. The resulting whole cell extracts were applied to a Mono Q 5/50 GL column . Proteins were eluted with a NaCl gradient from 50 mM to 500 mM in 15 column volumes, then 1 M for 5 column volumes. Fractions containing eluted 6 × His-tagged H1 variants were pooled, desalted on desalting columns , and then applied to an HiTrap Chelating HP Column  charged with Niv/v formic acid  and mobile phase B (MPB) consisted of ACN  with 0.1 % v/v formic acid. The HPLC gradient was ramped from 2 to 40 % MPB from 5 to 155 min, followed by a high organic wash and column equilibration period. The heated capillary temperature and electrospray voltage were set to 175 °C and 2.0 kV. The top 5 molecular ions (2+/3+ charges) were selected for CID fragmentation in a data-dependent fashion. Molecular ions were analyzed in the Orbitrap . Molecular ions were selected and subjected to CID fragmentation in the linear ion trap . Dynamic exclusion parameters were enabled . Protein identifications were determined using the MassMatrix search engine (v 2.4.2) and the complete, reviewed Swiss-Prot entries from UniProt  [Tryptic peptides were desalted online using a μ-precolumn  and separated based on hydrophobicity at a flow rate of 2 μL/min . Peptides were introduced into a ThermoFisher LTQ Orbitrap XL mass spectrometer  with the aid of micro/nanospray ionization source . Mobile phase A (MPA) consisted of HPLC water with 0.1 % ly 2013) –65. Searly 2013) . Proteinw/v TCA, then resolved and visualized on western blots.Whole cell extracts of U2OS cells expressing 6 × His-tagged H1 variants were applied to a Superose 6 10/300 GL column . The column was washed with 1.5 column volumes of NP40 lysis buffer. Proteins in each collected fraction were precipitated with 20 % U2OS cells seeded on cover slips were fixed in 4 % paraformaldehyde for 20 min at room temperature and then washed with PBS. The washed cells were permeabilized with 0.5 % Triton X-100 in PBS for 15 min, then washed with PBS three times, and blocked with blocking buffer  for 1 h. After blocking, the cells were incubated with primary antibodies diluted in blocking buffer overnight followed by three washes in PBST, and incubated with secondary antibodies for 1 h. Then the cover slips with cells were washed three times in PBS, and mounted onto microscope slides with Vectashield mounting serum containing DAPI (Vector Laboratories). The slides were examined using Axioskop 40 microscope. Immunofluorescence pictures were taken with an Axiocam HRC camera and processed in Zen 2 Pro software.6 staining were standardized against CTCF of DAPI staining. Regression analysis was carried out using Microsoft Excel built-in statistical functions.Corrected total cell fluorescence (CTCF) was quantified using ImageJ as previously described . CTCF ofp ≤ 0.05 (*), p ≤ 0.01(**), and p ≤ 0.001(***).RNAs were extracted from U2OS cells using Trizol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. The resulting RNAs were used to prepare cDNAs using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instruction. Levels of gene expression were measured using TaqMan Gene Expression Assays , TagMan Universal PCR Master Mix (Applied Biosystems) and the ABI 7300 sequence detector as previously described [


The composition and structure of nascent complex ions have been studied. During electrochemical reduction of [Rh(NH3)4Cl2]+ complexes, the synthesis of Rh nanoparticles, as well as Fe, Cu, and Mo nanocoatings, were realized.The electrochemical behavior of rhodium at low-temperature carbamide-chloride and acetamide-chloride melts was investigated. It was found that, during rhodium anodic dissolution in carbamide and acetamide containing chloride melts, mixed complexes [Rh(NH Nanoparticles and materials based on them exhibit unique electrical, chemical, magnetic, optical, catalytic, and other properties. That is why such studies receive considerable attention  allowed6. For Rh (III) complex compounds characterized by the formation of octahedral configuration, the main state for octahedral complexes of Rh (III) is 1A1g(t2g)6, and a singlet excited state is 1T1g and 1T2g relating to the configuration (t2g)5(eg).The most stable oxidation state of rhodium is Rh (III) with the electron configuration [Kr] (4d)−1 and 37,000 cm−1 appear at electron spectra, and in acetamide-chloride melt—at 28,000 cm−1 and 36,000 cm−1  at 330 cm−1 was found. In accordance with the literature data [Liver [3)4Cl2]+ of D4h symmetry.During anodic dissolution of Rh in the urea-chloride melt, bands at 28,000 cma [Liver ], this i3)4Cl2]+ in urea-chloride and chloride-acetamide melts: 10 Dq = 31000 (30650) cm−1; B = 562 (500) cm−1; b = 0.78 (0.70). The strength of ligand field in the mixed complexes [Rh(NH3)4Cl2]+ is weaker than in pure ammonia complexes [Rh(NH3)6]3+, where 10 Dq = 33200 cm−1 [Galus [3)4Cl2]+.The following spectroscopic parameters were obtained for the complexes [Rh(NH3)4Cl2]+ in the investigated melts is electroactive, and its reduction is observed in the cathodic part of cyclograms as a maximum shown in Fig. ip/V1/2 from V1/2 The size of the Rh deposit particles is within 10–22 nm, but a significant portion of it is aggregated. The average size of the rhodium crystallites was evaluated by physical expansion of peaks and is about 5 nm. This indicates the formation of rhodium nanocomposites on the surface of metallic Fe, Cu, and Mo in the investigated melts. Rhodium coatings obtained from melts on the basis of urea and acetamide with soluble rhodium anode are uniform, of gray color, and 6.1-micron thick  complexes which are reduced in the diffusion mode, in one step to metal. The synthesis of Rh nanoparticles and nano-coatings at Fe, Cu, and Mo surface was realized.


Hepatocellular carcinoma (HCC) is the seventh most common malignancy and the third leading cause of cancer-related death worldwide with an extremely grim prognosis. Berberine (BBR) has been found to inhibit proliferation of human HCC cells, although the underlying mechanism(s) are unclear.Protein expression was detected by Western blots. Cell viability was determined by using the CellTiter Assay kit.We confirm that BBR treatment inhibits HepG2, Hep3B, and SNU-182 cell viability, and suggest that it regulates this proliferation via the modulation of multiple tumorigenesis-related genes protein expression. BBR treatment up-regulated protein expression of tumor suppressor genes, including Kruppel-like factor 6 (KLF6), activating transcription factor 3 (ATF3) and p21, while down-regulating the expression of selected oncogenes, including E2F transcription factor 1 (E2F1) and pituitary tumor transforming gene 1 (PTTG1). The specific extracellular signal–regulated kinases 1/2 (ERK1/2) inhibitor, PD98059, partially inhibited BBR effects including reduction of cell viability, and up-regulation of KLF6 and ATF3 expressions; although, PD98059 did not alter the down-regulation of E2F1 and PTTG1 expression by BBR.Our results suggest that BBR inhibits HCC cell viability by modulating multiple tumorigenesis-related genes, and that up-regulation of tumor suppressor genes by BBR is in part the result of ERK1/2 action. The results of this study augment our understanding of the mechanisms underlying the effect of BBR on hepatocellular cancers and provide further evidence as to the biological plausibility of this agent’s role in the treatment of these malignancies.The online version of this article (doi:10.1186/s12935-017-0429-3) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) is the seventh most common cancer and the third leading cause of cancer death worldwide, with few therapeutic options . The AmeCoptidis rhizoma, Phellodendron chinense schneid, and Phellodendron amurense [Berberine (BBR) is an isoquinoline alkaloid, which can be isolated from a variety of naturally occurring plants such as amurense . BBR hasamurense –12. BBR amurense , 13. In amurense , 14–19. amurense . In addiamurense .p21 [Many tumor suppressor genes and oncogenes are related to HCC tumorigenesis. Expression of tumor suppressor genes including Kruppel-like factor 6 (KLF6) , 22, actp21  have beep21 , 26, whip21 . In HCC,p21 . p21 hasp21 , 29. Altp21 , 31 and p21  are overp21 .The above findings suggest that BBR is a promising candidate for the treatment of HCC. However, the molecular mechanisms underlying the anti-neoplastic action of BBR in HCC are not fully understood. It is possible that BBR acts by modulating these tumor suppressor genes and oncogenes known to play a role in HCC, a hypothesis tested in the present study.2.HepG2 (Cat# HB-8065), Hep3B (Cat# HB-8064) and SNU-182 (Cat# CRL-2235) were purchased from ATCC . HepG2 and Hep3B cells were cultured in medium Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). SNU-182 cells were cultured in medium RPMI 1640 with 10% FBS. All cells were cultured at 37 °C in a humidified chamber with 95% air and 5% COBBR (Cat# B3251) and PD98059 (Cat# sc-3532) were purchased from Sigma-Aldrich  and Santa Cruz Biotechnology . The concentration of stocks are 10 mM in water for BBR and 25 mM in DMSO  for PD98059.5 per well overnight and then treated PD98059, BBR or both for 24 h. After treated, samples were lysed with a lysis buffer, and protein concentrations were determined by using coomassie blue method. Forty µg of total protein were separated on SDS-PAGE gels and then transferred to PVDF membranes. Membranes were immunoblotted with the appropriate primary antibodies   at 4 °C overnight. After washing, membranes were incubated with a secondary antibody , detected with chemiluminescence reagent  and exposed by autoradiography.Cells were seeded in 12 wells plate with cell number 1 × 102 incubator. After overnight incubation, cells were treated with or without BBR for 24 or 72 h in DMEM with 5% FBS. Prior to conducting the cell viability assay, cells were washed with PBS twice and incubated in PBS 100 μl/well. Twenty microliters of CellTiter solution was added to each well. Cells were incubated in CO2 incubator for 2 h. Absorbance was determined with a microplate reader at 490 nm.Cell viability was determined by using the CellTiter Assay (MTS) kit . Cells were trypsinized and seeded 5000 cell/well into 96-well plates and incubated overnight in DMEM with 10% FBS in COCells were trypsinized and seeded 40,000 cells/well into 12-well plates and incubated overnight in DMEM with 10% FBS in CO2 incubator. After overnight incubation, cells were treated with or without BBR for 72 h in DMEM with 5% FBS. After 72 h treatment, cells were trypsinized, and Cell number were counted by the hemocytometer under a microscope. Dead cells were excluded by Trypan Blue stain.Cells were pretreated with dimethyl sulfoxide  or PD98059 25 µM for 30 min then treated with or without BBR 100 µM for 24 h. Cell viability was analyzed by MTS assay. ERK1/2 phosphorylation, and BBR- regulated protein expression was analyzed by western blot.p < 0.05 was considered statistically significant. All experiments were repeated 3 times (n = 3). All values are presented as mean ± SEM.Comparisons of multiple groups were carried out by analysis of variance (ANOVA), followed by a post-test using the Fisher (among groups) or Dunnett (compared with control group) tests . A Three HCC cell lines were treated with BBR in concentrations of 0, 10, 20, 50 and 100 µM for 24 and 72 h. Cell viability was detected by MTS assay. After 24 h of treatment, BBR in concentrations of 50 and 100 μM inhibited HepG2 cell viability approximately 50 and 80% respectively, and BBR in concentrations of 20, 50 and 100 μM inhibited HepG2 cell viability approximately 40, 80 and 95%, respectively with 72 h treatment , ATF3 (p < 0.05) and p21 (p < 0.05) expression with stimulation reaching significance at a concentration of 100 μM in HepG2 cells  and PTTG1 (p < 0.05) expression in HepG2 cells , with PD98059 (25 μM) alone, or with PD98059 in combination with BBR, for 24 h. Treatment of PD98059 inhibited endogenous and BBR-stimulated ERK1/2 phosphorylation Fig. a.Fig. 3PPD98059 was able to partially, but significantly (p < 0.05) reduce the inhibition of cell viability by BBR Fig. b; howevePD98059 did not alter BBR-reduced PTTG1and E2F1 expression in HepG2 Fig. d, Hep3B Overall, we confirmed that BBR inhibited HepG2 and Hep3B cell proliferation , 18, 34.In addition to cell proliferation, BBR also regulates gene expression differently between these three HCC cell lines. BBR stimulated expression of three tumor suppressor genes, KLF6, ATF3 and p21, and reduced two oncogenes E2F1 and PTTG1 in HepG2 cells, while BBR just induced ATF3 and reduced E2F1 expression in Hep3B cells. As HepG2 expresses wild type p53 and Hep3B is a p53-deficient HCC cell line, these results suggest that BBR regulation of KLF6, p21 and PTTG1 expression is possibly p53 dependent. Indeed, BBR has been found to up-regulate miR-23a via regulation of p53 . In contThe ERK1/2-specific inhibitor, PD98059 partially blocked BBR-induced inhibition of cell proliferation in HCC cell lines, suggesting that activation of the ERK1/2 pathway is involved in BBR-inhibited cell proliferation. Indeed, Aspafilioside B, a steroidal saponin extracted from Asparagus filicinus and a known active cytotoxic component, has been shown to induce apoptosis via ERK1/2 activation in HepG2 cells . In addiDMSO, the solvent for PD98059, has been discovered to induce p21 expression in B cell lines . When weKLF6 has been reported to upregulate p21  and ATF3In conclusion, overall BBR inhibits cell proliferation of tested three HCC cell lines including HepG2, Hep3B and SNU-182. However, BBR-regulated protein expression of multiple tumorigenesis associated genes in these cell lines differently (summary in Table


Acinetobacter baumannii (AbIDH2) with a mass of 83 kDa was formerly recognized as a typical monomeric IDH. However, both size exclusion chromatography and analytical ultracentrifugation analysis indicated that AbIDH2 exists as a homodimer in solution. The crystallographic study of the substrate/coenzyme-free AbIDH2 gave a dimeric structure and each subunit contained a domain I and a domain II. The dimeric assembly is mainly stabilized by hydrophobic interactions  from the dimer’s interface platform, which centered around the three parallel helices  and one loop from the domain II. Kinetic analysis showed that the dimeric AbIDH2 showed much lower catalytic efficiency (0.39 μM−1·s−1) as compared to the typical monomeric IDHs (~15 μM−1·s−1). Key residues crucial for dimer formation were simultaneously changed to generate the mutant mAbIDH2. The disruption of the hydrophobic forces disassociated the dimeric AbIDH2, making mAbIDH2 a monomeric enzyme. mAbIDH2 sustained specific activity (21.9 ± 2 U/mg) comparable to AbIDH2 (25.4 ± 0.7 U/mg). However, mAbIDH2 proved to be a thermolabile enzyme, indicating that the thermostable dimeric AbIDH2 may have a physiological significance for the growth and pathogenesis of A. baumannii. Phylogenetic analysis demonstrated the existence of numerous AbIDH2 homologous proteins, thus expanding the monomeric IDH protein family.Monomeric isocitrate dehydrogenases (IDHs) have a single polypeptide sizing around 85 kDa. The IDH2 from the opportunistic bacterium  Two types of IDHs exist in nature according to the coenzyme specificity: NAD+-dependent IDHs  and NADP+-dependent IDHs . NADH generated by NAD-IDHs is the main component of energy metabolism, while NADPH generated by NADP-IDHs is an important source of reducing power. NADPH also serves as a defense against oxidative damage in vivo [Isocitrate dehydrogenase (IDH) catalyzes the NAD(P) in vivo .Four kinds of IDHs with different oligomeric states have been found in nature: monomeric IDHs, homo-dimeric IDHs, homo-tetrameric IDHs, and hetero-oligomeric IDHs ,5,6. Whe+, and they cannot use NAD+ for catalysis [+-specific monomeric IDHs identified from Campylobacter species, thus expanding the phylogenesis of the IDH protein family since both NAD+ and NADP+ specific enzymes exist among all the three IDH subfamilies [Acinetobacter species and Mycobacterium species. A previous work demonstrated that the IDH2 (741 aa) from Mycobacterium tuberculosis (MtIDH2) forms a stable dimer in solution [Acinetobacter baumannii (AbIDH2) to 3.0 Å. AbIDH2 is a homodimer, formed through the interactions between the two monomers’ surfaces. To our knowledge, AbIDH2 is the first dimeric structure reported to have originated from the typical monomeric IDH protein family.Monomeric IDHs have been characterized from various bacteria. It was once recognized that monomeric IDHs are highly specific to NADPatalysis ,10,11. Wfamilies . Interessolution . This inE. coli expression system and then purified through metal affinity chromatography  or Mn2+ (20.6 ± 3.1 μM), its catalytic efficiency for isocitrate was also 8-fold higher in the presence of Mn2+ compared to achieved that when Mg2+ was used as the cofactor (Kinetic characterization of the recombinant AbIDH2 demonstrated that its apparent ectively . No NAD+ing Mg2+ . Althougcofactor .+ (94 μM) when compared to the typical monomeric IDHs, such as Azotobacter vinelandii IDH (AvIDH) (5.8 μM), Corynebacterium glutamicum IDH (CgIDH) (4 μM), and Streptomyces lividans IDH (SlIDH) (2.4 μM) [−1·s−1) as compared with those of the well-characterized monomeric IDHs, such as AvIDH (15.9 μM−1·s−1), CgIDH (22 μM−1·s−1), and SlIDH (9.6 μM−1·s−1) [Escherichia coli IDH (4.7 μM−1·s−1), human cytosolic NADP-IDH (2.7 μM−1·s−1), and yeast cytosolic NADP-IDH (2.5 μM−1·s−1) [+ associating sites are conserved between AbIDH2 and monomeric IDHs  ,15,16. TM−1·s−1) ,15,16. FM−1·s−1) ,18. PrevM−1·s−1) ,19. DuriM−1·s−1) . Since tric IDHs , the douThe crystal structure of the substrate/coenzyme-free AbIDH2 was determined at 3.0 Å resolution in space group P4222 with two protomers associated in a dimer per asymmetric unit A. Each s+), yielding a higher RMSD value of 2.07 and 2.81 Å, respectively  to that of the wild-type enzyme (25.4 ± 0.7 U/mg). An analytical gel-filtration experiment demonstrated that the systematic mutations disrupted the AbIDH2 dimeric association, leading to the overwhelming presence of the monomeric mAbIDH2 in solution . These rA. baumannii could bear a much higher temperature than that needed for normal growth. Furthermore, A. baumannii could enter a viable nonreplicating state when exposed to the harsh environments where nutrients were low [A. baumannii proteins are thermostable in general or if it is a special trait of some few, like AbIDH2. In the latter case, it will be necessary to investigate whether AbIDH2 would contribute to the survival of the bacteria in unfavorable environments.We further compared the thermostability of the mutant and wild-type enzyme. The monomeric mAbIDH2 showed much worse stability as compared to the wild-type dimeric AbIDH2, demonstrated by the heat-inactivation study in which incubation at 42.5 °C for 20 min caused a dramatic 95% loss of mAbIDH2 activity while no activity loss was observed for AbIDH2 . Furtherwere low ,25. The A. baumannii (AbIDH2)  . The cryMycobacterium tuberculosis (MtIDH2), has also been biochemically identified as a homodimer composed of two subunits with a size of 86 kDa [Numerous IDHs with a size of around 85 kDa were grouped together with AbIDH2 during the rebuilding the phylogenetic tree of the type III IDH subfamily . As AbIDf 86 kDa . ConsistOur study suggested that both type III monomeric IDHs and dimeric IDHs are widely distributed in bacteria. Monomeric IDHs have been demonstrated to be the most efficient IDHs among the ancient IDH protein family ,11. The AbIDH2 gene from A. baumannii was cloned into pET28b . Plasmid was then transformed into E. coli BL21 (DE3) and the recombinant protein was overexpressed. The transformed cells were grown on Luria-Bertani (LB) agar plate containing 100 μg mL−1 kanamycin. A single colony was picked and grown in LB medium containing 100 μg mL−1 kanamycin until OD600 reached 0.6 at 37 °C. After induction by 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG), the cells were further grown for 20 h at 22 °C and harvested by centrifugation. E. coli BL21 cells were resuspended in lysis buffer containing 50 mM NaH2PO4 pH 7.5, 300 mM NaCl, and then disrupted by sonication. The recombinant AbIDH2 with 6× His-tag on its N-terminus was purified using BD TALON Metal Affinity Resin  according to the manufacturer’s instructions. Protein was further purified by gel filtration on a Hi-Load 16/60 Superdex 200 column . The major peak fractions were collected and concentrated for crystallization. Mutations were introduced into AbIDH2 by overlap extension, PCR-based, site-directed mutagenesis. Mutant AbIDH2 was expressed and purified as the wild-type AbIDH2.A full-length 2PO4 pH 7.5, 300 mM NaCl. Protein molecular mass was evaluated from a calibration curve using ovalbumin (45 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa).Analytical gel-filtration experiments were performed with a HiLoad™ 10/300 Superdex 200 column (Amersham Biosciences). A 1.5-mL aliquot of purified proteins were eluted using 50 mM NaHg. The test mode was based on the sedimentation velocity, and a continuous c(s) distribution was applied in the analysis mode. The derivative profiles were used to calculate the experimental sedimentation coefficient (sexp). The data were also analyzed using the Svedberg program [http://bbri.org/RASMB/rasmb.html) was used to calculate the partial specific volume (v2), solvent density (ρ), and viscosity (η). The corrected coefficient, s20,w, was calculated using the following equation: s20,w = sexp  /(1 − ρ·× v2).Analytical ultracentrifugation (AUC) analysis was conducted with a Beckman XL-A analytical ultracentrifuge, using a four-hole An-60 Ti rotor and a test time interval of 3 min. The reference loading volume was 410 μL, and the sample loading volume was 400 μL. In a typical experiment, 280 absorbance profiles were recorded at 20 °C and 45,000  program . The Sed2 or MnCl2, 1.5 mM DL-isocitrate, and 1.0 mM NADP+. An NADPH increase was detected at 340 nm with a thermostated Cary 300 UV-Vis spectrophotometer , using a molar extinction coefficient of 6.22 mM−1·cm−1. One unit of enzyme activity was defined as the reduction of 1 µM of NADP+ per minute. Protein concentrations were determined using the Bio-Rad protein assay kit  with bovine serum albumin as the standard. The Michaelis constant (Km) values of the wild-type and mutant enzymes for NAD+ and NADP+ were measured by fixing the isocitrate concentration at 1.0 mM with varying cofactor concentrations. Apparent maximum velocity (Vmax) and Km values were calculated by nonlinear regression using Prism 5.0 . All kinetic parameters were obtained from at least three measurements.IDH activity assays were carried out at 25 °C in buffer containing 35 mM Tris-HCl (pH 7.5), 2 mM MgCl2, and then were flash-frozen at 100 K in a nitrogen gas stream in the cryoprotectant consisting of the mother solution supplemented with 30% ethylene glycol. The crystals diffracted to 3.0 Å resolution and the data were collected at beam line 17U1 and 19U1 of the Shanghai Synchrotron Radiation Facility. The atomic coordinates of the C. glutamicum IDH (CgIDH) (PDB ID 2B0T) were used as the search model for the molecular replacement method by program PHASER [The recombinant AbIDH2 was purified and then concentrated to 10 mg/mL for crystal growing. Protein crystals were acquired by the vapor diffusion using the mother liquid consisting of 0.1 M HEPES pH 7.5, 14% 2-propanol, 20% glycerol, 10% PEG4000, and 40 mM MgClm PHASER . The modm PHASER . The strm PHASER . The cooThe structure-based amino acid sequence alignment was performed by using the Clustal X program and ESPript 3.0 web tool ,31. The


Plant anthocyanin biosynthesis is well understood, but the regulatory mechanism in purple foliage tea remains unclear. Using isobaric tag for relative and absolute quantification (iTRAQ), 815 differential proteins were identified in the leaves of Zijuan tea, among which 20 were associated with the regulation of anthocyanin metabolism. We found that the abundances of anthocyanin synthesis-related enzymes such as chalcone synthase, chalcone isomerase, dihydroflavonol 4-reductase and anthocyanin synthetase, as well as anthocyanin accumulation-related UDP-glucosyl transferase and ATP-binding cassette (ABC) transporters in the purple leaves were all significantly higher than those in the green leaves. The abundances of the transcription factors bHLH and HY5, regulating anthocyanin biosynthesis at transcriptional level were also obviously higher in purple leaves than those in green leaves. In addition, bifunctional 3-dehydroquinate dehydratase and chorismate mutase in purple leaves were distinctly higher in abundance compared to green leaves, which provided sufficient phenylalanine substrate for anthocyanin synthesis. Furthermore, lignin synthesis was found to be reduced due to the lower abundances of cinnamoyl-CoA reductase 1, peroxidase 15 and laccase-6, which resulted in increase of intermediates flow into anthocyanin synthesis pathway. The physiological data were consistent with proteomic results. These four aspects of biosynthetic regulation contribute to anthocyanin accumulation in purple leaves of Zijuan tea. Anthocyanins are natural water-soluble pigments belonging to the family of flavonoids. Thus far, more than 250 anthocyanins have been identified in plants . Anthocy−1 Dry Weight (DW) in Zijuan tea (Camellia sinensis var. kitamura), which is 10-fold higher than that of an ordinary tea  propanesulfonate (CHAPS), and 40 mmol·L−1 dithiothreitol (DTT)) in a water bath at 37 °C for 2.5 h. The homogenate was centrifuged at 17,000× g for 15 min at room temperature. The supernatant was collected, and the protein concentration was measured in accordance with a previously described procedure [Total protein concentration of the tea leaves was extracted in accordance with a previously described phenol extraction method . Proteinrocedure .−1 enzyme liquid was added to 100 µg of protein sample on the basis of trypsin concentration : protein = 1:20. The sample was incubated at 37 °C for 4 h. This step was repeated twice. After hydrolysis occurred, the resulting peptides were dried through vacuum centrifugation. The peptides were then re-dissolved in 0.5 mol·L−1 tetraethyl ammonium bromide. One unit of iTRAQ label was thawed at room temperature and reconstituted in 70 μL of isopropanol. Peptides from green and purple leaves were labeled with iTRAQ tag, respectively. The peptide samples were incubated for 2 h, pooled together, desalted using Sep-Pak Cartridge , and dried through vacuum centrifugation until use.After reductive alkylation was completed, 1 µg·µLv/v) acetonitrile (ACN) containing 10 mmol·L−1 KH2PO4, pH 2.8. The samples were gradiently eluted at a rate of 200 μL·min−1 with buffer A for 10 min, with 0–35% buffer B (25% (v/v) ACN containing 10 mmol·L−1 KH2PO4 and 350 mmol·L−1 KCl, pH 2.8) for 30 min, and with 35–80% buffer B for 2 min. Elution was monitored at an absorbance of 214 nm, and 30 fractions were collected. Each fraction was desalted in a Strata X column and dried through vacuum centrifugation.The peptide samples were separated using a polysulfoethyl SCX separation column (2.1 mm × 100 mm) in a Shimadzu LC-20AB liquid phase system . The peptide samples were re-dissolved in buffer A (25% (−1 with buffer A (2% (v/v) ACN and 0.1% (v/v) formic acid) for 15 min. Afterward, each eluted fraction was separated at a rate of 200 nL·min−1 with 5% (v/v) buffer D (98% (v/v) ACN and 0.1% (v/v) formic acid) for 1 min, a linear gradient of buffer D from 5% (v/v) to 35% (v/v) for 65 min, a linear gradient of buffer D from 35% (v/v) to 80% (v/v) for 5 min, and 80% (v/v) buffer D for 5 min.A solution of each fraction was injected into a 20AD HPLC system  combined with the liquid phase of the mass spectrometer and equipped with Micromass C18 column  for LC-MS/MS analysis. Each fraction (2.25 g) was eluted at a rate of 2 μL·minThe mass spectrum data were collected using TripleTOF 4600  with Nanospray III source (AB SCIEX) as an ion source and a radiator of a specific spraying needle  composed of a quartz material. The instrument of TripleTOF 4600 was run with peptide mass fingerprint recognition mode enabled, with the following parameter sets: electrospray ionization source, 2.5 kV; nitrous pressure, 30 psi; atmospheric pressure, 15 psi; outlet temperature, 150 °C; scan modes with a reflection and pulse frequency, 11 kHz; inspection frequency, 40 GHz; and normalized collision energy, 35 ± 5 eV; with at least twice the collision frequency of the same parent ion for 18 s.MS/MS data were analyzed using MASCOT  and compared with those in National Center for Biotechnology Information non-redundant protein sequences (NCBI-nr) database (release date: 2010 07 01) and Viridiplantae (Green Plants) databases. Data were also searched from the transcriptome database of Zijuan tea by using MASCOT. The engine parameters of MASCOT were set as follows: search type, MS/MS; threshold of ion score cutoff, 0.05 (with 95% confidence); MS/MS fragment mass tolerance, ±0.1 Da; enzyme-peptide tolerance in trypsin, 0.05 Da; monoisotopic mass values; peptide charge states, +2 and +3; and unrestricted protein mass.p < 0.05) in the two groups. Differentially expressed proteins were then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses.The relative intensities of the reported ions were used to determine the abundance of proteins in two groups, namely, green leaves (group G) and purple leaves (group P). Group G was used as a reference in Isobaric Labeling Multiple File Distiller and Identified Protein iTRAQ Statistic Builder. Proteins were considered differentially expressed when they exhibited more than 1.5-fold or less than 0.6-fold change in abundance  was mixed with 1.3 mL of 95% ethanol, 6 mL of 5% (v/v) vanillin–(hydrochloric) acid, and 0.2 mL of 20 g·L−1 ammonium iron (III) sulfate solution in 20 mol·L−1 HCl and incubated at 95 °C for 1 h. The absorbance of the mixture was determined at 546 nm. The proanthocyanidin concentrations in the sample were calculated on the basis of a previously established standard curve showing the correlation between proanthocyanidin concentrations and absorbances.Proanthocyanidin concentrations were determined by the method of Skerget et al.  with min−1 hydrochloric acid at 60 °C for 30 min. The extracts were further diluted with 0.1 mol·L−1 hydrochloric acid and ethanol to obtain a final volume of 25 mL. The absorbance of the extracts was determined at 530, 620 and 650 nm using a solution containing 0.1 mol·L−1 hydrochloric acid and ethanol as a blank control, and finally, the concentrations anthocyanin in the leaves were calculated [Anthocyanin concentrations were measured using the previously stated method described by Zheng et al.  with slilculated .v/v) ethanol at 80 °C for 10 min. The homogenate was centrifuged at 10,000× g for 10 min, and the supernatants were discarded. These steps were repeated thrice. The pellet was washed three times with ethanol and n-hexane (v:v = 1:1) and dried at 50 °C until absorbance analysis. The pellet was solubilized in 0.5 mL of 25% (v/v) acetyl bromide at 70 °C for 45 min. 0.9 mL of 2 mol·L−1 NaOH, 5 mL of acetic acid, and 0.1 mL of 7.5 mol·L−1 hydroxyamine hydrochloride were sequentially added to the resulting solution. The mixture was homogenized and centrifuged at 10,000× g for 10 min. The supernatants were collected; 2.9 mL acetic acid was added to 0.1 mL of the collected supernatant. Absorbance was determined at 280 nm, and the relative lignin concentrations were measured at A280·g−1 DW.Lignin concentrations were measured based on a method reported previously with slight modifications . The fre2 with a mortar and pestle, and then homogenized with 5 mL Na2HPO4/KH2PO4 buffer (pH 7.0) including 5 mmol·L−1 β-mercaptoethanol, 5% (w/v) polyvinyl-polypyrrolidine (PVPP), 50 mmol·L−1 ascorbic acid and 0.05% (v/v) Triton X-100. The homogenate was centrifuged at 20,000× g for 20 min at 4 °C. The supernatant was collected as the enzyme extract to measure the activities of phenylalanine ammonialyase (PAL), chalcone isomerase (CHI) and UDP-glycosyl transferase (UGT) in accordance with the protocol described by Huang et al. [Fresh leaf samples (1 g) were ground into powder using liquid Ng et al. . The act−1 Tris-buffered saline (TBS), 0.1% (v/v) Tween-20, pH 7.6). Afterward, the membranes were incubated with secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG) and immunoreactive bands developed with 3,3′-diaminobenzidine and quantified using software Image J  and three independent experiments were performed.Western blot was conducted to assess the different expression levels of CHS protein between green and purple leaves. Approximately 90 μg of protein samples was loaded onto 12% SDS-PAGE, processed through electrophoreses for 3 h, and transferred to polyvinylidene fluoride (PVDF) membranes  in accordance with a previously described method . Ponceau−1), and 3.6 μL of DNase-free water. Amplification was conducted using two-temperature cycling protocols with 30 s at 95 °C followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. After the completion of each amplification, melting curve analysis was performed with temperature ramping from 65 to 95 °C (rising by 0.5 °C per step with a 5 s rest) to confirm the specificity of the PCR. Three independent replicates of the samples were prepared. The relative expression level of each gene of the corresponding protein was normalized to Actin gene. A relative quantitative computing method (2Ct−ΔΔ method) was used to quantify gene expression [A modified cetyl trimethyl ammonium bromide (CTAB) method was employed to isolate RNA from the green and purple leaves of Zijuan tea . After tpression .p value less than 0.05 was considered statistically significant.Statistical analyses were performed using ANOVA to determine significant differences among group means. Data from three independent replicates of each treatment were presented as means ± standard deviation (SD). A PAL, CHS, CHI, DFR and ANS in purple leaves, were high due to activation of the transcription factors bHLH and HY5. This led to the high abundance or activity levels of their relative proteins (enzymes) which resulted in enhanced anthocyanin synthesis and accumulation in the cell vacuoles of the purple leaves. Furthermore, anthocyanin monomers can also be modified through glycosylation to form anthocyanins that accumulate in cell vacuoles through the regulation of UGT and ABC transporters in cells. A specific regulatory mechanism in anthocyanin accumulation was found in the leaves of Zijuan tea. The higher abundance levels of DHQ and CM implied that sufficient phenylalanine substrate was supplied for anthocyanin synthesis. Lignin synthesis was also reduced while CCR, POD and LAC abundances were decreased, which indicated that numerous intermediate metabolites flow to the anthocyanin synthesis pathway. This study proposed a potential regulatory network of anthocyanin metabolism in Zijuan tea.The regulations of anthocyanin biosynthesis were studied based on changes in protein abundances between the purple and green leaves of Zijuan tea using iTRAQ and physiological and biochemical analyses . The tra


The unusual structure Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° exhibits a high thermoelectric performance ZT = 1.61 at room temperature. The relatively high ZT value in contrast to that of previously reported Bi1.5Sb0.5Te3 materials and the Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 60° or 90° evidently reveals the crucial role of the unique interface and tilt-structure in favorably influencing carrier and phonon transport properties, resulting in a significantly improved ZT value. This method opens a new approach to optimize nano-structure film materials.The uniquely tilted nanopillar array favorably influence carrier and phonon transport properties. We present an innovative interfacial design concept and a novel tilt-structure of hierarchical Bi ZT = (S2σ/κ)T, where σ, S, κ, and T are electrical conductivity, Seebeck coefficient, thermal conductivity, and absolute temperature, respectively3. There exists a strong coupling of TE parameters κ, σ and S. Many research efforts to overcome the conventional κ-σ and σ-S trade-off have been made in attempts to obtain a high ZT value during recent years7. Theoretical and experimental analyses have shown that the low-dimensional structure can significantly optimize the transport properties of electrons and phonons, which are to break through the limitation of the electron-phonon coupling and provide an effective pathway past a low-dimensional structure material, such as, a ZT value of 3 was reported for the PbSeTe/PbTe quantum dot superlattice, and the record high ZT value of 2.4 was achieved for the Bi2Te3/Sb2Te3 superlattice. However, One-dimensional nanowires are predicted to exhibit a better TE property than superlattices10.Thermoelectric (TE) materials can directly interconvert thermal energy and electrical energy by on the basis of Seebeck effect and Peltier effect. TE energy conversion efficiency is determined by 2Te3 and its alloy are the best TE materials near room temperature, which are anisotropic with a layered structure. Their thermal and electrical conductivities along the a-axis (in the c-plane) are approximately two and four times higher, respectively, than those along the c-axis of Bi2Te3-based materials. But Seebeck coefficients are less dependent on the crystallography14. Therefore, an improved ZT value can be expected when utilizing the anisotropic thermal and electrical transport properties. Our previous results show that the hierarchical Sb2Te3 pillar array structure can selectively scatter phonon more than carrier, leading to a high ZT value15. In addition, we also find that unique nanowire array structuring can induce a change of the Fermi level of the Bi23 and favorably influence the phonon and carrier transport, thus dramatically enhancing a ZT result16. The previous studies have witnessed the feasibility of controlling novel microstructures to modify TE properties of Sb2Te3 and Bi2Te3-based alloys. However, it is noted that the hierarchical Sb2Te3 pillar array and the Bi23 nanowire array are grown perpendicular to the substrates and possess relatively high densities of interspaces, which degrade the in-plane transport properties to some extent. Stranz and Sun reported that a wafer-scale vertical nanopillar arrays or nanowire arrays can be realized by lithography and anisotropic etching for improving the performance of TE cross-plane devices as proposed recently18. But it is hardly to overcome a problem of carrier and phonon transport along the in-plane direction and measure cross-plane TE properties for vertical nanopillar arrays or nanowire arrays films. These adverse factors need to be further improved in films, enabling films to show better in-plane properties. Some vertically aligned nanowire arrays or nanopillar arrays have been synthesized by the electrochemical deposition with templates or the anisotropic etching and lithography method, however, this kind of hierarchical nanopillar arrays with tilt-structure have never been reported, let alone the tilted Bi1.5Sb0.5Te3 material. This motivates us to further explore the effect of tilt-structure on the hierarchical Bi1.5Sb0.5Te3 nanopillar arrays.Bi1.5Sb0.5Te3 nanopillar arrays based on the construction of one-dimensional nanowires. A simple thermal evaporation technique without using any templates was carried out, to our best knowledge, for the first time on the tilt-growth of hierarchical Bi1.5Sb0.5Te3 nanopillar arrays. The unusual multi-scale and multi-dimension structure Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° exhibits a stringkly high in-plane ZT = 1.61 at room temperature. It is believed that the interrelationship between the tilt-structure growth of films and the tilt angle of deposition substrates uncovered by this work may help to better understand unique tilt structuring of this kind of material. Furthermore, it provides a new avenue to control the structural configuration of materials with possible relevance to improvement of their properties.Hence, in this work, we aim to control the tilt angle of hierarchical Bi1.5Sb0.5Te3 nanopillar arrays with tilt angles of 45°, 60°, and 90° were studied by SEM, respectively. The SEM images  of the Bi1.5Sb0.5Te3, was obtained in hierarchical nanopillar arrays samples. A preferential orientation (0 1 5) peak was mainly observed in all nanopillar arrays. The intensity of (1 0 10) texture of the hierarchical nanopillar array with a tilt angle of 45° is dramatically strong. When the tilt angle becomes large to 60°, the intensity of (1 0 10) peak of the hierarchical nanopillar array becomes weak. Finally, the peak is disappeared in the hierarchical nanopillar array with a tilt angle of 90°. This seems to indicate that the tilt angles of the nanopillars to the substrate are associated with the (1 0 10) peak of nanopillar arrays. The atom lateral mobility increase on the surface due to a decrease in the angle of the deposition direction to the substrate plane may be responsible for the structure change. The growing grains can be sufficiently mobile to migrate to the preferred sites for crystallization growth.In order to gain insight into the crystal structure, nanopillar arrays were examined by XRD. Figure 1.5Sb0.5Te3 molecular structure was built through Vestal to further explore the deposition mechanism of the Bi1.5Sb0.5Te3 films. Figure 1.5Sb0.5Te3 molecular. The structure of Bi1.5Sb0.5Te3 is similar to that of Bi2Te3. In Bi1.5Sb0.5Te3 crystal, a quarter of Bi sites are occupied by Sb atoms. The (0 1 5) and (1 0 10) preferential orientations are depicted in the structure. As seen from Figs 1.5Sb0.5Te3 nanopillar arrays. Thin films of PVD deposited materials normally grow with the plane of highest atomic density or the plane with minimum surface free energy21. The (0 1 5) and (1 0 10) planes perhaps have relatively low surface free energy. As can be seen from Fig. 1.5Sb0.5Te3 films.Then the simulated model of Bi1.5Sb0.5Te3 nanopillars with a tilt angle of 45° varies continuously from the nanowire surface to the unique interface shown in Fig. The details of the specially hierarchical nanopillar array microstructure are observed in TEM and HRTEM images, as depicted in Fig. 1.5Sb0.5Te3 nanopillar arrays with tilt angles of 45°, 60° and 90° will be proposed and further investigated, respectively. The growth process of nanopillar arrays are illustrated in Fig. 22. Three primary modes of thin-film growth include: (1) Volmer-Weber (3D island formation), (2) Frank-van der Merwe (2D layer-by-layer), (3) Stranski-Krastanov (layer-plus-island), Stranski-Krastanov growth is an intermediary process characterized by both 2D layer and 3D island growth. Theoretically, neglecting the strain energy of films, the growth modes of thin films are determined by the free energy of the substrate surface (σs), the surface free energy of the heteroepitaxial layer (σh), and the interface free energy (σi). When the angle of the substrate plane to the horizontal plane is about 0°, that is, the substrate parallels to the horizontal plane, and the deposition direction is approximatively vertical to the substrate. In the initial nucleation stage, the inequality σs <σi + σh sets the condition for the epitaxial film, no wetting the substrate, which represents the Volmer-Weber growth. Thermal evaporation produces relatively high energy depositing particles due to which enhanced number of nucleation sites is produced, leading to fine grains and small island growth. Subsequently, a second nucleation and growth process happened on the already formed 3D islands, and the preferential direction of 3D islands is (0 1 5) (SEM images of (0 1 5)-preferential 3D islands are provided in Supporting Information, Figure 1.5Sb0.5Te3nanopillar array with hierarchical structure, which is similar to our previous result for the vertical Sb2Te3 pillar array with hierarchical structure15. With increasing the angle between the substrate plane and the horizontal plane to 30°, the angle of the deposition direction to the substrate plane is 60°. In this case, the deposited atoms or ions can obtain some energy for slightly lateral movement on the substrate surface, being equivalent to increase σs. The inequality σs >σi + σh sets the condition for the epitaxial film to wet the substrate. The 2D layer growth occurs and the seed crystal layer is formed. Next, atoms or ions are deposited on the surface of the seed crystal layer. As the layer thickness increases, the accumulated thermal stress and strain energy contribute to σi. Then, the tilted 3D island growth occurs (σs <σi + σh). The growth process happened on the already formed seed crystal layer, and the oriented direction of the seed crystal layer and tilt-structure 3D islands is [0 1 5] and [1 0 10]. Subsequently, a second nucleation and growth process happened on the already formed tilt-structure 3D islands. Under this condition, the tilt-structure crystal growth in the radial direction is faster than that of the planar direction. Thus, under the induced effect of (0 1 5) and (1 0 10) lattice planes, the key for fabricating the hierarchical Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 60° is to keep the relative high deposition rate to grow in the radial direction. Therefore, the angle of 60° between the deposition direction and the substrate plane is necessary experiment condition for fabricating the hierarchical Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 60°. When the angle between the substrate plane and the horizontal plane increases to 45°, that is, the angle of the deposition direction to the substrate plane is 45°  and (1 0 10) lattices and tilt-structure of nanopillar arrays provides the fastest channels for carriers transport. This gives a reasonable explanation why the carrier mobility and electrical conductivity were enhanced in the nanopillar array with a tilt angle of 45°.It is of considerable interest to be able to tune electronic transport properties by modifying the morphology and crystal structure of films. The in-plane electrical conductivities of hierarchical Bi1.5Sb0.5Te3 nanopillar arrays with tilt angles of 45°, 60° and 90° are presented, respectively, which are positive values for all samples, indicating a p-type semiconductor. It shows that the highest Seebeck coefficient reaches to 268 μV K−1 for the hierarchical nanopillar array with a tilt angle of 45° at the temperature of 200 °C. An outstanding increase in the Seebeck coefficient is observed with a decrease in tilt angles for the hierarchical nanopillar arrays. The Seebeck coefficient value in the present work is much higher in comparison to the reported results of bismuth antimony telluride materials31. The reason for this greatly difference is the change in the microstructure compared to the reported structure. This tilt-structure can possibly promote the carrier mobility in the in-plane direction, leading to large Seebeck coefficients.In Fig. S2σ versus temperature for hierarchical Bi1.5Sb0.5Te3 nanopillar arrays is plotted in Fig. 1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° slightly decrease firstly and then increase with increasing temperature. It exhibits that a strikingly high average power factor of 5.76 mW/m·K2 was obtained for the unique tilt-structure film between 30–200 °C, along with the maximum power factor value of 5.86 mW/m·K2 at 30 °C, which also implies that the S2σ nearly remains constant with temperature. In contrast to Sb2Te3-based and Bi2Te3-based materials31, whose S2σ values increase or decrease as the temperature increases, the temperature stability of S2σ of the hierarchical Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° are useful in practical use. It is clearly demonstrated that the hierarchical Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° shows a largely enhanced power factor compared with the hierarchical nanopillar array with a tilt angle of 60° or 90° and previous results32. There is no doubt that the tilt architecture significantly aids in achieving high TE properties, especially for the hierarchical nanopillar array with a tilt angle of 45°. However, the novel structure material was further considered to have better TE properties due to the greatly reduce thermal conductivities originating from lots of interfaces, numerous interspaces, multi-scaled grains, etc.The power factor κ values. The κ of the hierarchical Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° is 1.09 W m−1 K−1 (see Table −1 K−1) due to the influence of the tilt-structure of nanopillar arrays. However, the presented thermal conductivities are lower than the reported results for Bi-Sb-Te materials33. The excellent phonon transport properties are attributed to the comprehensive effects induced by the multi-scaled grains, thin nanowires, nano-scaled open gaps, coherent grain boundaries, rough surfaces, surface dangling bonds, antisite defects as well as other defects. These factors together are responsible for scattering phonon with a variety of wavelengths, leading to low thermal conductivity in unique nanopillar arrays, as SEM and TEM microstructure above.The uniquely hierarchical film is composed of nanopillar arrays based on the construction of 1D nanowires, which is expected to be low in-plane ZT value was calculated at room temperature, as shown in Table ZT of the hierarchical Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° was 1.61, which is superior to that of the nanopillar arrays with tilt angles of 60° and 90° and the reported results of Bi-Sb-Te film and bulk materials34, and comparable with the value (1.86) of the bulk Bi1.5Sb0.5Te3 material as reported recently35. Tilt-structuring opens great opportunities for an effective modification of the interrelated TE transport properties at hierarchical nanopillar arrays with tilt growth, where the size effect36, energy filtering effect37, and preferential route effect can have a profound influence on the TE transport properties. The different behavior of the hierarchical nanopillar array with tilt-structure in terms of thermal and electrical conduction strongly suggests that the hierarchical Bi1.5Sb0.5Te3 nanopillar arrays promise to be a most efficient structure for TE devices. The results of this study provide insights for the structural design and synthesis of TE materials, which will be very important for the development of functional materials in the future.According to the measured thermal conductivity, electrical conductivity, and Seebeck coefficient, the 1.5Sb0.5Te3 powder in five different regions of each nanopillar array sample, as shown in Table 1.5Sb0.5Te3 films. Note that the carrier concentration of bismuth antimony telluride depends on acceptor-like SbTe (BiTe) defects and donor-like VTe vacancies. The Te-excess samples can suppress the generation of VTe vacancies under a Te-rich condition. We believe that slightly Te-excess samples can provide an improvement in hole carrier concentration. It can be seen that the carrier mobility and carrier concentration exhibit different among these nanopillar arrays. The concentrations of carriers are 4.2 × 1019 cm−3, 5.3 × 1019 cm−3 and 6.7 × 1019 cm−3 for hierarchical Bi1.5Sb0.5Te3 nanopillar arrays with tilt angles of 45°, 60° and 90°, respectively. These films possess the value of carrier concentration 1019 cm−3. It is found that the optimal carrier concentration for the best power factor is approximately 1019 cm−3 in Bi-Sb-Te materials38. Besides, it clearly exhibits that the concentrations of carriers decrease owing to the formation of a relatively perfect crystalline structure at a proper deposition condition. Here, the hierarchical Bi1.5Sb0.5Te3 nanopillar arrays with an angle of 90° obtains the relatively high value of the carrier concentration possibly due to the formation of lots of defects, which negatively effects on Seebeck coefficients. On the other hand, the ordered nanopillar array with a tilt angle of 45° has a high mobility of 130 cm2/V·s, which is prominently higher than that of the nanopillar arrays with tilt angles of 60° and 90° (94 cm2/V·s and 69 cm2/V·s), respectively, as shown in Table 1.5Sb0.5Te3 nanopillar arrays with a tilt angle of 45°. Therefore, it is easy to understand that the (0 1 5) and (1 0 10)-oriented structure can remarkably increase the carrier mobility and enhance power factors in nanopillar arrays. Taking advantage of optimization to structure, the enhanced TE performance has been achieved by the new avenue to adjust the carrier and phonon transport. It is believed that this direction of research will inspire a flurry of interest in exploring effective approaches to fabricate such hierarchical tilt-structure TE materials using scalable, simple, and controllable synthesis processes.In order to further verify novel tilt-structure nanopillar arrays playing a greatly important role to optimize TE transport properties, the mobility and concentration of carriers were examined at room temperature. The composition analysis by EDX was at first considered due to the composition of films affecting the carrier concentration. The result of EDX confirms that the atomic ratios are quite similar to those of the Bi1.5Sb0.5Te3 nanopillar arrays with tilt angles of 45°, 60° and 90° based on the construction of one-dimensional nanowires have been fabricated by a simple thermal evaporation technique. The unusual multi-scale and multi-dimension structure Bi1.5Sb0.5Te3 nanopillar array with a tilt angle of 45° exhibits the highest Seebeck coefficientof 268 μV K−1 at 200 °C, the maximum power factor value of 5.86 mW/m·K2 at 30 °C, the average power factor of 5.76 mW/m·K2 between 30–200 °C, along with the strikingly high thermoelectric performance ZT = 1.61 at room temperature. The strong preferred (0 1 5) and (1 0 10) lattices and tilt-structure of nanopillar arrays provides the fastest channels for carriers transport, enhancing TE properties in the nanopillar array with a tilt angle of 45°. With optimizing tilt angles, crystal orientation, nanowires diameter, nano-scaled open gaps, and coherent surfaces, the TE performance is likely to be better. It provides a new control over the structural configuration of materials with relevance to improvement of their properties.Uniquely hierarchical Bi1.5Sb0.5Te3 nanopillar arrays with tilt-structure can be prepared by a simple vacuum thermal evaporation technique. A sketch of the vacuum thermal evaporation setup is provided in Supporting Information, as shown in Figure 1.5Sb0.5Te3 nanopillar arrays with tilt angles of 45°, 60°, and 90° on SiO2 glass substrates by the thermal evaporation technique, the corresponding angles between the substrate holder plane and the horizontal plane are supposed to be about 45°, 30°, and 0°, respectively. Due to Te deficiency easily occurred in Bi2Te3-based films, Te and Bi1.5Sb0.5Te3 powders  were put on the evaporating dish, and the evaporated current was 165 A for all hierarchical nanopillar arrays. Before deposition, the SiO2 substrate was at first cleaned by diluted nitric acid, and then acetone, and dried under the nitrogen gas flow. Next, the SiO2 substrate was loaded onto the sample holder. In ordered to remove oxygen, N2 gas was introduced into the chamber and vacuumized three times. The deposition temperature was set at 300 °C, the working pressure was maintained at 2 × 10−6 Torr in the film deposition process. The thickness of films could be controlled by changing the deposition time in our experiments.In this work, the hierarchical Biα radiation (λ = 0.154056 nm). A field emission scanning electron microscope  equipped with an energy dispersive x-ray spectroscope (EDX) were used to investigate the morphology and composition of all samples. The crystal structure of Bi1.5Sb0.5Te3 nanopillar array was performed with the aid of a high-resolution transmission electron microscope (HRTEM) (Tecnai G2 F20S-Twin). The film thickness was measured by surface profilometry . The Seebeck coefficients and electrical conductivities of all films were simultaneously examined by ZEM-3 . A Laser PIT  was used to collect the in-plane thermal conductivity data at room temperature. The principle of the measurement method for the thermal conductivity is described in ref.39. The carrier mobility and carrier concentration were determined by a four-probe method using Hall effects measurement system (ECOPIA HMS-3000) at room temperature. All tests for transport properties were repeated at least 5 times. The errors are 5% for thermal conductivities, 4% for electrical conductivities, 5% for Seebeck coefficients, and 10% for ZT values.The crystal structure characterizations for the nanopillar arrays were measured using x-ray diffraction (XRD) on a Rigaku D/MAX 2200 PC automatic x-ray diffraction with Cu KTilt-structure and high-performance of hierarchical Bi1.5Sb0.5Te3 nanopillar arrays


Aedes albopictus is the dominant Aedes mosquito species and the main vector of dengue in China. Epidemiologically, dengue mainly occurs in Guangdong Province; it does not occur or rarely occurs in other areas of mainland China. This distribution may be associated with climate, mosquito density, and other factors in different regions; however, the effect of temperature on the vector competence of Ae. albopictus for dengue viruses (DENV) remains unclear. In this study, Ae. albopictus was orally infected with dengue virus 2 (DENV-2) and reared at constant temperatures  and a fluctuating temperature (28–23–18°C). The infection status of the midguts, ovaries, and salivary glands of each mosquito was detected by polymerase chain reaction (PCR) at 0, 5, 10, and 15 days post-infection (dpi). DENV-2 RNA copies from positive tissues were quantified by quantitative real time PCR (qRT-PCR). At 18°C, DENV-2 proliferated slowly in the midgut of Ae. albopictus, and the virus could not spread to the salivary glands. At 23 and 28°C, DENV-2 was detected in the ovaries and salivary glands at 10 dpi. The rates of infection, dissemination, population transmission, and DENV-2 copies at 28°C were higher than those at 23°C at any time point. At 32°C, the extrinsic incubation period (EIP) for DENV-2 in Ae. albopictus was only 5 dpi, and the vector competence was the highest among all the temperatures. Compared with 28°C, at 28–23–18°C, the positive rate and the amount of DENV-2 in the salivary glands were significantly lower. Therefore, temperature is an important factor affecting the vector competence of Ae. albopictus for DENV-2. Within the suitable temperature range, the replication of DENV-2 in Ae. albopictus accelerated, and the EIP was shorter with a higher temperature. Our results provide a guide for vector control and an experimental basis for differences in the spatial distribution of dengue cases.Dengue is a mosquito-borne disease that has been an epidemic in China for many years.  Dengue virus (DENV) is a mosquito-borne flavivirus that can cause a series of diseases, including dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) . It has Temperature is an important factor influencing the ecological habits, vector competence, and extrinsic incubation period (EIP) of mosquitoes for DENV . CompareAedes aegypti and Aedes albopictus . Eggs in de-chlorinated water were hatched to larvae. One hundred larvae per liter of water were fed turtle food with 0.1 g each day for the first 4 days and then 0.2 g each day until they pupated. Pupae were transferred to cages (20 cm × 20 cm × 35 cm). After emergence, adults were provided with 10% glucose solution or defibrinated sheep blood  for egg production.2 culture flask were inoculated with DENV-2 at a multiplicity of infection (MOI) of 1. After gentle shaking for 15 min, the culture flask was incubated at 37°C, 5% CO2 for 2 days until obvious cytopathic effects. The supernatant was harvested after centrifugation at 1,500 × g for 5 min, separated into 0.5-mL aliquots, and frozen at -80°C.Dengue virus 2  was provided by the Key Laboratory of Tropical Disease Control of Sun Yat-sen University . C6/36 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and maintained at 28°C. Cells grown in a 75-cm50) (5 cells per well and cultured at 28°C for 24 h. The control solution (RPMI-1640 containing 2% FBS) and 10-fold viral dilutions (10-1–10-11) were inoculated into C6/36 cells in eight wells. The microtiter plate was placed at 37°C, 5% CO2 for 2 h, and then the culture per well was replaced with RPMI-1640 supplemented with 2% FBS. The plate was incubated at 37°C, 5% CO2 until the cytopathic effect did not continue to increase. Results were calculated according to the Karber method (Dengue virus 2 titer was determined by 50% tissue culture infective dose (TCID50) . Brieflyr method .10TCID50/mL. The DENV-2 supernatant was collected and mixed with defibrinated sheep blood at a ratio of 2:1. The blood meal was maintained at 37°C for 30 min and transferred into a Hemotek blood reservoir unit . Five- to seven-day-old female Ae. albopictus mosquitoes were glucose starved for 12–24 h and allowed to feed on the infectious blood meal for 30 min. After anesthesia with CO2, fully engorged mosquitoes were removed and placed into 250-mL paper cups covered with gauze (10 mosquitoes/cup). Mosquitoes were placed in different HP400GS incubators  precisely set at 18, 23, 28, and 32°C. Another group of mosquitoes was placed at the fluctuating temperature . The control group was set for each temperature. DENV-2 stock solution was replaced with RPMI-1640 containing 2% FBS in the control group, the remaining steps were the same as mosquitoes engorged with the infectious blood meal. All treatments were maintained at 80% relative humidity and 16 h:8 h (light:dark) photoperiod, and all mosquitoes were fed 10% glucose solution.Mosquito infection was conducted in a Biological Safety Level 2 lab. Two days before infection, frozen DENV-2 stock was passaged once more through the C6/36 cells. The titer of the fresh virus was 7.375–7.875 logThe midgut, ovaries, and salivary glands of each mosquito from the abovementioned temperatures were dissected and detected at 0, 5, 10, and 15 days post-infection (dpi). The sample size collected from each temperature condition was 3–6 mosquitoes at 0 dpi and 10–16 mosquitoes at each additional time point. The experiment was independently repeated three to five times.TM Reverse Transcription System  were followed.The legs and wings of each mosquito were removed and washed three times in PBS. Disposable insect microneedles were used to separate the midgut, ovaries, and salivary glands of each mosquito under an anatomical lens. Tissues were washed three times in PBS droplets and then transferred to 50 μL of TRIzol  in 1.5-mL Eppendorf tubes. Total RNA was extracted according to TRIzol manufacturer’s protocol. cDNA was synthesized using a DENV-2-specific primer (5′-TGGTCTTTCCCAGCGTCAAT-3′), and the recommendations of the GoScriptPolymerase chain reaction (PCR) was used to detect DENV-2 in the tissues. A pair of primers was synthesized as described previously  . The tarAe. albopictus for DENV-2 transmission was evaluated by infection rate (IR), dissemination rate (DR), and population transmission rate (PTR), as follows .The amount of DENV-2 in the positive tissues of mosquitoes was further detected by absolute quantitative real-time PCR (qRT-PCR). The plasmid standard was constructed as previously described . In brie® selected master mix, 1 μL of each primer (10 μM), 2 μL cDNA or the plasmid standard, and 6 μL RNase-free water. The reaction was performed in the 7500 Real-Time PCR System as follows: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 1 min. Melting curves were given at 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. A standard curve was established by 10-fold dilutions of the plasmid standard (8.71 × 102–8.71 × 107). The result of qRT-PCR was ascertained by using non-template, negative (mosquito infected with C6/36 cells) and positive control (mosquito infected with DENV-2 at 0 dpi). Its minimum detecting amount is 87.1 copies/reaction of DENV-2. Each sample was conducted in three replicates, and the results were determined by the melting curve and cycle threshold values.The qRT-PCR reaction mixture per well contained 10 μL SYBRAe. albopictus for DENV-2 was analyzed using logistic regression. IR, DR, and PTR were separately compared at different temperatures or different time points. P-value was corrected by Bonferroni adjustments. Chi-square (and Fisher’s exact) tests were used to determine the difference in vector competence between 28°C and the fluctuating temperature (28-23-18°C). The amounts of DENV-2 in the tissues were log-transformed and then analyzed by post hoc Tukey’s LSD tests of variance analysis for the constant-temperature groups. Student’s t-test was used to compare the DENV-2 titer of the tissues between 28°C and (28-23-18°C). P < 0.05 was considered statistically significant.All statistical analyses were performed with SPSS 20.0 . Under constant temperature, the vector competence of Ae. albopictus females reared at 18, 23, 28, and 32°C were used to measure the rates of infection, dissemination, and transmission of DENV-2. At 0 dpi, the IRs of mosquitoes were 100% at all temperatures, indicating that all mosquitoes ingested the blood meal containing DENV-2 . After the infectious blood meal was digested, the IRs of Ae. albopictus at 18, 23, and 28°C were significantly reduced at 5 dpi compared with 0 dpi  and then gradually increased at 10 and 15 dpi . However, Ae. albopictus maintained an invariably high IR at 32°C, and no significant difference was present at any time point . At 5 dpi, the IR of Ae. albopictus was obviously lower at 18°C than that at the other three temperatures . IRs at 23 and 28°C showed no difference; however, they were lower than the IR at 32°C . At 10 and 15 dpi, IRs at 23, 28, and 32°C were not significantly different; however, they were higher than the IRs at 18°C .In this study, a total of 602 z = 12.085, 15.546, P < 0.01), and no difference occurred between 10 and 15 dpi . At 23 and 28°C, DENV-2 detected in the ovaries started 10 dpi, and the DR at 15 dpi was higher than that at 10 dpi . At 10 dpi, the DR arranged in descending order was 32, 28, and 23°C, and there were significant differences between any two temperatures . At 15 dpi, the DR was still lower at 23°C than that at 28°C  and 32°C ; however, no difference appeared between 28 and 32°C . No positive ovaries were detected at 18°C during the experiment .Dengue virus 2 was disseminated to the ovaries as early as 5 dpi at 32°C; however, the DR was low at this point. DRs at 10 and 15 dpi were significantly higher than those at 5 dpi . At 10 dpi, the PTR was lower at 23°C than those at 28 and 32°C , and the PTR at 28°C was lower than that at 32°C . Similarly, the PTR at 15 dpi was highest at 32°C among all temperatures. At 18°C, DENV-2 in the salivary glands was negative at all time points of the experiment .The infection status in the salivary glands was similar to that in the ovaries. Detection of DENV-2 in the salivary glands was 5 dpi at 32°C and 10 dpi at 23 and 28°C, and the PTR increased gradually over time  and fluctuating temperature (28–23–18°C) conditions. IRs at 0, 5, and 10 dpi showed no significant differences between 28 and 28–23–18°C; however, the IR at 28°C was higher than that at 28–23–18°C at 15 dpi  . Whether Ae. albopictus was reared at 28 or 28–23–18°C, positive ovaries and salivary glands were detected at 10 dpi. The DR at 10 and 15 dpi showed no difference between mosquitoes from constant and fluctuating temperature conditions . At 10 dpi, the PTR at 28°C was the same as that at 28–23–18°C. However, the PTR at 28°C was significantly higher than that at 28–23–18°C at 15 dpi  .We separately compared the IR, DR, and PTR of Ae. albopictus were further detected by qRT-PCR. At 0 dpi, DENV-2 titer showed no significant differences among all temperature groups . At 18°C, DENV-2 copies (log10) of the midguts decreased markedly from 0 (6.33 ± 0.77) to 5 dpi (4.17 ± 0.37) and then increased slowly over time. Until 15 dpi, the level of DENV-2 was still lower than at 0 dpi (P < 0.05) . Similarly, DENV-2 of the midguts at 23°C was lower at 5 dpi than that at 0 dpi, followed by a rapid recovery. The amount of DENV-2 at 10 dpi was close to that at 0 dpi, and no difference was found between 15 and 10 dpi (P > 0.05). At 28°C, DENV-2 showed a slow increasing trend in the midguts of Ae. Albopictus mosquitoes. DENV-2 copies (log10) at 10 and 15 dpi were higher than those at 0 and 5 dpi (P < 0.05); however, there was no difference between 0 and 5 dpi or between 10 and 15 dpi (P > 0.05). At 32°C, DENV-2 copies in the midguts increased rapidly to 8.18 ± 0.66 (log10) before 5 dpi and then trended toward stability .The amounts of DENV-2 in the midguts, ovaries, and salivary glands of Figure 3B). The viral titration (log10 copies) was 5.75 ± 0.83 at 5 dpi, and reached its peak at 10 dpi (7.34 ± 0.74). There was no significant difference between 10 and 15 dpi (P > 0.05). DENV-2 in the ovaries at 23 and 28°C started from 10 dpi. DENV-2 copies were lower at 23°C than those at 28°C; these values were significantly lower than those at 32°C by this time (P < 0.05) . After that, the virus in the ovaries at 28°C increased quickly from 10 to 15 dpi; however, it remained stable at 23°C . The variation trend of DENV-2 titer in the salivary glands was the same as in the ovaries . DENV-2 copies (log10) in the salivary glands at 32°C were higher than those at 23 and 28°C at any time point, and they were higher at 28°C than those at 23°C . DENV-2 was not detected in the ovaries or salivary glands at 18°C .Dengue virus 2 detected in the ovaries was the earliest at 32°C  . In the ovaries, the DENV-2 titer at 28 and 28–23–18°C showed no significant difference at 10 dpi, although it was higher at 28°C than that at 28–23–18°C at 15 dpi (P < 0.05) . In the salivary glands, DENV-2 copies (log10) at 28°C were obviously higher than those at 28–23–18°C at 10 and 15 dpi (P < 0.05) .The amounts of DENV-2 in the midguts, ovaries, and salivary glands of Ae. albopictus for the DENV under different temperatures would provide guidance for vector control. Our results showed that DENV-2 was confined to the midguts of Ae. albopictus at 18°C and could invade the salivary glands between 23 and 32°C. Shorter EIPs and higher transmission rates were detected with higher temperatures.In recent years, the epidemic area of dengue has been expanding with the acceleration of climate warming, trade globalization, and urbanization . MosquitThe lower critical value under which mosquitoes could not spread the virus was reported to be 18°C . In our Ae. albopictus mosquitoes and the amount of DENV-2 in the tissues were higher at 28 than those at 23°C except for 0 dpi. The titer of DENV was a key factor affecting mosquito infection. When the log10 plasma viremia level of DENV was between 4 and 10, the susceptibility of Aedes was enhanced with the increase of virus titer, and Ae. aegypti and Ae. albopictus could not be infected when the log10 plasma viremia level was below 4 . Whether DENV-2 can be transmitted through mosquito bites requires further study.The EIP at 23 and 28°C was consistent (10 dpi). However, the IR, TR, and PTR of  below 4 . At 23°CAe. albopictus was gradually shortened with rising temperature; therefore, more hosts would be infected by Ae. albopictus bites in the limited reproductive cycle. At 32°C, DENV-2 could spread to the ovaries and salivary glands of Ae. albopictus by 5 dpi. The annual average temperature was higher than 30°C from July to October in Guangzhou, and the high density of Ae. albopictus and the short EIP caused an outbreak of dengue during this season. In this study, the upper temperature limit was set at 32°C because pre-experimental results showed that the mortality rate of Ae. albopictus was extremely high and the mosquitoes would not survive until the end of the experiment.The EIP of DENV-2 in Ae. albopictus reared at constant (28°C) and fluctuating (28–23–18°C) temperatures. The results showed that, compared with the amount of DENV-2 in the salivary glands at 28°C, the amount of DENV-2 in the salivary glands at 28–23–18°C was significantly decreased. In China, dengue cases occur mainly in warm southern cities, and few cases are present in northern cities. In addition, Guangzhou and Shenzhen are both economically developed, densely populated cities in Guangdong Province; however, the number of cases of dengue was very different in 2014: with 96% of cases concentrated in Guangzhou. We speculated that diurnal temperature ranges in northern cities and Shenzhen belonging to coastal areas were large. These fluctuations affected the proliferation and spread of the DENV in mosquitoes.In addition, we compared the vector competence and DENV-2 titer of Ae. albopictus at different temperatures, a previous study showed that the vector competence of Ae. aegypti for different serotypes of DENV (DENV-1 or DENV-2) was similar under similar temperature conditions  to detect the vector competence of nditions . In addiZhL and X-GC designed the study. ZhL, ZZ, ZeL, TZ, ZJ, JG, and KW carried out data acquisition and analysis. ZeL wrote the paper. X-GC supervised the study. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AM and handling Editor declared their shared affiliation


The in vitro anticancer activities for these obtained compounds were evaluated against human cancer cell lines , which demonstrated that some of these heterocyclic pyrimidine derivatives exhibited significantly good cytotoxic activities against all tested cell lines compared with 5-fluorouracil (5-FU), especially, compound 3b exhibited high potential growth inhibitory activities against all tested cell lines with the IC50 values of 5.41 ± 1.34, 5.65 ± 1.02 and 10.64 ± 1.49 μM, respectively, which might be used as promising lead scaffold for discovery of novel anticancer agents.A series of steroidal[17,16- With the constant increase in cancer mortality, cancer has gradually become one of the most complicated diseases that threaten public health of humans2It is well known that pyrimidine core has wide occurrence in nature567891011121314161718192021222325262728303133d]pyrimidines derivatives 3a-p were synthesized according to the method shown in Due to the above description and our ongoing interest in the discovery of novel functional heterocycles, we wish to integrate the structural features of pyrimidines and DHEA scaffold to a core structure as shown in d]pyrimidines were synthesized via a sequence convenient transformation. The synthetic route for the preparation of these steroidalpyrimidines derivatives 3a-p is outlined in In this study, a series of heterocyclic steroidalpyrimidines 3a-p under the condition of base catalysis. Especially, the products for all these two steps are easy to separate and column chromatography isn’t required. All the newly prepared steroidalpyrimidines derivatives 3a-p and the intermediates 2a-p gave satisfactory analyses mainly including 1H NMR, 13C NMR and ESI-MS spectrum, and their chemical structures and basic physiochemical properties were summarized in the As shown in 3a-p were determined by their 1H NMR, 13C NMR and mass spectroscopy, and all these spectral data were in good agreement with their structures. For 1H NMR spectrum, all assignments of the signals are based on their chemical shifts and intensity patterns. As shown in the representative 1H NMR spectrum  for compounds 3a-p was measured on a Waters ACQUITY UPLC® H-CLASS PDA (Waters®) instrument, and the ion peak or adduct ions of the compounds were explored. According to the experimental results, the ESI-MS of compounds 3a-p exhibit the obvious molecular peak [M + H]+ with high abundance (100%) in the positive ion mode. In addition, all feature peaks present in the 1H NMR and 13C NMR spectra for target derivatives are described in All structures of target molecules spectrum , all 1H d]pyrimidines derivatives 3a-p and the steroidal intermediates 2a-p were screened for their potential in vitro cytotoxic activity against HepG2 (human hepatocellular liver carcinoma), Huh-7 (human heptoma), and SGC-7901 (human gastric cancer) cell lines by the standard MTT assay50 value represents the drug concentration required to inhibit cell growth by 50%.All obtained steroidalpyrimidines derivatives 3a-p displayed good cytotoxic activities than the corresponding steroidal intermediates 2a-p. Notably, the compounds 3a, 3b, 3d, 3e, 3f, 3g, 3h, and 3l exhibited excellent inhibitory activities against all three cell lines with 70–82% growth inhibition at the concentration of 40 μg/mL compared to the positive control 5-fluorouracil (58.6–70.5%). From 3a, 3b, 3d, 3e, 3f, 3g, 3h, and 3l also presented better inhibitory activities than the natural compound DHEA (57.9–69.4%), which indicated these heterocyclic steroidalpyrimidines can be used as a potential lead compounds for designing of novel anticancer drugs.Generally, as indicated in 3a, 3b, 3d, 3e, 3f, 3g, 3h, and 3l had good cytotoxic activities compared to 5-fluorouracil  against all three cell lines. Especially, we also can find that compounds 3b, 3g, 3h, 3i and 3l exhibited a certain selective inhibition  against all three cancer cell lines than the reference 3b showed the highest inhibitory effect on HepG2, Huh-7 and SGC-7901 cell lines, with an IC50 values of 5.41 ± 1.34, 5.65 ± 1.02 and 10.64 ± 1.49 μM, respectively.Meanwhile, the bioassay revealed many of these heterocyclic compounds mainly including rouracil , so in o3b, 3d, 3g, 3l and 5-fluorouracil have been presented in 3b containing an ortho-chlorophenyl unit  and aromatic enones (2a-p), respectively 3Ph and 2-CF3Ph group exhibited higher activity than the compounds bearing other substituents. From 3Ph group (2l and 3l) all present good inhibitory activities in these two systems, which may be due to the steric size of trisubstituted phenyl group is favourable for the binding to receptor. Taking into account these findings, it can be speculated it would have been more interesting to test the ortho-hydroxy and 3,4,5-trihydroxy substituted derivative, and the special properties of hydroxyl group will be helpful to increase the activity. These preliminary structure-activity relationships were to identify the target steroidalpyrimidines derivatives that could serve as potential lead antitumor molecules for drug discovery, and the further structural optimization based on these obtained SAR are well under way in our laboratory.As indicated in d]pyrimidines derived from dehydroepiandrosterone were designed and synthesized via a sequence transformation, and their in vitro inhibitory activities against cell proliferation were evaluated. From the present data it was found that some of these heterocyclic steroidalpyrimidines displayed significantly good cytotoxic activities against HepG2, Huh-7 and SGC-7901 cell lines compared to the reference d]pyrimidines will be investigated in due courses.Sixteen steroidal method3650) of each molecule for the different cell lines were also measured. All biological evaluation was performed in triplicate on three independent experiments, and measurement data were expressed as the mean ± S. D.The How to cite this article: Ke, S. et al. Steroidalpyrimidines derived from dehydroepiandrosterone: A convenient synthesis, antiproliferation activity, structure-activity relationships, and role of heterocyclic moiety. Sci. Rep.7, 44439; doi: 10.1038/srep44439 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.