P. vietnamensis var. fuscidiscus, called “Yesanqi” in Chinese, is a new variety of P. vietnamensis, which was first found in Jinping County, the southern part of Yunnan Province, China. Compared with other Panax plants, this species contains higher content of ocotillol-type saponin, majonoside R2. Despite the pharmacological importance of ocotillol-type saponins, little is known about their biosynthesis in plants. Hence, P. vietnamensis var. fuscidiscus is a suitable medicinal herbal plant species to study biosynthesis of ocotillol-type saponins. In addition, the available genomic information of this important herbal plant is lacking.P. vietnamensis var. fuscidiscus transcriptome, Illumina HiSeq™ 2000 sequencing platform was employed. We produced 114,703,210 clean reads, assembled into 126,758 unigenes, with an average length of 1,304 bp and N50 of 2,108 bp. Among these 126,758 unigenes, 85,214 unigenes (67.23%) were annotated based on the information available from the public databases. The transcripts encoding the known enzymes involved in triterpenoid saponins biosynthesis were identified in our Illumina dataset. A full-length cDNA of three Squalene epoxidase (SE) genes were obtained using reverse transcription PCR (RT-PCR) and the expression patterns of ten unigenes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Furthermore, 15 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely to involve in triterpenoid saponins biosynthesis pathway were discovered from transcriptome sequencing of P. vietnamensis var. fuscidiscus. We further analyzed the data and found 21,320 simple sequence repeats (SSRs), 30 primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions. Meanwhile, five major triterpene saponins in roots of P. vietnamensis var. fuscidicus were determined using high performance liquid chromatography (HPLC) and evaporative light scattering detector (ELSD).To investigate the P. vietnamensis var. fuscidiscus provide new insights into the identification of putative genes involved in triterpenoid saponins biosynthesis pathway. This will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level. The SSR markers identified and developed in this study show genetic diversity for this important crop and will contribute to marker-assisted breeding for P. vietnamensis var. fuscidiscus.The genomic resources generated from The online version of this article (doi:10.1186/s12864-015-1332-8) contains supplementary material, which is available to authorized users. Panax species belong to Araliaceae family. The Panax genus comprises approximately 14 species, more than 150 naturally occurring ginsenosides have been isolated from different parts of plants [Panax species, such as P. ginseng, P. quinquefolium, and P. notoginseng mainly contain protopanaxadiol-type and protopanaxatriol-type saponins, the other species like P. japonicus and P. zingiberensis, contain a large amounts of oleanolic acid saponins [P. vietnamensis have been found particularly accumulates surprisingly high content of ocotillol-type saponins, mainly majonoside R2, which is as high as 5.3% of the dried rhizome and exhibited anti-tumor and hepatocytoprotective activities [Ginsenosides are triterpenoid saponins found exclusively in saponins ,3, all otivities .Panax species were detected using de novo sequencing and transcriptome analysis, especially in P. ginseng, P. quinquefolius, and P. notoginseng [P. vietnamensis is the only species found in the narrow habitat in central Vietnam with high content of ocotillol-type saponins, which is also in the list of endanger species.2,3-oxidosqualene (OS), a precursor of terpenoids is synthesized via the mevalonic acid (MVA) pathway . After toginseng -22. Despoginseng . P. viet) gene, because they epoxidized the similar double bonds of squalene or protopanaxatriol  by SE [Arabidopsis, OSC, lupeol synthase (LUP1), directly converts DOS to oxacyclic triterpenoid epoxydammarane [P. vietnamensis, which catalyzed the cyclization of DOS , a total of 114,703,210 clean reads were obtained. The Q20 percentage (sequencing error rate < 1%) and GC percentage were 97.23% and 43.25%, respectively. An overview of the sequencing and assembly statistics are shown in Table de novo assembled using the Trinity program into 161,443 contigs consisting of 218,944,221 bp. The size of the contigs ranged from 201 to 15,880 bp, with a mean length of 1,356 bp and N50 length of 2,087 bp. Among these contigs, 82,699 (51.23%) were longer than 1000 bp, and 46,915 (29.06%) contigs were shorter than 500 bp. Using paired-end joining and gap-filling methods, these contigs were further assembled into126,758 unigenes with an average length of 1,304 bp and an N50 length of 2,108 bp. There were 60,741 unigenes (47.92%) longer than 1,000 bp, and 28,676 unigenes (22.62%) longer than 2,000 bp  were longer than 1,000 bp  were 10 were dP. notoginseng orthologs length against coverage depth  were annotated based on the information available from public databases including NCBI non-redundant protein (Nr), Swiss-Prot protein, Cluster of Orthologous Groups (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Table % were an−50), and 21.35% unigenes had high similarity (greater than 80%) , followed by Arabidopsis thaliana (11.60%), Glycine max (10.97%), and Medicago truncatula (9.14%)  classification was used to classify the functions of all unigenes. A total of 43,163 unigenes were assigned to one or more gene ontology categories, 88,984 unigenes were from the cellular component, 79,483 unigenes from the biological process, and 51,542 unigenes from the molecular function. Under the biological process category: GO classification belongs to metabolic process , cellular process , and response to stimulus . In the cellular component group, unique sequences related to cell , cell part , organelle , and organelle part  were found. For the molecular function category, binding  and catalytic activity  represented the majority of unique sequences  using Pfam_Scan program. A total of 3,602 conserved domains/families were identified from 46,649 unigenes  , followed by replication, recombination and repair , transcription , and signal transduction mechanisms   were annotated with KEGG and were assigned to 269 KEGG pathways  of the primer pairs successfully amplified clear and repeatable bands. Among the 29 successful primer pairs, 24 (80.00%) primer pairs produced PCR amplicons at the expected size, and 4 (13.33%) primer pairs generated PCR fragments longer than expected. We also found 15 (50.00%) primer pairs exhibited polymorphisms  ranged from 0.25 to 0.52, with an average value of 0.42; the effective number of alleles (Ne) ranged from 0.20 to 0.41, with a mean level of 0.34; Shannon’s information index (I) varied from 0.16 to 0.33, with an average value of 0.27; the number of polymorphic loci (NP) ranged from 0.80 to 4.40, with a mean level of 2.5; the percentage of polymorphic loci (PPB) ranged from 8.89 to 48.89, with a mean level of 28.15; and polymorphism information content (PIC) values ranged from 0.29 to 0.50 with an average of 0.44. These results indicated that there is a good genetic diversity existed among 13 P. vietnamensis var. fuscidiscus accessions.Thirty SSR primer pairs were randomly selected and synthesized to evaluate the amplification efficiency and polymorphism in 13 P. vietnamensis var. fuscidiscus accessions  clustering method was used to perform genetic correlation analysis among the 13 s Figure . The coe1 in P. vietnamensis var. fuscidiscus.The transcripts encoding all the known enzymes involved in triterpenoid saponin pathway were discovered from this Illumina transcriptome dataset, including AACT (acetyl-CoA acetyltransferase), HMGS (HMG-CoA synthase), HMGR (HMG-CoA reductase), MVK , PMK , MVD , GGPPS (geranylgeranyl pyrophosphate synthase), FPPS (farnesyl diphosphate synthase), IPPI (isopentenyl diphosphate isomerase), SS , SE, β-AS (β-amyrin synthase), DS (dammarenediol-II synthase), D12H (dammarenediol 12-hydroxylase), P6H (protopanaxadiol 6-hydroxylase), and β-A28O (β-amyrin 28-oxidase) Table . The dis2 is the main ginsenoside in P. vietnamensis var. fuscidiscus, so we focused on the discovery of the putative genes that might be involved in ocotillol-type ginsenoside biosynthesis. As mentioned above, the formation of ocotillol needs SE and OSC with “new” functions. Generally, SE catalyzes the epoxidation of squalene to OS in terpenoid biosynthesis, but in P. vietnamensis var. fuscidicus, SE might catalyze the epoxidation of terminal olefin of protpanaxatriol or OS , and a series of relevant studies are currently underway to determine the function of them. Many genes encoding OSCs have been isolated in plants; including those encode β-AS, DS, lupeol synthase (LUS), and cycloartenol synthase (CAS) [Majonoside RS Figure . Moreovese (CAS) ,37-39. EM. truncatula (CYP716A12) and V. vinifera (CYP716A15 and CYP716A17) are multifunctional oxidases, with β-A28O, α-amyrin 28-oxidase and lupeol 28-oxidase activities [P. ginseng, three CYP716A subfamily members have been isolated and characterized functionally, encode P6H (CYP716A47), D12H (CYP716A53v2) and β-A28O (CYP716A52v), respectively [Glycyrrhiza uralensis) CYP88D6 catalyze C-11 oxidation of β-amyrin in glycyrrhizin biosynthesis [M. truncatula CYP72A63 catalyze C-30 oxidation of β-amyrin [G. max CYP93E1, licorice CYP93E2 and CYP93E3 catalyze the C-24 hydroxylation of β-amyrin and sophoradiol in soyasaponin biosynthesis [Avena strigosa) CYP51H10 is able to catalyze both hydroxylation and epoxidation of β-amyrin to produce 12, 13β-epoxy-3β, 16β-dihydroxy-oleanane [M. truncatula CYP72A61v2 and CYP72A68v2 catalyze C-22 of 24-OH-β-amyrin and C-23 of oleanolic acid, respectively [Identification of specific CYP450 enzymes responsible for the production of particular metabolites is difficult due to its large numbers . Howevertivities ,42. In Pectively -15. Licoynthesis , while Gβ-amyrin . Both G.ynthesis ,45,46. OH CYP716A, D12H  are highly homologous to A. thaliana thalianol hydroxylase (AtCYP708A2). Unigene0038 is homologous to AsCYP51H10, unigene0042080 is homologous to GuCYP93E3 and AtCYP705A5, unigene0039295 and unigene0039296 are homologous to MtCYP72A67v2 and MtCYP72A8v2  was cloned from P. notoginseng [M. truncatula UGT71G1 [P. vietnamensis var. fuscidicus, 282 unigenes were found to encode UGTs , unigene0071620 is highly homologous to Barbarea vulgaris UGT73C11 and UGT73C10, which catalyze sapogenin 3-O-glucosylation [P. vietnamensis var. fuscidicus. Besides, unigene005064, unigene0031030, and unigene0031036 have close relationship to Solanum aculeatissimum steroidal saponin UDP-glucosyltransferase SaGT4A [M. truncatula UGT73F3 [Saponaria vaccaria UGT74M1, which is a triterpene carboxylic acid glucosyltransferase [UGTs catalyze the glucosylation of C-3, C-12, C-20 hydroxyl, and C28-carboxyl for the biosynthesis of ginsenosides in oginseng , which h UGT71G1 . In cDNAsylation , suggeste SaGT4A , M. trun UGT73F3 , MtUGT73 UGT73F3 , and soy UGT73F3 , indicatnsferase , suggestSE genes have different expression patterns in different tissues, similar to what was found in previous studies [The RT-qPCR analysis was used to investigate the tissue-specific expression patterns of 10 unigenes related to ginsenoside biosynthesis in this species. The expression pattern of these genes is shown in Figure  studies . Thus, wP. vietnamensis var. fuscidicus. Herein, the content of five major triterpene saponins in roots of P. vietnamensis var. fuscidicus was determined. High performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was employed for quantitative analysis of majonoside R2, due to its low UV absorptivity. As shown in Figure P. vietnamensis var. fuscidicus is about 68 mg/g, indicated that majonoside R2 were rich in the roots of this species. Quantitative analysis of other four triterpene saponins in the roots of this herb were performed using high performance liquid chromatography (HPLC). As shown in Figure The content of main component is the most widely used indicator to measure the quality of herb, so quantitative analysis of main component has important practical significance. According to previous research , majonosP. vietnamensis var. fuscidiscus exhibited remarkable disease resistance, and contains higher levels of ocotillol-type saponins. Thus, P. vietnamensis var. fuscidiscus is a suitable material for the study of ocotillol-type saponins biosynthesis and improvements of Panax plants. Because of the fact that P. vietnamensis var. fuscidiscus is a newly discovered variety of P. vietnamensis, no genomic information was available for this species. This is the first study performed on transcriptome sequencing of P. vietnamensis var. fuscidiscus using Illumina next-generation sequencing. In total, 126,758 unigenes were obtained. The large number of transcripts provided in this study not only facilitates the study of ocotillol-type saponins biosynthesis but also could provide opportunities to engineer microorganisms for the de novo production of active ingredients. Furthermore, numerous SSRs were identified and will be very useful for marker-assisted selection breeding of this herb.No specific permits were required for the described field studies. No specific permissions were required for these locations and activities. The location is not privately-owned or protected in any way and the field studies did not involve endangered or protected species.P. vietnamensis var. fuscidiscus plants were collected from Jinping County, Yunnan province, southwest of China , in May 2013. After morphological and molecular identification according the reference [SE gene cloning and RT-qPCR analysis. All samples were frozen immediately in liquid nitrogen and stored at −80°C until use.Four-year-old eference , the rooThe total RNA was extracted from the mixed sample by using Trizol reagent , following RNA purification by RNeasy MiniElute Cleanup Kit , according to the manufacture’s protocol. The RIN (RNA integrity number) values of the isolated RNA were determined by using Agilent 2100 Bioanalyzer . Samples with RIN of more than 8 were used for further analysis. The construction of the libraries and the RNA-Seq were performed by CapitalBio Corporation . Firstly, poly (A) mRNA was purified from 20 μg of total RNA using Oligo(dT) magnetic beads. Then, mRNA was fragmented into smaller pieces (200–700 bp), which were used for first-strand cDNA synthesis with reverse transcriptase and random hexamer-primer. The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase. The short double-stranded cDNA fragments are purified with QiaQuick PCR extraction kit  and resolved with EB buffer. These cDNA fragments underwent an end-repair process and poly(A) was added and then ligated with the Illumina paired-end sequencing adaptors. Subsequently, Ligation products were purified with magnetic beads and separated by agarose gel electrophoresis. A range of cDNA fragments (200 ± 25 bp) were excised from the gel and selected for PCR amplification as templates. The cDNA library was constructed with a fragment-length range of 200 bp (±25 bp). Final, the cDNA library was sequenced on a paried-end flow cell using Illumina HiSeq™ 2000 platform.de novo assembled using Trinity program [Before assembly, raw reads with adaptors and unknown nucleotides above 5% or those that were of low quality  were removed to obtain clean reads using a custom Perl script. Then the clean reads were  program  with def program  to form http://www.ncbi.nlm.nih.gov/), Swiss-Prot database (http://www.expasy.ch/sprot) and the Clusters of Orthologous Groups database (http://www.ncbi.nlm.nih.gov/COG/) [http://blast.ncbi.nlm.nih.gov/Blast.cgi), respectively, with an e-value of 1e−5. A Perl script was written to assign the functional class to unigenes. Unigenes were also compared with KEGG [−10. A Perl script was used to retrieve KEGG Orthology (KO) information from blast result and then established pathway associations between unigenes and database. Based on the results of Nr database annotation, we use Blast2GO program [Functional annotations were performed by sequence comparison with public databases included the NCBI non-redundant nucleotide database , non-redundant protein database  (ov/COG/)  using BLith KEGG  using BL program  to perfo program  software program  using Pf−5). Unigenes aligned to a higher priority database will not be aligned to lower priority database. The best alignment results were used to determine the sequence direction of unigenes. When a unigene could not be aligned to any database, ESTScan [The coding sequence (CDS) for unigene was predicted by BlastX and ESTscan. The unigene sequences were searched against the Nr, COG, KEGG and Swiss-Prot protein databases using BLASTX . We searched for SSRs with motifs ranging from mono- to hexa-nucleotides in size. The minimum of repeat units were set as follows: ten repeat units for mono-nucleotide, six for di-nucleotides, and five for tri-, tetra-, penta- and hexa-nucelotides. Primer pairs were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) with default parameters.Potential SSR markers were detected among the 126,758 unigenes using the MISA tool , 2 μL 10 × EasyTaq buffer, 1 μL of each primer (10 μM), 13 μL ddH2O, and 1 μL template DNA (approx. 10 ng/μL). PCR was performed as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at different Tm depending on the gene, extension for 30 s at 72°C, and a final step of elongation at 72°C for 5 min. The separation of alleles was performed on 8% polyacrylamide gel. PCR products were mixed with an equal volume of loading buffer. The mixture was denatured at 95°C for 5 min before loading onto the gel.A total of 30 primer pairs , effective number of alleles (Ne), Shannon’s information index (I), number of polymorphic loci (NP) and percentage of polymorphic loci (PPB). Allelic data were used to calculate the polymorphism Information Content (PIC) of each SSR marker by using the formula: PIC =1–∑pi2  [P. vietnamensis var. fuscidiscus accessions was constructed by the UPGMA (un-weighted pair group method with arithmetic mean) clustering method.The presence of each single band was coded as 1 and its absence as 0 in a data matrix. Base on the binary data matrix, popgene program version 1.32  was use h locus) . By NTSYh locus) , JaccardTMII 1st Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. The RT-PCR products were used as template for cloning of PvfSE1, PvfSE2 and PvfSE3. The full-length cDNA sequences of PvfSE1, PvfSE2 and PvfSE3 were obtained from our transcriptome data. The specific primers  and UDP-glycosyltransferase (UGT) from P. vietnamensis var. fuscidiscus were extracted individually using Trizol Kit  following the manufacturer’s protocol. Subsequently, RNA was treated with 4 × gDNA wiperMix at 42°C for 2 min to remove DNA. The purified RNA (1ug) was reverse transcribed to cDNA using HiScript QRT SuperMix for qPCR . The qPCR reactions were performed in a 20 μl volume composed of 2 μl of cDNA, 0.4 μl of each primer, and 10 μl 2 × SYBR Green Master mix (TaKaRa) in Roche LightCycler 2.0 system . PCR amplification was performed under the following conditions: 30 s at 94°C, followed by 45 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s. Three technical replications were performed for all quantitative PCRs. The phosphomevalonate kinase (PMK) gene, which was found in our transcriptome database, was chosen as reference gene control for normalization after the expressions of three reference genes  were compared in different tissues. The relative changes in gene expression levels were calculated using the 2-△△Ct method.Ten unigenes with potential roles in ginsenoside biosynthesis were chosen for validation using RT-qPCR with gene specific primers designed with Primer3 software. All the primers sequences used for the RT-qPCR analysis are shown in Additional file P. vietnamensis var. fuscidiscus roots (0.11 g) were extracted by sonication with 50 ml of methanol for 45 min, let cool, then weighed and the weight of methanol to complement the weight loss, shake, with 0.45 μm microporous membrane filtration, and 10 μL of filtrate was analyzed by HPLC-ELSD. For majonoside R2 determination, a Shimadzu LC 20A HPLC system  with a Sedex 75 evaporative light scattering detector  was used. Chromatographic separation was performed on an Waters symmetry shield™RP10  column maintained at 30°C. The mobile phase was acetonitrile-water , and the flow rate was 1 μL/min. The drift tube temperature of ELSD was set at 40°C and nebulizer nitrogen gas flow-rate was 1.5 l/min and gain of 9. Authentic majonoside R2 was provided by Yunnan Institute for Food and Drug Control .The dried powder of P. vietnamensis var. fuscidiscus roots (0.6 g) were extracted with 40 mL of 100% MeOH for 30 min and sonicated for 60 min and then diluted to 50 mL with MeOH. The methanol extract was filtered through a 0.45 μm membrane filter and 10 μL of filtrate was directly injected into the HPLC system.In brief, the dried powder of 18  was used and the column temperature was maintained at 30 °C. The flow rate was fixed at 1 mL/min, and the mobile phase consisted of acetonitrile (A) and water (B) and separation was achieved using the following gradient system: 85% B at 0 min, 80% B at 5 min, 77% B at 30 min, 60% B at 50 min, and 60% B at 55 min. Detection was performed at 203 nm for the remaining four triterpene saponins. Authentic ginsenoside Rg1, Rb1, notoginsenoside R1, and ginsenoside Rd were purchased from J&K Scientific Ltd .Quantitative analysis of the remaining four triterpene saponins  in the roots of this herb was performed on Agilent 1260 HPLC systems . The chromatographic column Agilent Zorbar SB-C


S. cerevisiae that Cdc42p associates with secretory vesicles in vivo.The activation and localization of the Rho-family GTPase Cdc42p at one pole of a cell is necessary for maintaining an axis of polarized growth in many animal and fungal cells. How the asymmetric distribution of this key regulator of polarized morphogenesis is maintained is not fully understood, though divergent models have emerged from a congruence of multiple studies, including one that posits a role for polarized secretion. Here we show with  S. cerevisiae, Cdc42p must associate with and be transiently activated at a single site on the plasma membrane to establish an axis of polarized growth that leads to bud formation S. cerevisiaeHow a cell establishes and maintains an axis of polarized growth is a key question in developmental biology. Failure of this process can result in aberrant cell division as well as developmental abnormalities in vitroin vivo observations. For example, a GFP-labeling study with astrocytes showed Cdc42p associated with vesicles in vivoS. cerevisiae secretory vesicles in vivo. Our EM-level analysis revealed this association, which is further confirmed by a complementary cell fractionation scheme in which Cdc42p was identified on immuno-isolated secretory vesicles.How polarized secretion contributes to the maintenance of a Cdc42p cap at the bud tip is not known. One model posits that secretory vesicles deliver Cdc42p to the bud tip, the site of polarized growth S. cerevisiaein vivo occurrence of this association, we prepared EM thin sections of whole cells for immunogold labeling, probing them with an antibody that binds Cdc42p  whether Cdc42p localized to one of the following membrane compartments in bud; plasma membrane, 80–100 nm diameter vesicles, or endoplasmic reticulum. When more than one of these membrane compartments were found within a 30 nm radius of the gold particle, the extended length of an antibody (1° and 2°)-gold complex in vivo in S. cerevisiae.Several studies have suggested that the small Rho-family GTPase Cdc42p associates with secretory vesicles in s Cdc42p . We foun2. Exclusive of the cell wall, buds had, on average, twice as many gold particles as mothers of the same cell  was seen in buds and mother cells, respectively. Therefore, association of Cdc42p with vesicles in vivo is neither strain nor temperature-dependent.The association of Cdc42p with secretory vesicles occurs in wild-type cells. To exclude the possibilities that Cdc42p association with secretory vesicles occurs only in r Cdc42p . We foun at 37°C . This diS. cerevisiae, we probed immuno-isolated secretory vesicles for Cdc42p with the same antibody used for our immuno-localization experiments.To further validate that Cdc42p associates with secretory vesicles in tssec6-4 cells cultured at 37°C. In the first step, membranes and membrane compartments were separated on a Nycodenz-sorbitol buoyant density gradient  supplied the reagents for media.Rich medium (YPD) containing 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose was used for growing NY17, 600), then split between two flasks incubated at 25 or 37°C for 1 h. To 10 O.D. units of log-phase culture in 50 mL conical tubes, 2X buffered-fixative  paraformaldehyde , 0.8% (v/v) glutaraldehyde , 160 mM potassium phosphate buffer, pH 6.7) was rapidly added, starting a 60 min room temperature incubation. Fixed cells were then harvested by centrifugation at 1500×g for 8 min at room temperature. Pellets were washed gently with 10 mL of sorbitol-phosphate buffer , followed by centrifugation at 1500×g for 3 min. Pellets were resuspended as before, though in 5 mL 80 mM potassium phosphate buffer containing 0.25 M sorbitol. After resuspension, cells were transferred to a glass test tube and centrifuged as above. Pellets were then resuspended in 5 mL of 1% (w/v) NaIO4 for 10 min at room temperature, centrifuged at 1500×g for 3 min, washed with deionized water, and centrifuged again. Pellets were then resuspended in 5 mL of 50 mM NH4Cl, centrifuged again, and resuspended in 3 mL 30% ethanol, preceding serial dehydration in 50, 70, 80, 85, 90, 95, 100, and 100% ethanol. Unless specified otherwise, all incubations were 5 min at room temperature, with gentle rocking. After dehydration, cells were gently pelleted and resuspended in 1.5 mL of 2∶1 ethanol: LR White resin  and nutated for 1 h, followed by overnight incubation in 1∶1 ethanol: resin mixture. Cells were then gently pelleted using a fixed angle VS-15 microcentrifuge  and resuspended in 1 mL 1∶2 ethanol: resin and nutated 2 h, followed by two 2 h incubations in 100% LR White. Resin-infiltrated cells were then transferred to a size 4 gelatin capsule , which was then nested in a size 00 gelatin capsule. The capsule was kept uncapped for 30 min to allow cells to settle before additional resin was added to completely fill the capsule. To harden the resin, capsules were baked at 45°C for 24 h.Cells were cultured in rich medium (YPD) at 25°C to mid log phase (∼0.25 O.D.) Aurion BSA-C , followed by three successive 10 min washes at room temperature with 0.1% Tween 20  diluted in IB. Sections were then incubated at room temperature for 1 h with secondary antibody, goat anti-rabbit IgG, conjugated to 10 nm gold particles  diluted 1∶50 in IB, followed by three successive washes with 0.1% Tween 20 in IB as described above. A final wash used deionized water. After the final wash, grids were wicked dry with Whatman filter paper. For contrast, sections were stained with 2% (w/v) aqueous uranyl acetate  for 4 min at room temperature, followed by 3 quick rinses with deionized water and dried as before.After the embedding, ∼85 nm sections were cut with a diamond knife and placed on 200 mesh nickel grids. Sections were hydrated with PBS and then incubated in 10 mM glycine (in PBS) for 10 min, followed by 15 min incubation with Blocking Solution . The sections were then incubated overnight at 4°C, in a moist chamber, in a 15 µL droplet of primary antibody . The method of Boyd et al. Cell fractions enriched in secretory vesicles or other membrane compartments were obtained on Nycodenz-sorbitol buoyant density gradients as described previously in Alfaro ) and mouse IgG,  served as controls for binding specificity. To bind antibodies to beads, 50 µL Dynabeads, washed thrice with 250 µL ice-cold PBS, were mixed with 1 µL antibody diluted in 250 µL PBS. After incubation at 25°C for 20 min with rotation, the beads were washed twice with PBS, once with PBS containing 1 mg/mL BSA, followed by two more washes with PBS. The beads were then mixed with 400 µL of a pooled density gradient fraction (400 µL each of fractions 9–15 were mixed to form a “pooled” fraction), which was diluted further by addition of 600 µL Tris-sorbitol buffer . Rotational mixing for 2 h at 4°C followed. Dynabeads were then concentrated with a magnet. Supernatants were mixed 1∶1 with 4X Sample Buffer To increase the homogeneity of vesicle preparations, vesicles were immuno-isolated from buoyant density gradient fractions with monoclonal antibody against Sec4p (mAb1.2.3) bound to Protein G-Dynabeads . Monoclonal antibody Pma1p (40B7)  were bound, as described above, to 20 µL M-500 beads. The antibody-bound beads were mixed for 2 h, at 4°C, with pooled buoyant density gradient fractions that contain exocytic vesicles, followed by three washes of 250 µL Tris-sorbitol buffer. The beads were then washed with 40 mM potassium phosphate buffer, pH 7.2 containing 0.8 M sorbitol, followed by fixation at room temperature for 1 h using the same phosphate-sorbitol buffer though containing 3% (v/v) paraformaldehyde  and 1% (v/v) glutaraldehyde . Following fixation, the beads were washed twice with 1 mL phosphate-sorbitol buffer and then mixed with 1 mL cold 40 mM potassium phosphate buffer, pH 7.2 containing 0.5% (v/v) osmium tetroxide and 0.8% (w/v) potassium ferricyanide. Beads were then incubated on ice for 1 h, washed 3 times with 1 mL EM-grade deionized water, and finally mixed with 1 mL of 0.5% (w/v) uranyl acetate solution prior to incubation at 4°C, overnight, in the dark. Beads were then pelleted using a magnet and washed twice with 1 mL deionized water. The samples were then dehydrated with increasing concentrations of ethanol; 1 mL 5% ethanol for 30 min, then 10, 20, 40, 80 and 100% ethanol for 15 min each, with the final wash made with 0.5 mL Spurr’s resin  prepared in ethanol. All these and subsequent incubations were made at room temperature. Dehydrated beads were then mixed gently with 0.5 mL 5% Spurr’s resin for 2 h, followed by 20% resin overnight. The next day, beads were mixed with 0.4 mL 33% resin for 6 h and then overnight in 0.4 mL 50% resin. The following day, the beads were mixed with 0.4 mL 66% resin and nutated for 4 h, with a final overnight incubation with 100% gently layered on the beads. Resin was then decanted and 0.4 mL 100% resin added for 5 h. To polymerize the resin, the tubes were heated at 70°C for at least 2 d. After the embedding, ∼85 nm sections were cut with a diamond knife and placed on 200 mesh copper grids. Staining was done with lead citrate and 3% uranyl acetate. After air-drying the grids, sections were viewed as described above.


PCP removal rate was measured under different factors such as pH, energy intensity, SPS, H2O2 concentration, Tert- butyl alcohol (TBA) and chemical oxygen demand (COD). The concentration changes of PCP were determined using spectrophotometer and HPLC spectra, respectively. The best removal PCP rate obtained in condition of pH of 11, 0.02 mol L−1 of SPS, 0.2 mol L−1 of H2O2 and energy intensity of 600 W. Moreover, COD removals in MW/H2O2 and MW/SPS process were 83% and 94%, respectively, also TBA test decreased 15% and 3% of PCP removal in MW/SPS and MW/H2O2 processes respectively. Experimental results indicated that sulfate radical was stronger than hydroxyl radical and examinations order reaction was in first order. In this study, was cleared that MW/SPS process was more effective than MW/H2O2 process in PCP removal.Pentachlorophenol (PCP) is one of the most fungicides and pesticides used in wood protection. Poisoning from PCP may be happened in dermal absorption, and respiration or ingestion. With regard to health and environmental effects of PCP, many methods were studied for its removal. Microwave assisted other methods are environmental friendly, safety, and economical method, therefore, in this study; a modified domestic microwave assisted hydrogen peroxide (MW/H Exposur or SPS)  can mine or SPS) ,10,11. Tte Na2S2O or SPS [ or SPS) . MW irra or SPS) ,13,14. B species .2O2 could be a good option for the MW oxidation technique. In this study, due to an environmental-friendly in addition to highly efficient method and low existence of specific work in this condition, analysis of the PCP removal by MW/H2O2 and MW/SPS under various kinds of parameters was performed and in the end, the effectiveness of MW/SPS and MW/H2O2 processes in the PCP removal was compared.Similar to hydroxyl radicals, sulphate radicals react with organics by electron transfer, hydrogen abstraction, or addition mechanisms ,17. Acco6Cl5ONa) with 98% purity was used without further purification. The characteristics of the PCP included of boiling point: 309-310C0, mass molar: 288.32 g mol−1. PCP solution was prepared by dissolving PCP in NaOH solution to accelerate its dissolution [−1 were used as oxidants.Sodium salt PCP, which is the sodium salt of PCP CCl5ONa wisolution . HydrogeUnder atmospheric pressure, all of the experiments were performed and triplicated in modified domestic microwave oven  , SPS and H2O2 dose , PCP concentration , effect of Tert-butyl alcohol (TBA ) with 0.04 mol L−1 concentration, and COD (344 mg L−1) were determined. Changes of PCP concentration were detected using spectrophotometer according to , energy intensity , and HPs 254 nm .2O2, its amount was 42, 53 and 56% respectively  × 107 M−1S−1 respectively. It is cleared that the reaction rate constant of Eq. (2) is more than Eq. (1). According these equations in all pHs and alkaline pH, both SO40− and OH0 are possibility responsible for degradation of organic contaminants, but previous studies have shown that in pHs of 3–10, amount of hydroxyl radical is more than sulfate radical and in pH > 10.5 amount of sulfate radical is more than hydroxyl [2O2 in alkaline pH, amount of OH0 and other radicals participating in PCP removal is more than other pHs (Eqs (3) to (8)) [Under MW/SPS, the rate constants for Eqs. (1) and (2) are < 2 × 10hydroxyl ,27. Accohydroxyl . In simi−3 and .5 ± 1 ×  to (8)) ,28.−1, PCP removal efficiency was increased (56 to 94%). But with increasing the initial SPS concentration from 0.02 to 0.05 mol L−1 PCP removal rate was decreased from 94 to 49% respectively. Furthermore under MW/H2O2, PCP removal efficiency for 0.01 to 0.05 mol L−1 of H2O2 was 12.5% to 75% respectively. PCP removal (87%) was stabled in doses of 0.2 and 0.3 mol L−1 of H2O2 (data not show). Therefore, optimal doses of SPS and H2O2 were 0.02 and 0.2 mol L−1 respectively. Shih et al. reported that, in extremely high initial concentration, SO40− reacted with persulfate according to the following equation [From Figure equation .40− to S2O80− reducing the oxidizing power for PCP removal [2O2 concentration in MW/H2O2 system, quenching of OH° radicals is happened to produce HO2° radicals [So that an over-dose of persulfate transformed the SO removal ,31. Alsoradicals ,11,15.2O2, have a decreasing effect in the organic compounds removal efficiency [Therefore, existences of a scavenger of OH° radicals, such as Hficiency ,17.2O2 system amount of PCP removal was 20, 80 and 87%, respectively. PCP removal efficiency did not change for higher power (>600 W). Subsequently, the microwave irradiation of 600 W was chosen for further experiments. According to other studies, removal efficiency can only increase to a limited extent [The test results shown in Figure d extent  and degrd extent ,34. In td extent ,36.−1 TBA (OH0 scavenger) added to MW/SPS and MW/H2O2. The results shown that the degradation rate of PCP was decreased 15% and 3% under MW/SPS/TBA and MW/H2O2/TBA respectively .In this study, 0.04 mol L2O2/TBA results show that OH0 is activation initiator and isn’t dominant radical (its role was only 3%). Based on following equations, MW is able to dissociate H2O2 to many radicals as well as OH0[2O2 system, O2 is dominant radical [MW/Hll as OH0,28,38. A radical .2O2 demonstrated that the PCP removal follows first-order kinetics law . In this study, the HPLC spectra and COD results showed a similar trend in mineralization and the lack of toxic intermediates and by products [Results abstained from COD removal showed that MW/SPS and MW/Hy Figure . Intermeproducts ,39.2O2 processes could efficiently degrade refractory compounds at strong alkaline, via radical production. MW/SPS in PCP removal was more effective than MW/H2O2, because SPS is dissociated and activated more easily than hydrogen peroxide. Addition of SPS and H2O2 doses during MW process enhances the rate of PCP degradation, except when the radical scavenging effects of SPS and H2O2. Results obtained from radical scavenger test showed that OH° had only an initiator role, and had not a dominant role and order reaction in both of systems was in first order. Also, the microwave degradation is able to mineralize refractory compounds without any toxic byproduct. The microwave degradation has many advantages such as convenience, safety, economy and high efficiency. Accordingly these methods, especially WM/SPS, have a better prospect in future for removal of other chlorinated organic compounds such as Aldrin, Dieldrin and Lindane, in alkaline pH.MW/SPS and MW/H2O2: Hydrogen Peroxide; MW: Microwave; K: Reaction constant.PCP: Pentachlorophenol; AOPs: Advanced oxidation processes; TBA: Tert- butyl alcohol; COD: Chemical oxygen demand; SPS: Sodium Persulfate; HThe authors declare that they have no competing interests.All authors studied and approved the final manuscript.


TP53 gene mutation is widely known as one of the determinants of impaired chemosensitivity. p53 is a tumor-suppressor protein in humans encoded by the TP53 gene. Some studies have shown that TP53 gene mutation and accumulation of the p53 protein are closely related with serum anti-p53 antibody positivity. This study aimed to evaluate the predictive significance of the serum p53 antibody status in metastatic colorectal cancer (mCRC) patients treated with fluoropyrimidine, oxaliplatin, plus bevacizumab as first-line chemotherapy.KRAS genotype was revealed at the beginning of treatment. Before chemotherapy initiation, the serum p53 antibody level was quantified by enzyme-linked immunosorbent assay using MESACUP® anti-p53 test kits. The cutoff value for positivity was 1.3 U/mL, as calculated previously. The KRAS genotype of the tumor samples was analyzed using the Luminex® assay.Ninety patients treated with fluoropyrimidine, oxaliplatin plus bevacizumab as first-line chemotherapy were enrolled, including 70 whose P = 0.61). The corresponding values for median progression-free survival were 13.3 months and 14.6 months , respectively.Overall response rates of Response Evaluation Criteria in Solid Tumors criteria were 77.7 % (42/54) in anti-p53–negative patients and 69.4 % (25/36) in anti-p53–positive patients. The odds ratio was 1.07. Median overall survival was 36.1 months in the anti-p53–positive patients, and not available in the anti-p53–negative patients (hazard ratio, 0.81; 95 % confidence interval, 0.37–1.77; Serum anti-p53 antibody positivity did not predict chemoresistance in mCRC treated with fluoropyrimidine, oxaliplatin, plus bevacizumab as first-line chemotherapy. TP53 mutations were proposed as the driver mutations in colorectal carcinogenesis . The corresponding values for median PFS were 13.3 months and 14.6 months , respectively . PFS was 13.36 months, and 13.3 months (Table P = 0.99) .ORR according to RECIST criteria was 77.7 % (14/18) and 76.1 % 32/42) in the p53 protein negative tumors and the p53 protein positive tumors, respectively. The odds ratio was 1.09.OS was 33.5 months in the p53 protein negative tumors, and NA in the p53 protein positive tumors  patients, ORR according to RECIST was 90.9 % and 83.8 % in the anti-p53–negative patients and anti-p53–positive patients, respectively. Median OS was 35.6 months in all patients, 35.6 months in the anti-p53–negative patients, and NA in the anti-p53–positive patients . The corresponding values for median PFS were 14.6 months in total, 17.9 months, and 16.7 months, respectively   patients, ORR according to RECIST was 69.2 % (9/13) and 76.9 % (10/13) in the anti-p53–negative patients and anti-p53–positive patients, respectively. Median OS was 33.8 months in all patients, 13.8 months in the anti-p53–negative patients, and 15.8 months in the anti-p53–positive patients . The corresponding values for median PFS were 14.6 months, 34.3 months, and 26.6 months, respectively  .In the KRAS wild-type n = 2 patientIn univariate analysis, peritoneal metastasis and multiple metastasis were also significant predictors of OS. On the other hand, lung metastasis and lymph node metastasis were also significant predictors of PFS. In the multivariate analysis, peritoneal metastasis was significant predictors of OS and lung metastasis and lymph node metastasis were significant predictors of PFS. However both anti-p53 antibody and IHC of p53 protein negativity did not yield any independent predictive factors . Moderate correlation  was found to exist between anti-p53 antibody positivity and the TP53 mutation [TP53 has been associated with decreased sensitivity to agents inhibiting DNA synthesis, such as 5-FU [The mechanism underlying anti-p53 auto-antibody production has yet to be revealed but is thought to be associated with the presence of the mutation . Mutatio as 5-FU . These gTP53 tumor-suppressor gene may have clinical relevance to the efficacy of anti-VEGF agents, such as bevacizumab.As mentioned above, preclinical research has indicated that disruptions in the Ras/Raf/MEK/ERK pathway or inactivation of the However, in this retrospective study, we did not assess mCRC patients who were more likely to respond to bevacizumab therapy.TP53 mutation alone is insufficient to trigger anti-p53 antibody production. Only 20–50 % of patients which detectable TP53 mutations produce detectable anti-p53 antibodies [There are some reasons to explain the results of this study. First, An Anti-p53 antibody is not normally produced wild type p53 protein induces tolerance of the host. However tibodies . This istibodies . Second tibodies . Third, tibodies .TP53 or KRAS merit discussion. Indeed, the question is whether anti-p53 antibodies are a reliable parameter for the TP 53 mutation status. These antibodies have high specificity but lack sensitivity [TP53 mutations negate p53 protein synthesis and accumulation.The methods used to determine the mutational status of sitivity . They haIn this study we also investigate whether IHC of p53 protein was the predictive factor of chemosensitivity or not, however there was no relationship between IHC of p53 protein and clinical outcomes.TP53 as it applies to CRC. In previous studies, perioperative variations in serum anti-p53 antibody levels have been shown to predict OS (Table Other techniques, such as sequencing and functional assays, have been developed to determine the mutation status of OS Table  [12, 14–TP53 mutation using sequencing method which is one of the main methods of detect TP53 mutation. When we assess the relationship between TP53 gene mutation and chemoresistance in mCRC patients, we should use other methodologies such as sequencing and functional assays, apart from the anti-p53 antibody status.This study had some limitations. Because of its retrospective and single-center nature, an unknown bias may exist in the findings. Furthermore, we didn’t measure Serum anti-p53 antibody positivity did not predict chemoresistance in mCRC treated with fluoropyrimidine, oxaliplatin, plus bevacizumab at first-line chemotherapy. We believe that if we want to know the relationship between the anti-p53 antibody status and chemosensitivity, we should use other methodologies like sequencing, and functional assays, apart from the anti-p53 antibody status.


Although molecule-based bottom-up nanosheets manufactured directly from molecular components can exhibit greater structural diversity than top-down nanosheets, the bottom-up nanosheets reported thus far lack useful functionalities. Here we show the design and synthesis of a bottom-up nanosheet featuring a photoactive bis(dipyrrinato)zinc(II) complex motif. A liquid/liquid interfacial synthesis between a three-way dipyrrin ligand and zinc(II) ions results in a multi-layer nanosheet, whereas an air/liquid interfacial reaction produces a single-layer or few-layer nanosheet with domain sizes of >10 μm on one side. The bis(dipyrrinato)zinc(II) metal complex nanosheet is easy to deposit on various substrates using the Langmuir–Schäfer process. The nanosheet deposited on a transparent SnO The creation of functional 2D bottom-up nanosheets woven from molecular components remains a large challenge. Here, a bottom-up nanosheet featuring a photofunctional bis(dipyrrinato)zinc(II) complex motif is synthesized using interfacial syntheses, enabling a photoelectric conversion system. Nanosheets with two-dimensional polymeric structures have recently attracted significant attention. Graphene, a prominent nanosheet with various attractive properties, has been investigated extensively for applications in electronics1257891012131416171819108Another type of nanosheets, namely molecule-based bottom-up nanosheets, is emerging. This series of nanosheets is fabricated directly from atomic, ionic and molecular components. The concept was proposed nearly a century ago, although it has only recently been realized222324262931323335363738404142434445Given this background, we have sought to create functional bottom-up nanosheets, including an electrically conductive nickel bis(dithiolene) nanosheet4748505253N1 synthesized from three-way dipyrrin ligand L1 and zinc(II) acetate. To verify the macroscopic formation of N1, the first process considered was a liquid/liquid interfacial synthesis−2 mol l−1) and a dichloromethane solution of L1  substrate it appears as a transparent film  substrate modified with 1,1,1,3,3,3-hexamethyldisilazane (HMDS/Si(111)) was observed to be 700 nm by atomic force microscopy . The thickness of N1 may be controlled by the concentration of L1 in dichloromethane and may span 6–800 nm (5–670 layers) using L1 solutions with concentrations of 1.0 × 10−6 to 5.0 × 10−4 mol l−1 .ent film . Multi-lopy AFM, , which c mol l−1 . We note texture . In factN1. 4748L1 (7.4 × 10−5 mol l−1) was gently dropped onto the surface of an aqueous phase containing zinc(II) acetate (5.0 × 10−2 mol l−1) at ambient temperature. After prompt evaporation of dichloromethane, spontaneous air/water interfacial complexation occurred between L1 and zinc(II) ions, and single-layer N1 was produced at the interface. The resultant single-layer N1 was almost invisible to the naked eye but could be transferred onto various substrates via horizontal deposition known as the Langmuir–Schäfer method  to assess its constituent elements (N and Zn) and their bonding properties zinc(II) complex M1 (N1 deposited on highly ordered pyrolitic graphite (HOPG) were also subjected to XPS. Ligand L1 did not show a Zn 2p3/2 peak, whereas the peak was present at similar binding energies in the spectra of M1, multi-layer N1 and single-layer N1 . The spectrum of L1 features two N 1s peaks (400.0 and 398.4 eV), assignable to pyrrolic and iminic nitrogen atoms, respectivelyM1 shows a single N 1s peak at 398.8 eV, which originates from its coordination to the zinc(II) centre, which makes the two nitrogen atoms equivalent to one another. Both multi-layer N1 and single-layer N1 displayed a single peak for N 1s at almost the same binding energy as M1 (399.0 eV). In addition, the abundance ratio calculated from the peak area corrected using the photoionization cross-section is consistent with the ideal value of N:Zn=4:1 . These findings indicate that the bis(dipyrrinato)zinc(II) complex motif formed quantitatively in the nanosheet. We note that coordination of H2O molecule(s) to the zinc(II) centre to form a square–pyramidal or trans-octahedral coordination sphere may be excluded by the fact that multi-layer N1 on oxygen-free HOPG lacked an O 1s peak in XPS (XPS was conducted for single-layer operties . For refmplex M1  and multk in XPS . Also nok in XPS  exhibitek in XPS . This reN1. N1 on an HMDS/Si(111) substrate. The phase image clearly distinguishes single-layer N1 and the bare substrate: the nanosheet possesses a phase value that is lower by 2.4°. The height image shows a domain with one side >10-μm long, which is a noteworthy size for bottom-up nanosheets29303132333435363738N1 was scratched with the AFM tip at a force of 8.6 × 102 nN. N1, leaving an intact HMDS/Si(111) surface. A cross-section analysis traversing one of the scratched edges demonstrated that N1 was 1.2-nm thick (4 nN) resulted in a more drastic change in the AFM image (N1 and the HMDS/Si(111) surface were destroyed.The AFM analysis revealed the flat sheet texture and morphology of single-layer nm thick , which iFM image . In thisN1 with larger domain sizes was fabricated by increasing the amount of L1  revealed that single-layer rom HOPG . The in-HOPG  ion is known to induce a redshift in the 1π–π* bandM1 displayed a 49-nm wavelength shift relative to L1. Few-layer N1 also showed an absorption band in the visible region, with the absorption maximum being closer to that of M1 than to that of L1, which also indicates that the complexation of L1 with zinc(II) ions was complete. Using the 1π–π* band as a probe, single-layer N1 was accumulated stepwise on a quartz substrate. The single-layer nanosheet was fabricated on the air/water interface of a Langmuir–Blodgett trough, which was deposited repeatedly on a quartz substrate at a constant surface pressure using the Langmuir–Schäfer method. 1π–π* band at 500 nm is proportional to the number of deposition processes zinc(II) complex sensitizer despite its excellent light absorption ability. We then studied the relationship between the quantum yield of the photoelectric conversion and thickness of N1 , which decreases gradually with the growth of the thickness of N1, leading to negligible photoresponses at thicknesses of over ~300 layers. For the photocurrent, the maximum is located at ~100–150 layers zinc(II) complex sensitizers: plain zinc(II) complex M2, which was dropcasted onto a SnO2 electrode to form a physisorbed film  and manipulability (to facilitate deposition and layering) of N1 is also advantageous for potential applications.s set up . Triethat up zinc(II) complex motif was fabricated. A nanosheet with atomic thickness was prepared via an air/water interfacial synthesis, during which spontaneous complexation proceeded between a three-way dipyrrin ligand and zinc(II) ions at the interface. The nanosheet was identified using ultraviolet/visible spectroscopy and XPS, which revealed the complete formation of the bis(dipyrrinato)zinc(II) complex motif. The single-layer nanosheet was confirmed by AFM via a scratch experiment. The domain size of the single-layer nanosheet reached 10 μm on one side, which is large for bottom-up nanosheet materials. Repeated deposition of the single-layer nanosheet on a flat substrate resulted in its quantitative layering. The nanosheet efficiently collectes visible light at around 500 nm, and that physisorbed on a transparent SnOH-pyrrol-2-ylidene)methyl)-3,5-dimethyl-1H-pyrroleH-pyrrol-2-ylidene)(phenyl)methyl)-1H-pyrroleN1, pretreatments of substrates and photoelectric conversion were supplied from Kanto Chemical Co., Inc. as high-performance liquid chromatography grade and were used as received. Water was purified using a Milli-Q purification system (Merck KGaA). Tetra-n-butylammonium perchlorate as a supporting electrolyte was purified by recrystallization from ethanol, which was dried in vacuo. Solvents for organic syntheses were purified using a solvent purification system . The other chemicals were general grades and were used as received. All procedures were conducted under an ambient condition otherwise stated.5′-(4-Formylphenyl)--4,4′′-dicarbaldehyde1H (500 or 400 MHz) and 13C (125 or 100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker-DRX500, JEOL ECX-400 or JEOL AL-400 spectrometer. Fast atom bombardment mass spectrometry (FAB-MS) and electrospray ionization time-of-flight spectrometry were conducted using a JEOL JMS-700 MStation and Micromass LCT Premier XE mass spectrometer, respectively.1H NMR : δ 7.95 , 7.80 , 7.61 , 6.56 , 6.19 , 2.47 ; 13C NMR : δ 165.18, 146.78, 141.99, 137.88, 135.32, 132.12, 130.51, 128.89, 127.12, 116.54, 112.11, 105.97 and 18.21; HR-FAB-MS (m/z): [M]+ calculated for C57H48N6, 816.3940; found, 816.3924.5′-(4-Formylphenyl)--4,4′′-dicarbaldehyde  and 2-methylpyrrole  were dissolved in dichloromethane (100 ml) under a nitrogen atmosphere. One drop of trifluoroacetic acid was added, and the solution changed from light yellow to bright red and was stirred at room temperature for 3 h. When complete consumption of the aldehyde was confirmed by thin-layer chromatography, a solution of chloranil  in dichloromethane was added, and the resultant mixture was stirred for an additional 15 min. The reaction mixture was washed with water, dried over magnesium sulfate, filtered and evaporated. The crude product was purified by column chromatography on aluminum oxide (activity II–III) with dichloromethane as an eluent to yield a deep-yellow powder . L1 was dissolved in dichloromethane to a concentration of 1.0 × 10−4 mol l−1, and 10 ml of the solution was poured into the vial. Then, pure water (10 ml) was layered gently onto the organic phase, which served as a buffer layer. After 2 h, aqueous zinc(II) acetate solution  was slowly added to the buffer layer. The reaction system was left undisturbed for 4 days to obtain an orange film at the liquid/liquid interface. The resulting multi-layer N1 could be deposited on various substrates.A 50-ml cylindrical glass vial (3.2 cm in diameter) was used as the reaction container. L1  were dissolved in dichloromethane (5 ml) in a glass vial. The reaction mixture was allowed to stand at room temperature for 1 day. The resultant product was filtered and washed with dichloromethane, ethanol and water to yield a brown solid  were dissolved in N,N-dimethylformamide (5 ml) in a glass vial. After sealing the vial tightly, the reaction mixture was heated at 105 °C for 1 day in an oil bath. After being cooled down to room temperature, the resultant product was filtered and washed with dichloromethane, N,N-dimethylformamide and water to obtain a brown solid (Two types of reaction were performed. Stoichiometric amounts of zinc(II) acetate dihydrate  and wn solid . In the wn solid .−1, 30 ml) was poured into the vial. A small amount of a dichloromethane solution of L1 was then sprinkled gently on the surface of the aqueous phase: single-layer N1 was synthesized using 5.0 μl of L1 solution (7.4 × 10−5 mol l−1), whereas 20 μl of L1 solution (7.4 × 10−5 mol l−1) was used for the few-layer nanosheet. After spontaneous evaporation of the organic solvent, the reaction system was left undisturbed, such that N1 was produced at the air/liquid interface. Single-layer or few-layer N1 was then transferred onto substrates using the Langmuir–Schäfer method.A 50-ml glass vial (3.2 cm in diameter) was used as the reaction container. Aqueous zinc(II) acetate (0.05 mol lH-pyrrol-2-ylidene)methyl)-3,5-dimethyl-1H-pyrrole , and the reaction mixture was stirred overnight at room temperature. Methanol (20 ml) was added to recrystallize the product as an orange solid . 1H NMR : δ 7.21 , 7.11 , 5.91 , 2.16 , 2.04  and 1.28 ; 13C NMR : δ 156.07, 143.26, 143.06, 139.18, 135.95, 134.27, 127.98, 127.88, 119.74, 19.33, 16.13 and 14.70; HR-FAB-MS (m/z): [M]+ calculated for C42H46N4Zn, 670.3014; found, 670.3011.Zinc(II) acetate dihydrate  and triethylamine (0.11 ml) were added to a dichloromethane solution (20 ml) of 2-(phenyl)methyl)-1H-pyrrole  in methanol (10 ml) was poured into a suspension of anhydrous zinc(II) acetate  in methanol (3 ml). The mixture was stirred overnight at room temperature. The reaction mixture was filtered, and the resultant solid was washed with methanol. Filtration yielded a light orange solid . 1H NMR : δ 7.50−7.39 , 6.55 , 6.18  and 2.12 ; 13C NMR : δ 159.44, 144.71, 139.52, 139.39, 133.22, 130.89, 128.17, 127.14, 117.10 and 16.62; HR-FAB-MS (m/z): [M]+ calculated for C42H46N4Zn, 558.1762; found, 558.1782.A solution of 2-methyl-5- δ 7.41 , 7.26 , 6.48 , 6.17 , 4.18 , 3.05 , 2.71 , 2.46  and 1.28 ; 13C NMR  δ 172.84, 153.67, 140.99, 139.91, 138.30, 135.49, 130.99, 128.96, 127.41, 117.35, 60.49, 35.81, 30.79, 16.29 and 14.27; HR-FAB-MS (m/z): [M]+ calculated for C22H24N2O2, 348.1829; found, 348.1838.To a dichloromethane (20 ml) solution of ethyl 3-(4-formylphenyl)propanoate  and 2-methylpyrrole , trifluoroacetic acid (10 μl) was added, under a nitrogen atmosphere, and the solution was stirred overnight at room temperature. Chloranil  was added and stirred for another 30 min. After evaporation of the solvent, the residue was passed through a short pad of aluminum oxide (activity II–III) using dichloromethane as an eluent. After evaporation of the solvent, the crude product was purified by column chromatography on aluminum oxide (activity II–III) with hexane/dichloromethane (1:1 v/v) as an eluent. The yellow band was collected and evaporated to yield ethyl 3-(4-((5-methyl-1H-pyrrol-2-yl)(5-methyl-2H-pyrrol-2-ylidene)methyl)phenyl)propanoate as an orange oil . H-pyrrol-2-yl)(5-methyl-2H-pyrrol-2-ylidene)methyl)phenyl)propanoate  was added to tetrahydrofuran (30 ml) and water (5 ml), and the mixture was stirred at room temperature for 5 min. Then, an aqueous solution (10 ml) of NaOH  was added, and the reaction mixture was stirred overnight at room temperature. After adjusting the pH of the reaction mixture to 4 using aqueous hydrochloric acid (1.0 × 10−4 mol l−1), the mixture was poured into water (50 ml) and washed with ether (20 ml × 3). The water layer was then treated with a solution of anhydrous zinc(II) acetate  in water (3 ml). The mixture was extracted with dichloromethane and dried over sodium sulfate. The solvent was evaporated under a reduced pressure to yield a dark orange solid of M3 . 1H NMR : δ 12.34 , 7.34–7.30 , 6.33 , 6.20 , 2.91 , 2.61  and 2.39 ; 13C NMR  δ 158.99, 144.79, 139.06, 133.29, 130.90, 130.59, 127.60, 117.85, 31.16, 25.62 and 16.49 (two quaternary carbons are missing because of low solubility or overlaps with other peaks); HR-ESI-MS (m/z): [M-H+] calculated for C40H37N4O4Zn, 701.2106; found, 701.2122.Ethyl 3-(4-((5-methyl-12 layer was sonicated in acetone (10 min), 2-propanol (15 min × 2), water (15 min) and ethanol (10 min). After rinsing the substrate in ethanol a couple of times, the substrate was immersed in an ethanol solution of HMDS (1 v/v%) for 1 h. The substrate was then washed with ethanol several times before being annealed at 130 °C for 30 min. The modified substrate was then sonicated in ethanol (15 min) and water (10 min) and was stored in water. Just before use, HMDS/Si(111) was sonicated in 2-propanol (15 min) and water (15 min), which was dried in vacuo overnight. ITO (on glass), quartz and SnO2  substrates were sonicated in acetone (10 min × 2), water (10 min × 2) and nonionic detergent in water (30 min). Then, the substrate was washed with water until the bubbles of the detergent disappeared before being sonicated in water (10 min × 3) and ethanol (10 min × 2). The cleaned substrate was stored in water, and dried by nitrogen blow just before use.HOPG was cleaved with a piece of cellophane tape to obtain a flat and clean surface just before use. Si(111) substrates were hydrophobized using HMDS. The procedure to form a HMDS/Si(111) surface is as follows. The Si(111) substrate covered with a natural SiO−1) was poured into the trough as a subphase, the trough was washed with ethanol. The surface of the aqueous solution was cleaned by suctioning the surface of the subphase several times on compressing the trough barrier. Then, a dichloromethane solution of L1  was dropped gently onto the surface of the subphase. Under this condition, the ideal coverage of single-layer N1 reached 90% of the trough area. After spontaneous evaporation of the organic solvent, the reaction system was left undisturbed for 4 h, such that single-layer N1 was produced at the air/liquid interface. The trough was then compressed to yield a trough area of 69.8 cm2  at a surface pressure of 0.04 mN m−1. Under these conditions, single-layer N1 was transferred onto a quartz substrate via the Langmuir–Schäfer method. The trough was then shrunk by the same area as the substrate, and the next transfer was conducted. This process was repeated to layer single-layer N1.A Langmuir–Blodgett trough  was used as a reaction container. Before a zinc(II) acetate aqueous solution (0.05 mol lN1 (thickness: 700 nm) was deposited on a quartz or ITO substrate. The modified substrate was immersed in a dichloromethane solution of Rhodamine B (5.0 × 10−5 mol l−1) for 15 h and was then rinsed with dichloromethane to yield Rhodamine B-encapsulated multi-layer N1.Multi-layer 2 area. The spectra were analysed with MultiPak Software and standardized using the C 1s peak at 284.6 eV. AFM and STM were performed using an Agilent Technologies 5,500 scanning probe microscope under an ambient condition. AFM was performed in high-amplitude mode (tapping mode) with a silicon cantilever PPP-NCL (Nano World). The probe for STM  was cut from a wire using a nipper to obtain a sharp edge. An optical microscope image was taken using a VHX-100 (Keyence Corporation). A field-emission scanning electron microscopic image was collected using a JEOL JSM-7400FNT. Transmission electron microscopy images/SAED patterns were recorded at accelerating voltage of 75 kV using a Hitachi HF-2000 equipped with an AMT-CCD camera. The sample was prepared by depositing multi-layer N1 (thickness: 700 nm) on a carbon film supported by a copper grid  directly from the liquid/liquid interface. To acquire electron diffractions, we focused on the edge of multi-layer N1. To reproduce the obtained SAED pattern, three-dimensional structures of multi-layer N1, which comprise piles of single-layer N1, were considered  . Al Kα  was used as the X-ray source, and the beam was focused on a 100-μmare Ltd) . UltraviN1 with thicknesses ≤155 layers was deposited on a transparent SnO2 electrode using the repeated Langmuir–Schäfer procedure shown in N1 with thicknesses of >155 layers was fabricated directly using the liquid/liquid interfacial synthesis . The modified SnO2 electrode was used as a working electrode (photoanode). Silver and platinum wires were employed as reference and counter electrodes, respectively. The resulting three-electrode system was immersed in a homemade photoelectrochemical cell (n-butylammonium perchlorate (0.1 mol l−1) containing TEOA (0.05 mol l−1) as a sacrificial donor reagent. The cell was sealed and deoxygenized by argon bubbling for 30 min. Monochromatic light for the action spectrum shown in 2, which was determined by a fluorocarbon rubber o-ring  and np is the mole of photons absorbed by the sensitizer per unit time (in mol s−1). ne and np were calculated using where i is the current flow (in A), F is the Faraday constant (9.65 × 104 C mol−1), W is the photon flux of incident light (in J s−1), λ is the wavelength of the irradiated light (5.00 × 10−7 m), A is the absorbance at the irradiated wavelength, NA is the Avogadro constant (6.02 × 1023 mol−1), h is the Planck constant (6.63 × 10−34 Js) and c is the velocity of light (3.00 × 108 m s−1). A representative data set for the determination of φ for N1 is shown in i was calculated using where iL is the average light current for the first cycle (10 s) and iD is the average dark current just before the illumination of light. A photon counter  was employed for the quantification of W. For every sample, W was measured independently. A typical value for W was ~0.20 mW. When i was below the measurable level (<1.0 nA), W was increased to 1.0–2.0 mW such that the photocurrent signal was amplified. For referential mononuclear complex M2, film formation was conducted as follows: two droplets from a disposable Pasteur pipette of an acetonitrile solution of M2 (1.86 × 10−5 mol l−1) were dripped onto a SnO2 substrate (~0.8 cm2). The substrate was then dried under vacuum for 10 min to allow the solvent to evaporate. The modified SnO2 electrode was subjected to photoelectric conversion, following the procedure for N1 except that aqueous 0.1 mol l−1 sodium sulfate was used as an electrolyte solution; this change prevented the M2 film from redissolution. Referential mononuclear complex M3 was immobilized on a SnO2 electrode through the carboxy group using the self-assembled monolayer procedure2 substrate was immersed in a dimethylsulfoxide solution of M3 (1.96 × 10−3 mol l−1) for 1 day at room temperature, and the decorated substrate was rinsed with dimethylsulfoxide and dried by an argon blow. The modified SnO2 electrode was subjected to photoelectric conversion using the same procedure as N1.where L1. K.H., Q.L. and T.N. performed the fabrication of N1 and M1. T.N. and M.T. synthesized M2 and M3, respectively. Y.K. optimized the three-dimensional lattice of multi-layer N1 at the molecular mechanics level of theory. K.H. conducted the characterization of N1. T.Y. and K.H. investigated the photoelectric conversion of N1, M2 and M3. R.S. and K.H. co-wrote the manuscript.R.S. conceived and directed the project. R.S., H.N., K.H. and W.-Y.W. initiated the project. S.K., Q.L. and W.-Y.W. synthesized ligand How to cite this article: Sakamoto, R. et al. A photofunctional bottom-up bis(dipyrrinato)zinc(II) complex nanosheet. Nat. Commun. 6:6713 doi: 10.1038/ncomms7713 (2015).Supplementary Figures 1-14 and Supplementary References


AbstractParatullbergia Womersley, 1930 is recorded for the first time from China. Paratullbergiachangfengensissp. n. from Shanghai is described and illustrated. It is characterized by the presence of 1+1 pseudocelli on thoracic segment I, with two pairs of pseudocelli on each of thoracic segments II and III, presence of seta px on abdominal segment IV, seta a2 and p4 on abdominal segment V as microsetae, and less differentiated sensory seta p3 on abdominal segment V. Both sexes present. The new species can be easily distinguished from its congeners by the presence of pseudocelli on thoracic segment I. An updated key to the world species of the genus Paratullbergia is provided.The genus  Tullbergiidae Bagnall, 1947 contains a group of tiny euedaphic collembolans with approximately 200 species reported in the world , Shanghai, China.Specimens were collected by Berlese-Tullgren funnels and preserved in 80% ethanol. The material was mounted on slides in Hoyer’s solution and dried in an oven at 45 °C for identification. Drawings were done with the aid of a phase contrast microscope. The type specimens are deposited in Abbreviations used in the descriptions: thoracic segmentTh.; abdominal segmentAbd.; antennal segmentAnt.; sensillums; postantennal organPAO; anterior setaea; medial setaem; posterior setaep; pleural setaepl; pseudocellipso.Taxon classificationAnimaliaCollembolaTullbergiidaehttp://zoobank.org/2E6BA83A-245D-4B94-89DA-99257167E83CSNHM), China, Shanghai, extracted from soil samples of broad-leaved forest of Changfeng Park, PageBreak121°23'E31°13'N , 15-III-2015, coll. Y. Bu & Y. Gao. Paratypes, 2 females  (SNHM), data same as holotype. Other materials: 2 juveniles and 1 male subadult , data same as holotype.Holotype, male (No. Changfeng2-2) . Both females and males were present. Setae well differentiated into micro- and macrosetae Fig. , others Head seta a0 present 20–22 µm), c1 absent, oc2 as macroseta (25–30 µm), and sd5 as mesoseta (20–25 µm)  shorter than head (130–150 µm). 5 µm shorLegs without clavate tibiotarsal hairs Fig. . Coxa, tTh. II-III, and lateral sensory setae s 37–39 µm long  and p4 (19–21 µm) as microsetae  and shape of sensory seta p3 on abdominal segment V (setiform vs. flame-like). The nine existing species of the genus Paratullbergia can be distinguished by the following key.The presence of pseudocelli on thoracic segment I easily distinguishes


Clathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.  •The endocytic pioneer protein Eps15 engages AP-2 and Fcho1/2 noncompetitively•Structural analysis shows arrayed DPF motif triad in Eps15 for Fcho1/2 μHD binding•DPF-based codes direct transient Fcho1/2⋅Eps15/R⋅AP-2 ternary complex formation•In ternary complex, Fcho1 interdomain linker primes AP-2 for cargo capture How clathrin-coated vesicles initiate at the plasma membrane is uncertain. Ma et al. show through structural and biochemical analyses that a three-way interaction between early-arriving endocytic factors Eps15/R and Fcho1/2, and the adaptor AP-2, promotes membrane deposition of AP-2 in open conformation for sustained clathrin-coat assembly and cargo loading. Rapid i2)  and on d2) .The lack of a tightly coupled energy input suggests that arrival of AP-2, pioneers, and CLASPs at the plasma membrane is a stochastic phenomenon , in partFcho1 and Fcho2 likely play a key role in local AP-2 restructuring, as they are among the earliest proteins to mark a nascent CCS  and affeFcho1 and Fcho2 have numerous binding partners . The chi615TNLDFFQSDPFVGSDPFKDDPF) of EPS15 sufficient for engaging Fcho1/2 (629DPFKDDPF) of the EPS15 μHD-binding element is nearly identical to a small portion of the CLASP DAB2 (The association of muniscin μHDs with EPS15 requires the unstructured C terminus (residues 595–896) of the protein , rich in Fcho1/2 A–1C. FCH Fcho1/2 D. This E Fcho1/2 C. The diASP DAB2  and 1C tASP DAB2 . Yet EPSASP DAB2 F, suggesASP DAB2 C (MulkeaASP DAB2 .623DPF→APA mutation is introduced into the longer 615–636 form (2DPF), while not interfering effectively with EPS15/R or intersectin 1 binding, prohibits the engagement of the weaker-binding proteins, including DAB2 (3DPFX2DPF) essentially eliminates all partner binding. A 623DPF→APA substitution in the full (residues 615–637) tandem fusion diminishes EPS15/R and intersectin binding more efficiently than does the short fusion (residues 626–637). This again implicates the proximal section of the EPS15 sequence in higher-affinity binding. Neither HRB nor CALM have related DPFX2DPF motifs within the unstructured polypeptide regions, but are Phe rich, suggesting that the weaker affinity of these FCHO1 μHD partners likely reflects use of variant but related interaction motif(s).The role of the proximal half of the mapped DPF tract on μHD engagement is highlighted by the substantially reduced apparent affinity of cytosolic Fcho2 for the GST-EPS15 fusion protein when either residues 615–625 (TNLDFFQSDPF) are removed A or a 62636 form B. No bin636 form E. In agr636 form F affectsing DAB2 G. By conAn ectopic yet experimentally tractable manifestation of μHD partner engagement occurs in cultured cells in the context of a customized membrane-anchored Tac-FCHO1 μHD fusion protein . TransmeET615-EG637) to the zebrafish Fcho1 μHD  with a GAGA spacer crystallized and diffracted to 2.4 Å. The structure was solved by single-wavelength anomalous dispersion using an Xe derivative . Simultaneous alteration of the nearby conserved residues N735A + R740A has a different and weaker inhibitory effect: durable EPS15/R binding still persists, but HRB and CALM associations are lost  binding for the A629E or P627D + A629E substitutions  compared with a single DPF⋅α-appendage interaction (KD ∼ 200 μM)   favors i 200 μM) A. On the2. A local but temporary DPF-mediated three-component nanocluster would allow AP-2 to linger in the vicinity of Fcho1/2 to advance CCS assembly by encounters with the muniscin effector linker (step 3). This interaction leads to conformational rearrangement of AP-2 to an “open” or active form Psociated ; that ovsociated ; and thasociated . As the sociated I , 200 mM sodium citrate, 22% polyethylene glycol 3350, and 10 mM DTT and were cryoprotected in 22% glycerol before freezing. A Xe derivative was used for phasing .Pull-down assays entail mixing glutathione-Sepharose-immobilized GST-fusion proteins with a cell extract or purified proteins, washing, and analysis of resulting supernatant and pellet fractions. For SDS-PAGE and blots, five times more of each pellet versus supernatant fraction was loaded. ITC involves repeated injection of EPS15 peptides into a temperature-controlled solution of μHD utilizing a Nano ITC instrument (TA Instruments).Prior to transferrin addition, HeLa cells were incubated for 60 min at 37°C in a starvation medium of DMEM supplemented with 25 mM HEPES and 0.5% BSA of to clear receptors of apotransferrin. Fluorescently labeled Alexa Fluor transferrin (25 μg/ml) was added in prewarmed (37°C) medium and internalization was for either 2 or 10 min at 37°C. For the transferrin pulse-chase uptake assays, starved cells were incubated with fluorescent transferrin at 37°C for 2 min and then immediately chilled on ice and washed with PBS as described by 2) for comparison.Confocal imaging was on an inverted Olympus FV1000 microscope equipped with a PlanApo N (60 ×/1.42 NA) oil objective. Excitation and emission wavelengths were preprogrammed by the instrument running the FV10-ASW software, and emission signals in the different channels were collected in the sequential scan mode. Exported TIFF file images were imported into Adobe Photoshop CS4 for minor adjustments to brightness and/or contrast. Quantitation of transferrin uptake in confocal image files was performed using the linescan application within MetaMorph (Molecular Devices). The extent of transferrin internalization in confocal image stacks that were collected following the pulse-chase format was analyzed using Fiji software . Images Starved cells in glass-bottomed MatTek dishes were imaged by TIRFM. Four separate positions were selected for each experiment and images were collected every 5 s for 5 min. The surface residence times of positively labeled structures were calculated using Imaris (Bitplane). The “spots” function was used to delineate AP-2 (β2-YFP)-positive structures in selected ROIs using local contrast thresholding of the TIRFM datasets. Spots were uniformly sized at slightly greater than diffraction limit (267 nm) for the 100 × 1.49 NA TIRFM objective and the wavelength of light collected. Spot persistence was tracked over time (5 s image separation) to determine duration of residency of individual structures at the cell surface. In addition, live-cell TIRFM images and immunofluorescence confocal images from fixed cells were analyzed by using Fiji and MetaMorph. Raw AP-2 channel images were background corrected. For AP-2 TIRFM datasets, 2D deconvolution was then applied to these images in MetaMorph to increase the signal-to-noise ratio. Further analysis was carried out using Fiji; individual cells were marked and saved separately. Deconvoluted images were smoothed and thresholded manually to create a mask, and masks used on the corresponding background-corrected images to calculate values for the size/area of the AP-2 puncta.Conceptualization, L.M.T; Methodology, D.J.O. and L.M.T; Software, A.J.M; Validation, L.M., P.K.U., A.G.W., A.L., A.J., D.J.O., and L.M.T.; Formal Analysis, A.J.M., A.G.W., S.S.H., and S.C.W.; Investigation, L.M., P.K.U., A.G.W., A.L., A.J., A.J.M., D.J.O., S.S.H., T.P.-S., L.M.T., and S.C.W.; Resources, S.S.H., A.J., A.L., L.M., A.J.M., D.J.O., T.P.-S., L.M.T., P.K.U., S.C.W., and A.G.W.; Writing – Original Draft, D.J.O. and L.M.T.; Writing – Review & Editing, D.J.O., L.M.T., and P.K.U.; Visualization, D.J.O., L.M.T., P.K.U., and A.G.W.; Supervision, D.J.O. and L.M.T; Funding Acquisition, D.J.O. and L.M.T.


We describe turn-on fluorescent probes based on azide groupreduction on the fluorogenic ‘naphthalene’ moiety tofluorescent amines and intracellular hydrogen sulfide detection without the use ofan organic solvent. L1 and L2 weresynthetically modified to functional groups with comparable solubility on theN-imide site, showing a marked change in turn-on fluorescent intensity in responseto hydrogen sulfide in both PBS buffer and living cells. The probes were readilyemployed to assess intracellular hydrogen sulfide level changes by imagingendogenous hydrogen sulfide signal in RAW264.7 cells incubated withL1 and L2. Expanding theuse of L1 to complex and heterogeneous biological settings,we successfully visualized hydrogen sulfide detection in the yolk, brain and spinalcord of living zebrafish embryos, thereby providing a powerful approach for liveimaging for investigating chemical signaling in complex multicellular systems.Hydrogen sulfide (H The current workdescribes the synthesis of a highly water-soluble fluorescent probeL1 for selective hydrogen sulfide detection, comparingthe optical and biological properties, such as fluorescent intensity and cytotoxicity inliving cells along with the relatively lower solubility of L2.Finally, we report the visualization of bright fluorescent signal through theexogenous-responsive hydrogen sulfide detection in live zebrafish.Moreover, the catabolism of hydrogen sulfide is quick, which can result in continuouschanges in its concentration, leading to difficulty in the accurate analysisL1 and L2 is outlined in46Hydrogen sulfide participates in nucleophilic substitution as a reactive nucleophilein biological systems. A number of hydrogen sulfide probes based on the reduction ofaromatic azide show a delayed response time (>20 min) towardhydrogen sulfide24L1 and itsreaction with hydrogen sulfide using NaHS (a common hydrogen sulfide source) in PBSbuffer  at 37 °C. Allexperiments for L1 were conducted without the use of DMSOas a co-solvent, because L1 displays remarkable solubilityin aqueous buffer solution. Naphthalimide-based structure itself is essentially non-or low fluorescent in aqueous solution. As shown in L1 exhibited two noticeableabsorption bands at approximately 350–400 nm and400–500 nm. The probe exhibited absorbance originating fromthe naphthalene moiety at 350–400 nm, as followed by anobvious increase of new absorbance peak at 435 nm after treatment withhydrogen sulfide. The large red shift of 60 nm in the absorptionbehavior induced a color change of the solution from colorless to yellow  inaqueous media when excited at the λmax (457 nm)of L1. The consequential bright green fluorescentenhancement was also observed by 457 nm laser irradiation along with theincreased absorbance varied from 0.028 to 1.9, was observed over 30 min of reaction timewithout any background correction  found in mammalianblood101114Corresponding to the concentration-dependent increase, the dynamic simulation of thefluorescence response for o 200 μM . Bothpro 200 μM . AlthougL1 and L2 with hydrogensulfide, the selectivity profile of the probes was determined for hydrogen sulfidetoward various biologically relevant species, such as sulfur, oxygen, and nitrogenspecies (RSONS). We investigated the fluorescence response by hydrogen sulfide forL1 only and for the mixed solution ofL1 and analytes. Sulfur-containing inorganic ions,an inorganic salt (NaH2PO4), an organosulfur compound(α-lipoic acid), reactive oxygen species (H2O2), areactive nitrogen species , thiols  and L-ascorbic acid were used as analytes and proved to bechemically inert toward the probes. Based on the previous reports using hydrogensulfide as a reductant for azideL1 would have a high selectivity for hydrogensulfide over RSONS, including biologically relevant thiols. As shown in L1, another selectivity test of thefluorescence response was conducted by comparing L1 andL2 on the basis of fluorescence titration for variousanalytes. As expected, the fluorescent properties demonstrated the remarkableselectivity of both probes for hydrogen sulfide over the biologically relevantspecies; noticeable responses were not observed from other anions , and after24 h, the cell viability remained at approximately 90%, demonstratingthat both probes were minimally cytotoxic toward cultured cell lines . The celobserved . Then, tectively . InteresL1(5 μM) for 1 h at 37 °Cwas followed by the addition of different concentrations of NaHS  and then incubation for another 1 h.After removing the excess NaHS, the cells were subsequently imaged using a confocalfluorescence microscope.Incubation of RAW264.7 cells with L1 as a control showed no fluorescence, at505–605 nm under excitation of 488 nm. However,in the presence of L1 and NaHS, RAW264.7 cells showedstrong fluorescence at only 50 μM NaHS. The fluorescenceintensity increased with increases in the NaHS concentration. These resultsdemonstrate that L1 has potential in visualizing hydrogensulfide in living cells, which can likely be extended to assays involving biologicalfluids such as serum, blood, or tissue homogenates. Also, the availability of thiswater-soluble fluorescent probe will significantly help the effort of makingbiocompatible fluorescent sensors for the detection of hydrogen sulfide in livingcells.As shown in L1 as an in vivo hydrogensulfide reporter, we next examined its endogenous detection using zebrafish embryos.By taking advantage of their transparency, we treated L1into the developing zebrafish embryos at 24 h postfertilization.Incubation of 5 μM L1 withzebrafish embryos elicited fluorescent signals mainly in the yolk , a frequently usedinhibitor against cystathionine-β-synthase (CBS), a key enzymefor hydrogen sulfide synthesisL1 incubation  that were tested sinceno obvious deformity or survivability were found upon treatment  was synthesized in our laboratory.4-Bromo-1,8-naphthalic anhydride was purchased from TCI .2-[2-(2-Aminoethoxy)ethoxy]ethanol and sodium azide were purchased fromSigma-Aldrich. A mouse leukemic monocyte macrophage cell line (RAW264.7) wasobtained from the cell bank of the ATCC. L1 was prepared in dimethyl sulfoxide(DMSO) and stored at −18 °C in the dark. Allother reagents and chemicals were from commercial sources, were of analyticalreagent grade, and were used without further purification. The progress of thereactions was monitored by TLC on precoated Merck silica gel plates (60F254).6-Azido-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione  andL2 (32 μL) stock solutionvia micropipette, the UV-vis absorption(λabs = 340–400 nmand 400–500 nm) and fluorescence spectrawas recorded. Aqueous stock solutions of NaHS, L-cysteine, homocysteine,glutathione, L-ascorbic acid, α-lipoic acid,NaS2O3, Na2SO3,Na2SO4, SCN−,NaH2PO4, NO, NO2, NO3 andH2O2 was then injected via micropipette. The reactioncuvettes were incubated at 37 °C during theexperiment.Spectroscopic measurements were performed in PBS buffer at 37 °C. Stock solution ofL1was recorded after incubation at 37 °C in PBS buffer. Then L1 was treated withNaHS at various concentrations , and the fluorescence spectra were measuredafter incubation for 90 min at 37 °C. Eachdata point represents at least three trials. A linear regression was constructedusing the background-corrected fluorescence measurements, and the detectionlimit was determined to be concentration at which the fluorescence equals thatof [blank + 3σ]. The detection limit wascalculated with the following equation: Detectionlimit = 3 σ/k,k = the slop of emission intensity versus NaHSconcentration graph, σ = the standard deviation of 7blank measures.The fluorescence of seven blank cuvettes containing L1 were determined byusing Rhodamine 6G (ΦF = 0.95 inethanol) as a fluorescence standard. The quantum yield was calculated using thefollowing equation:Fluorescence quantum yields for F is the fluorescence quantum yield, A is theabsorbance at the excitation wavelength, F is the area under the correctedemission curve, and n is the refractive index of the solvents used. Subscripts Sand X refer to the standard and to the unknown, respectively.where Φ1 wasachieved as a light yellow solid .1H-NMR : δ8.56 , 8.43 , 8.31 , 7.95 , 7.78 , 4.42 , 3.86 . 13C-NMR: δ 163.52, 163.49,133.14, 131.98, 131.15, 130.98, 130.29, 130.22, 128.70, 127.97, 122.71, 121.86,72.48, 70.39, 70.04, 67.91, 61.64, 39.20. HRMS (m/z); Calcd for[M + Na]+ 430.0261, found430.0268.4-Bromo-1,8-naphthalic anhydride was dissolved in ethanol (9.3 mL) and2-[2-(2-aminoethoxy)ethoxy]ethanol  was added and stirred in the refluxing ethanol at80 °C for 2 h. TLC showed the consumption ofstarting materials at this stage. The reaction mixture was cooled, and thesolvent was evaporated. The product was purified by column chromatography onsilica gel using ethyl acetate/hexane (3:1) as the eluent. Product 1 was dissolvedin 11.6 mL of dry DMF, and NaN3  was added. After stirring the mixture for6 h at 80 °C while monitoring TLC, thesolution was diluted with H2O and extracted with ethyl acetate. Theorganic layer was separated and dried over Na2SO4. Theproduct was concentrated in vacuo, affording L1 as ayellow solid . 1H-NMR: δ 8.51 , 8.43 , 8.29 , 7.66 , 7.35 , 4.40 , 3.85 . 13C-NMR: δ 163.81, 163.36,143.25, 132.05, 131.57, 128.80, 126.68, 123.96, 122.18, 118.43, 114.51, 77.10,72.45, 70.38, 70.00, 67.92, 61.61, 39.00. HRMS (m/z); Calcd for[M + Na]+ 393.1169, found393.1170.0.5695 g (1.3949 mmol) of product 1H-NMR: δ 8.66 , 8.57 , 8.42, 8.05 , 7.86 , 13C-NMR: δ 163.60, 133.16,131.96, 131.15, 131.06, 130.61, 130.13, 128.04, 123.16, 122.30, 76.98, 40.35,30.14, 20.37, 13.79. HRMS (m/z); Calcd for[M + Na]+ 354.0, found 354.02.4-Bromo-1,8-naphthalic anhydride  wasdissolved in ethanol (80 mL), and butylamine  was added. The reaction mixture was stirred in therefluxing ethanol for 24 h. After cooling to room temperature, themixture was filtered and dried under reduced pressure to yield4.60 g (96%) of a yellow solid. 2 was dissolved in50 mL of dry DMF, and NaN3  was added. After stirring the mixture for6 h at 80 °C, the solution was diluted withH2O and extracted with EtOAc. The organic phase was washed withH2O and brine, dried over Na2SO4, andconcentrated in vacuo. The residue was placed at room temperature overnight, andthen, ether was added (to the residue yellow solid and triturated at roomtemperature) The yellow solid was filtered off and dried in vacuo to giveL2 .1H-NMR : δ8.63 , 8.58, 8.43 , 7.75 , 7.47 , 13C-NMR: δ 163.95, 163.53,143.32, 132.12, 131.61, 129.12, 126.81, 124.32, 122.67, 122.67, 118.97, 114.62,76.98, 40.24, 30.19, 20.35, 13.80. HRMS (m/z); Calcd for[M + Na]+ 317.1, found 317.1.2 g (6.02 mmol) of product 2 and 95% air at37 °C. After 24 h, the cover slips wererinsed 3 times with Dulbecco’s Phosphate Buffered Saline (DPBS) toremove the media and were then cultured in DPBS for later use. For theverification procedure, 5 μM ofL1 was added to the above cellular samples andincubated for 30 min. Then, the samples were rinsed 3 times withDPBS. The cells were incubated with NaHS  in the medium for 60 min. Prior toimaging, the cells were washed 3 times with DPBS, and the fluorescence imageswere acquired on a confocal microscope (Olympus Fluoview 1000) using anoil-immersion 60× objective.RAW264.7 murine macrophages were cultured in Dulbecco’s ModifiedEagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)in an atmosphere of 5% CO2 in air at37 °C. Following incubation for 24 h,L1 and L2 (5% DMSO as aco-solvent for only L2) were added to the abovecellular samples plates. After incubation for 30 min,10 μL of CCK-8 solution  was added toeach plate well, and the cells were further incubated for 30 min.The absorbance at 450 nm was measured with a microplate reader(SpectraMax M2 / Molecular devices).HeLa cells were plated in flat-bottomed, 96-well plates at a the density of5,000 cells/well in 200 μL of DMEM  supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin in ahumidified incubator in 5% COet al.L1 solution with two concentrations of5 μM or 25 μM for30 min in E3 egg water at room temperature, again with three time(5 min each) washes with E3 egg water afterwards. The embryos wereembedded alive in the 2.5% methyl cellulose, and fluorescence signals werevisualized under the Olympus SZX16 stereo microscope equipped with theexcitation filter GFP-A illuminated using a mercury lamp .Images were captured using Olympus XC10 camera. All zebrafish husbandry andanimal care were carried out in accordance with guidelines from the KoreaResearch Institute of Bioscience and Biotechnology (KRIBB) and all experimentalprotocols were approved by KRIBB-IACUC .Wild type adult zebrafish (AB line) reared at 28 °C withthe light cycle of 14 h light/10 h dark weregroup-mated. Spawned eggs were staged according to Kimmel How to cite this article: Choi, S.-A. et al. Structural effects ofnaphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in livezebrafish. Sci. Rep.6, 26203; doi: 10.1038/srep26203 (2016).


The modified electrode displayed favorable sensitivity, good reproducibility and stability, which suggests that CMS is promising for fabricating third-generation bioelectrochemical sensors.By using the hydrothermal method, carbon microspheres (CMS) were fabricated and used for electrode modification. The characteristics of CMS were investigated using various techniques. The biocompatible sensing platform was built by immobilizing hemoglobin (Hb) on the micrometer-sized CMS-modified electrode with a layer of chitosan membrane. On the cyclic voltammogram, a couple of quasi-reversible cathodic and anodic peaks appeared, showing that direct electrochemistry of Hb with the working electrode was achieved. The catalytic reduction peak currents of the bioelectrode to trichloroacetic acid was established in the linear range of 2.0~70.0 mmol·L Recently, the electrochemical behavior of proteins has roused great interest, and the results can be applied to the study of electron transfer mechanisms in biosystems and the construction of third-generation electrochemical biosensors or biofuel cells ,2. Howevet al. proposed a hydrothermal technique for the fabrication of colloidal spheres from aqueous cyclodextrin solution 3−/4− mixture solution and the results are shown in 6]3−/4− with increased responses.Electrochemical behaviors were verified by cyclic voltammetry in the 0.1 mol·LetR) value of CILE was found to be 37.97 Ω (curve c) and that of CTS/CILE increased to 54.75 Ω (curve d), proving that the interfacial resistance was increased with the existence of unconductive CTS film. On CTS/Hb/CILE (curve e) the etR was 82.83 Ω, which could be ascribed to the existence of Hb further increasing the interfacial resistance. While on CTS/CMS/CILE (curve a) and CTS/CMS-Hb/CILE (curve b) the etR decreased to 10.76 Ω and 15.25 Ω, respectively, showing that the existence of CMS decreased the resistance. CMS is a carbon material with good conductivity that can quicken the electron transfer.p as 87 mV. The formal peak potential (E0′) was calculated as −0.184 V (vs. SCE). The ratio of the cathodic (Ipc) and the anodic (Ipa) peak current was found to be 1.06. To explore the best CV responses of Hb on CTS/CMS-Hb/CILE, the amount of CMS cast on the electrode was optimized in the control experiments with a concentration range of 0.05 to 5.0 mg·mL−1. The highest redox currents appeared at 0.5 mg·mL−1 CMS, which was used for electrode modification.υ) on voltammetric responses of CTS/CMS-Hb/CILE was checked. A couple of redox peak appeared at different scan rates with the regression equations of peak currents and υ as Ipc(μA) = 92.5·υ (V·s−1) + 6.9  and Ipa(μA) = −78.0 υ·(V·s−1) − 10.0  (Γ*) of electroactive Hb by the formula (Γ* = Q/nAF). By subtracting the background current, the peak currents of Hb were integrated with the Γ* value found to be 9.06 × 10−10 mol·cm−2, which was larger than the theoretical monolayer coverage (1.89 × 10−11 mol·cm−2) .65 s−1)  and Hb-I−1) [−1) , indicat0′E and pH was found to be E0′(mV) = −44.25·pH − 54.73 (γ = 0.998). The slope value (−44.25 mV·pH−1) was smaller than the theoretical value (−59.0 mV·pH−1) for a one-proton and one-electron transfer process [The influence of buffer pH on electrochemical behaviors of CTS/CMS-Hb/CILE was studied in different PBS and the results are displayed in  process ,34. At pThe redox-protein-based biosensor exhibits excellent electrocatalytic ability for TCA, which is an important analytical target in biochemistry and environmental chemistry. As shown in −1) − 4.89 . The linear range and the detection limit were obtained as 2.0 ~ 70.0 mmol·L−1 and 0.30 mmol·L−1 (3σ), respectively. The cathodic peak current reached a stable value when the TCA concentration exceeded 70.0 mmol·L−1, indicating a typical Michaelis-Menten kinetic process. The Lineweaver-Burk equation of 1/Iss = (1/Imax) (Mapp/C1 + K) [MappK) at a value of 1.60 mmol·L−1, which was smaller than those of published values [The catalytic cathodic peak current at −0.243 V depended linearly on the TCA concentration with the equation of Iss(μA) = 3.04 C (mmol·LKMapp/C)  was usedd values ,37,38,39To verify its application, CTS/CMS-Hb/CILE was used for determining of TCA in lab water with the standard addition method. As shown in −1 TCA independently. An acceptable relative standard deviation (RSD) of 2.15% was determined. CTS/CMS-Hb/CILE was put into a 4 °C refrigerator for a certain period to check the storing stability. Every 5 days, the peak response of CTS/CMS-Hb/CILE to 10.0 mmol·L−1 TCA was tested, which decreased by 3.1% after 10 days and 7.5% for 25-days storage, proving the relative good stability of CTS/CMS-Hb/CILE.The reproducibility was studied by applying six Hb-modified electrodes to the determination of 10.0 mmol·LCMS was prepared using a hydrothermal method and direct electrochemistry of Hb was carried out on CMS-modified CILE. A couple of well-defined redox peaks could be seen on CTS/CMS-Hb/CILE, indicating the acceleration of direct electron transfer of Hb due to high conductivity, large surface area and good biocompatibility of CMS. The immobilized Hb molecules kept their original structure and exerted good electrocatalytic ability for the reduction of TCA. As compared with other types of redox-protein-modified electrodes for TCA detection ,37,38,39