Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. Circular plasmids exist as extrachromosomal copies and are not integrated into chromosomes. Plasmid vectors normally contain a marker gene conferring amino acid autotrophy, so that transformants can be selected in selective media (such as EMM and SD) lacking the corresponding amino acid. The effect of expression from plasmids varies in individual cells and colonies, because the copy number of plasmids varies in individual cells. This situation may cause an inconsistency in statistical quantification of the phenotype induced by the expression from plasmids. We therefore need to pay special attention to compare the expression level among colonies when careful quantification is necessary.In the long history of fission yeast studies, plasmids have been widely used to express external genes . We may also need any of fluorescent protein tag genes to fuse with the GOI. Those elements are termed ‘modules’ in this study.For integration of external genes, we need to construct plasmids containing the following gene elements: a gene of interest (GOI), a promoter and a terminator to express the GOI, a selectable marker gene for colony selection, and sequences to target the entire construct to a certain part of chromosomes (see figure 1a). It is then linearized and introduced into S. pombe cells so that homologous recombination takes place.All of those necessary modules need to be cloned in the proper order in a circular plasmid, which we call an ‘integration plasmid’ a. It is S. pombe. Those include simple plasmids , which enable integration of a cloned gene fragment at the leu1+ or ura4+ locus [nmt promoters (Pnmt1/Pnmt41/Pnmt81) have been cloned in advance to promote expression of a GOI [arg1, his3 and lys1) as target sites of integration, and introduced three antibiotic resistance genes as a selection marker (kan/hph/bsd), as well as the ura4+ gene [A number of plasmids for integration have been constructed and have greatly contributed to studies using of a GOI . Some ofa4+ gene ,7. More Thus, development of those modules expanded the scope of use of integration plasmids. Users normally choose plasmids suitable for their own experiments from the catalogue of those ‘ready-made’ plasmids, and insert a GOI into them. From the current catalogue, however, it is often hard to find an optimal integration plasmid that suffices our demand for promoters, tags, selection markers and so on. This is because a growing number of new modules (such as new fluorescent proteins and selection marker genes) have been invented. Accordingly, the number of integration plasmids that we need to prepare surges, in order to fulfil the collection of integration plasmids, considering combination of many kinds of modules. To make such an integration plasmid, we may need to modify one of the existing plasmids by repeating DNA ligation and plasmid minipreps several times until all the DNA fragments are connected in the right order. It is therefore beneficial to build up materials and methods that enable us to create those constructs easily and systematically.The Golden Gate shuffling method is a powerful method developed by Marillonnet and co-workers –11, whic2.2.1.S. pombe studies, such as Pnmt1, Pnmt41 and Pnmt81 promoter modules, and kanMX, hphMX and natMX marker modules.Here, we present protocols for one-step plasmid construction, based on the Golden Gate DNA shuffling method. We also present a library of plasmids harbouring various modules. We call them ‘module plasmids’, which can be used as entry plasmids for the Golden Gate reaction. Modules in the library include a number of representative gene elements that are frequently used in We also introduce new ‘target modules’ for gene targeting. A target module must be present in an integration plasmid to induce homologous recombination.2.2.a needs to be constructed. It needs to contain a number of gene modules that are necessary to achieve expression of a GOI and its integration. The example plasmid in figure 1a is to express a ‘GOI’ (module) in S. pombe cells. To visualize protein localization of the gene product, a fluorescent protein tag (an ‘FPtag’ module) is to be fused in frame at the C-terminal end of the GOI. The fusion gene construct is to be expressed under a promoter (a ‘pro’ module). To target the gene construct to a part of S. pombe chromosomes, the sequence of the target site (‘target’ module) needs to be cloned in the plasmid. A selection marker gene (a ‘marker’ module) is necessary for selection of proper S. pombe transformants. The ‘vector’ module contains a vector backbone including the ampicillin resistance gene (AmpR), which is useful to amplify integration plasmids using E. coli.The outline of the integration method used in this study is as follows. For integration of a gene construct, an ‘integration plasmid’ as shown in figure 1S. pombe transformation. The target modules we provide in this study contain an FseI restriction site in their middle. Digestion with FseI splits the target module into two: tar.F and tar.R (a). When the fragment is introduced into yeast cells using standard S. pombe transformation methods .The integration plasmid needs to be linearized in the middle of the target module prior to nd tar.R a. When tds (e.g. ), the liachinery a.2.3.b). The method has been invented by Marillonnet and co-workers [BsaI, for connection of many DNA fragments in a desired order.To create integration plasmids, we applied the Golden Gate shuffling method b. The me-workers . It usesa, for instance, each of six modules needs to be prepared in advance, either in a plasmid or in a fragment: a promoter module ((1), figure 1b), a GOI module (2), a FPtag module (3), a module for the selective marker gene ((4); this module also contains a terminator for the GOI but is omitted in figure 1b for simplicity), a target module to induce homologous recombination (5) and the vector module (6). All modules except (2) have been cloned in the pCR-Blunt II-TOPO vector, and the GOI module (2) has been amplified through PCR. Note that a BsaI site flanks each end of all the modules . The restriction enzyme has the unique property that enables ordered connection of many restriction fragments using a single enzyme. This is explained as follows.To make the integration plasmid in figure 1EcoRI, recognizes and cleaves the sequence 5′-G!GATCC-3′ . The cohesive end created by EcoRI is therefore unique (c). BsaI, on the other hand, specifically recognizes 5′-GGTCTC-3′. Notably, the enzyme cleaves the sequence right after it: the sequence for recognition and digestion is therefore 5′-GGTCTCn!NNNN-3′, in which ‘n’ and ‘N’ represent any nucleotide (c). BsaI thus creates DNA fragments with a variety of overhang sequences , but their religation is limited only to among those two with the same overhang sequence. By modulating the sequences, we can selectively connect those fragments in an order we desire.First, a standard restriction enzyme, e unique c. BsaI, cleotide c. BsaI tBsaI sites with ‘a’ and ‘b’ , as these two sites are designed to have distinct overhang sequences that are specific to the sites. Next, the GOI module (2) is flanked by two distinct sequences ‘b’ and ‘c’, in which b is same as the overhang b in the module (1).These cohesive ends with various overhang sequences are named as ‘a’, ‘b’, etc. For instance, a promoter module has two BsaI, cohesive ends a, b, c, etc. are generated and exposed, but only DNA fragments that contain the same overhang can be ligated efficiently. For instance, the b site of (1) can be connected with the b site in the GOI fragment (2). Similarly, c in (2) can be exclusively ligated with c in (3). Moreover, digestion with BsaI results in loss of its recognition sequence (5′-GGTCTC-3′) at one end. Once ligation between two fragments without the recognition sequence is made, the site is no longer re-digestible (c). Using this unique property, we can connect many DNA fragments in a selective manner, with only a single enzyme BsaI. In the example, these six modules can be connected in the order we designed, which yields the integration plasmid .After digestion with gestible c. Using asmid 7, b.BsaI and DNA ligase is subsequently added to the mixture, to repeat cutting and ligation by the thermal cycler . The sample is then introduced into E. coli and correct transformants are selected with ampicillin-containing plates, as in standard methods of cloning.The experimental procedure for the Golden Gate reaction is simple. Prepare all of (1)–(6) and then mix them in a tube. 2.4.E. coli, and named pBMod in this study. All module plasmids we created are systematically numbered and listed in Here we created a number of common modules cloned in plasmids so that researchers can share those frequently used modules. All the modules we created (except for the GOI) were cloned into the plasmid vector pCR-Blunt II-TOPO (Invitrogen), which confers kanamycin resistance to BsaI site (the recognition site with a cohesive end) at the 5′ end, in order to flank the GOI with two distinct cohesive ends. Cloning of modules into vectors is not necessarily required.In many cases, therefore, the only material you need to prepare by yourselves is the GOI module (2). This can be amplified from a template sequence (such as genome or cDNA) using a pair of oligonucleotide primers, both of which contain a Also, researchers may want to use other modules that are not yet cloned in this study . Those new modules can be prepared similarly, either as plasmids or as fragments. The detailed instructions will be described below.2.5.a illustrates construction of a typical integration plasmid to express a GOI fused with an FP protein in frame at its C-terminus (GOI-FP). It consists of six modules  connected in this order.Figure 2es I–VI, a connecta,b) is the group for promoter modules, where ‘(ab)’ indicates names of two cohesive ends  generated by BsaI digestion ’ a,b is tha, the fusion gene of GOI (group II. GOI (bc)) and GFP at the C-terminus ) via a linker sequence is placed under the adh1 promoter ) and the terminator ). To target the gene construct to S. pombe chromosomes, a sequence from a region on the chromosome I ) is used. For selection of transformants after integration, an antibiotic selection marker gene, kan, is used . The vector backbone ) is derived from pFA6a, which has been commonly used as a PCR template for standard methods for gene targeting [In the example of figure 2argeting ,22–25.2.6.b illustrates a catalogue of modules we present in this study (except for the GOI).Figure 2Promoter (ab) modulesI. nmt promoter , widely used as artificial promoters to control expression of external genes. Briefly, those were derived from the promoter of the endogenous nmt1 gene [We have chosen seven promoters that are commonly used in fission yeast studies: Pnmt1 (#1), Pnmt41 (#2), Pnmt81 (#3), Padh1 (#4), Padh41 (#5), Padh81 (#6) and Purg1 (#7). Those include a series of the mt1 gene . Pnmt41 mt1 gene . The tramt1 gene ).adh1 promoter variants : Padh1 is derived from the promoter region of the endogenous adh1 gene, whereas Padh41 and Padh81 are mutant versions of Padh1 with weaker promoter activities [urg1 promoter (Purg1) is an inducible promoter, which is activated in response to uracil [A series of the tivities ,27. The o uracil .As we mentioned earlier, it is possible to make new modules for other promoters  ,28,29. AGOI (bc) module: for C-terminal taggingII. This module is supposed to vary depending on individual researchers. How to prepare your own modules for your GOI will be described below . When anFPtag-C (cd): fluorescent protein modules for C-terminal tagging of GOI (bc)III. b and The C-terminus of the GOI (bc) can be tagged with one of the FPtag-C (cd) modules listed in III . The JB19F sequence consists of 19 bases, which lacks the first base of the 20-bp Fwd sequence commonly used in pFA6a-based plasmids [GGATCCCCGGGTTAATTAAgGGAGCAGGTGCTGGTGCTGGTGCTGGAGCATTTTCCGTCCCCATTACAACAGCTAGC-3′ . The translated amino acid sequence is RIPGLIKGAGAGAGAGAFSVPITTAS.We made modules for GFP (#8), mCherry (#9) and ECFP (#10). Each FPtag module has a linker sequence with the JB19F sequence a,b. The plasmids . The act. FPcontrol-C (bd): control constructs without GOI (bc)IIIab and figure 2b).We may need to construct control strains at the same time, which has the FPtag module without the GOI. In this case, control FPtag modules , we can achieve such tagging by including the tag sequence into an oligonucleotide primer to amplify the GOI. We present sequences of the primers for C-terminal FLAG and HA tagging. All information for designing oligonucleotides is summarized in Thus, by modifying sequences of the cohesive ends, we can customize which modules to include or to exclude.Terminator and selection marker modulesIV, IVa and IVb. adh1 gene (Tadh1) to express external genes [kan, hph, nat and bsd, which confer resistance to the antibiotics geneticin (G418), hygromycin B, clonNAT and blasticidin S, respectively [In order to terminate transcription of GOI, a module containing a terminator is required. In general, researchers tend to use the terminator of the al genes . Next, sectively ,19,24,25b and figure 2b).As we place a selection marker gene normally right after the terminator for the GOI, we prepared modules which contain both Tadh1 and a selection marker tandemly aligned; for example, Tadh1 + kan (#20), Tadh1 + hph (#21), Tadh1 + nat (#22) and Tadh1 + bsd (#23) in the group IV. terminator + marker (de) (figure 2b). The terminator module  is flanked by cohesive ends ‘d’ and ‘g’. In this study, we present only Tadh1 cloned in pCR-Blunt II-TOPO. If you need another terminator, simply prepare the DNA fragment through PCR, with additional cohesive ends ‘d’ and ‘g’ ) and the selection marker modules (IVb. Marker (ge)) b. The tekan (#25), hph (#26), nat (#27) and bsd (#28), as well as ura4+ (#29) and LEU2 (#30) .Selection marker modules without a terminator (IVb. Marker (ge)) are flanked by cohesive ends ‘g’ and ‘e’. In this study, we present six selection marker modules without a terminator for GOI: Target (ef) modulesV. S. pombe cells. The ‘co2’ (#31) sequence in figure 2a is derived from a region on the S. pombe chromosome I . Similarly, ‘z2’ (#32) is from a region on the chromosome II. ‘lys1’ (#33) and ‘arg1’ (#34) are derived from the endogenous lys1 (chromosome I) and arg1 (chromosome III) genes, respectively. How these modules were designed is described later  moduleVI. b) [BsaI recognition sites in pFA6a .All modules above can be connected to the pFA6a-based vector module , figure 2b) . Note thab) + GOI (bc) + pBMod-GFP (cd) + pBMod-Tadh-kan (de) + pBMod-co2 (ef) + pBMod-AmpVec (fa).When designing an integration plasmid, make sure that all modules of your choice can be properly ligated in the desired order. Alphabetical names of the cohesive ends need to circularize, starting and ending with ‘a’: pBMod-Padh1 , whose expression is regulated by the adh1 promoter and terminator.Expression of an N-terminally tagged protein (GOI) can be achieved in a similar way. Figure 3a,b; tables Here, all modules for C-terminal tagging introduced in figure 3FPtag-N (bc) modulesVII. The second module for N-terminal tagging should be an FPtag (bc) module. It has a fluorescent protein gene followed by the linker sequence, which is flanked by cohesive ends ‘b’ and ‘c’ at the 5′- and 3′-ends, respectively b.FPcontrol-N (bd) modules and GOI with FLAG/HA tag-N (bd) modulesVIIa. b). Modules for N-terminus FLAG/HA epitope tagging can be also prepared through PCR with an oligonucleotide primer containing the sequence of FLAG or HA epitope. Alternatively, such small epitope tags may be prepared through an oligonucleotide annealing method (see 2.9.2 for details). Similar to modules in the group IIIa, each module of the group contains an FPtag flanked by cohesive ends ‘b’ and ‘d’. See As a control, another strain expressing an FPtag without the GOI may need to be constructed. For that purpose, modules belonging to group VIIa can be used b. ModuleGOI (cd) modulesVIII. b).The third module is a GOI (cd) module. This should be prepared through PCR, with adding cohesive ends ‘c’ and ‘d’ at the 5′- and 3′-ends, respectively b.2.8.BsaI (not HF) from New England Biolab (#R0535S). Similar enzymes by other suppliers may also work. We strongly recommend diluting each module element (including linear modules such as the GOI) in 20 fmol µl−1 . The concentration appears critical for efficient ligation. One microlitre (20 fmol) of each module is taken and mixed in a PCR tube. Fill up to 16 µl with ddH2O.A concrete protocol for the Golden Gate reaction  is as foBsaI. The total volume will be 20 µl. The tube can be placed on a thermal cycler to perform all the reaction steps automatically. The Golden Gate reaction comprises basically two major repetitive steps followed by the termination reaction (b). The first step is DNA digestion by BsaI , and the second step is ligation . Repeat these steps up to 50 times in total. Conditions such as temperature and duration of each step may need to be varied for optimization according to instructions of suppliers of enzymes used therein. Then shift up to 50°C for 10 min followed by the heat inactivation step . The sample can be kept at 4–15°C.Add 2 µl of 10× buffer for ligase followed by 0.5 µl of T4 DNA ligase and 1.5 µl of reaction b. The fiE. coli transformation. We normally use 2 µl of the reaction sample for transformation. Colonies on LB + Amp plates could be subjected to minipreps. Note that pCR-Blunt II-TOPO used as an entry vector carries the kanamycin resistance gene, so that unreacted entry vectors never grow on LB + Amp. This eliminates contamination of colonies with unreacted module plasmids.The sample can be directly used for BsaI digestion and ligation, because once two cohesive ends without BsaI recognition sites are connected, the site cannot be redigested (a). Correct clones must have all the modules connected in the right order we expect, and cloned in the AmpR-bearing vector . This can be confirmed by digestion with NotI (figures a and 3a).Successfully ligated clones survive and accumulate during 50 cycles of repetitive digested a. Correcector 7, b. This c figures a and 3a.S. pombe. First, the plasmid is linearized with FseI (a). Appropriate host strains of S. pombe are to be used for transformation with the linearized DNA, following a standard protocol using lithium acetate [c). After transformation, choose stable colonies that can grow on the solid medium containing appropriate antibiotics, or minimal medium without an appropriate supplement (leucine or uracil), depending on the marker gene you use.A successful plasmid is then subjected to transformation of ith FseI a. Approp acetate . Homologlys1 or arg1 target module is used, correct transformants should confer both antibiotics resistance and a lysine or arginine auxotroph, respectively. This can be tested with minimal media lacking lysine or arginine, respectively. When co2 and z2 are used, colony PCR can be done to double-check correct insertion of the digested plasmid.Correct integration of the DNA fragment into the target site can be verified in the following ways. When either Thus, we can make an integrant using the Golden Gate method and modules presented in this study.2.9.2.9.1.BsaI site (both recognition site and cohesive end) at the 5′-end must be prepared. For example, in the case of C-terminal FP tagging (b), forward and reverse primers must have ‘b’ and ‘c’ at their 5′-ends, respectively.Next we introduce how to prepare the GOI modules. The GOI fragment must display cohesive ends at both ends. PCR is the best way to achieve this. A pair of oligonucleotides including the sequence of the  tagging b, forwarb), forward and reverse primers should have ‘c’ and ‘d’, respectively. If you do not need any FPtag to fuse, the cohesive ends ‘b’ and ‘d’ should be added. Actual sequences of ‘b’, ‘c’ and ‘d’ are shown in figure 2c and On the other hand, in the case of N-terminal FP tagging b, forwarATGT-(gene-specific)-3′, as shown in BsaI, whereas the bold TATG becomes the cohesive end (b), which is exposed when the amplified double strand DNA is digested with BsaI. The single base ‘a’ inserted in between serves as a spacer. The underlined ATG is expected to serve as the initiation codon (for methionine) of the GOI, as the TATG directly connects the 3′-end of the promoter module, which exposes the complementary cohesive end.For easy oligonucleotide designing, we show a table summarizing which types of oligonucleotide primers you need . For insTCAG-(gene-specific)-3′, where ‘ttt’ could be any other three bases, and the bold TCAG is the cohesive end (c) for the neighbouring FP module to be connected, which exposes the complementary cohesive end. Note that it is essential to not include termination codons at the end of the GOI in the oligonucleotide, when in-frame fusion with FPtag-C (cd) is planned.Similarly, The amplified products may need purification through gel electrophoresis and a standard DNA purification protocol. The purified product can be subjected to the Golden Gate reaction together with other modules.2.9.2.E. coli, but cloning is not necessary. Instead, we recommend preparing linear DNA fragments with BsaI sites attached to both ends using PCR as is the case for GOI, so that it can be directly applied to the Golden Gate reaction.As we mentioned, we can use new modules that are not listed in Information for oligonucleotide primer sequences necessary to construct new modules is summarized in BsaI sites. This would further increase the flexibility of the system.As mentioned earlier, a small epitope tag such as FLAG or HA can be introduced through PCR with an oligonucleotide primer that contains the tag sequence (see 2.6.IIIa and 2.7.VIIa for details). Alternatively, a DNA fragment for a small epitope tag can be prepared by annealing two complementary synthetic oligonucleotides, which are designed to expose the required overhang after annealing . The fra2.10.lys1 on chromosome I, z2 on chromosome II and arg1 on chromosome III.Additional explanation for target modules is given below. In this study, we chose the following four sites as target sites for integration: co2 and lys1 target module as an example.lys1+ gene as the target site, and named the first half of the region as ‘lys1F’, and the latter half as ‘lys1R’ (a). We amplified DNA fragments of both lys1F and lys1R, and the lys1R DNA fragment was placed ahead of the lys1F fragment, with an FseI restriction site in between (b). Cohesive ends named ‘e’ and ‘f’ (figure 2c and figure 4b). Plasmids for other target modules presented in this study .First, we chose an approximately 1 kb region in the middle of  ‘lys1R’ a. We amp between b. Cohesi figures ; table 1d).Inverted connection of the lys1F and lys1R sequences is not necessarily required for integration itself, as other integration plasmids invented previously are not in this style . In thosfigure 42.11.As a proof of principle, we show examples of integrant strains made using the materials and methods presented in this study.nmt1 promoter (derived from #4), C-terminal GFP tag (#8), the kan selection marker (#20) and the co2 target sequence (#31). As a GOI, we used either of wild type and 10 truncated mutants of the cut7 gene (1.2–3.0 kb). In total, 11 Golden Gate reactions were performed. Transformation of E. coli cells using each reaction sample was performed, and some colonies grown on LB + Amp were chosen for minipreps. Plasmids were then digested with NotI to confirm successful ligation. Rates for successful ligation were: 1/1 (in three reactions), 1/2 (in one reaction) and 3/3 (in seven reactions), demonstrating that the Golden Gate reaction using the materials efficiently yields correct plasmids.First, we created integration plasmids to express the fusion protein of Cut7 with GFP, with the PS. pombe chromosome. Figure 5a is a schematic describing which modules were used. We prepared three reaction samples (b): 〈1〉 Pnmt1-driven lifeact-GFP with the selection marker kan; 〈2〉 Pnmt41-driven lifeact-GFP with kan; and 〈3〉 Pnmt1-driven lifeact-mCherry with the selection marker hph. Five modules  were mixed for one Golden Gate reaction.In the next example, we aim to visualize the actin cytoskeleton by the fusion gene of GFP or mCherry with lifeact ,34, whic samples b: 〈1〉 Pnles 1–5, a were mia). At the same time, the forward and reverse primers carried cohesive ends ‘b’ and ‘d’ at the 5′ end, respectively. The amplified product (2) was the in-frame fusion gene of lifeact-GFP (or lifeact-mCherry) with the linker, so that it could be connected both to a promoter module (with ‘b’ in 1) and to a terminator + marker module (with ‘d’ in 3) (a). Researchers thus may modify any parts of Golden Gate modules according to the requirement.In these cases, the GOI should correspond to the lifeact gene, but it was too short in sequence (51 bp) to prepare through PCR. Instead, the lifeact sequence was included in the forward oligonucleotide primer to amplify the GFP (or mCherry) gene with the linker, using the module plasmid for the GFP tag (#8)  as a template 2, a. At thed’ in 3) a. ResearE. coli colonies were seen on LB + Amp plates. Eight colonies from each transformant were chosen for plasmid minipreps, and digestion with NotI confirmed that 5, 6 and 2 out of 8 samples showed correct ligation for the integration plasmid 〈1〉, 〈2〉 and 〈3〉, respectively. Correct integration plasmids were then digested with FseI, and the linearized fragments were used for transformation of S. pombe cells expressing mCherry-tubulin or GFP-tubulin. Stable transformants grown in the selective medium were chosen for colony PCR to test whether accurate integration occurred. Integration was mostly accurate and efficient, as seven out of eight colonies for the strain 〈1〉 were positive (c). Positive colonies for each strain were cultured in rich medium overnight, and observed by microscopy. Actin patches, cables and rings could be visualized with lifeact-GFP and lifeact-mCherry in all three constructs by conventional widefield microscopy (b). The lifeact-GFP signal was also sufficiently robust to allow structured illumination microscopy (SIM), revealing individual actin cables in exquisite detail (d). With Pnmt1-driven lifeact-GFP actin patches could be viewed in vertical stacks taken of horizontally arrayed cells [e).After transformation of three Golden Gate samples, 30–150 transformed positive c. Positicroscopy b. The lie detail d. With Pin vivo. Thus, the Golden Gate method works with the modules provided in this study, as expected.These observations indicate that, at least in rich medium (YE5S) containing thiamine, Pnmt1 was better than Pnmt41 for visualization of the actin organization 3.3.1.S. pombe work, a feature particularly important in the context of large-scale phenotyping projects [This study presents materials and methods that enable systematic construction of many kinds of integration vectors. This provides a flexible, scalable way to capitalize on the ever-increasing number of fluorescent protein and selection marker gene modules available for projects –38.For efficient Golden Gate reaction, we compiled some technical tips in experimental procedures.et al. [The number of modules in a reaction does not matter for now: Golden Gate reaction works efficiently even with 5–8 DNA fragments of kilobase length. We have not tried reactions with more than nine modules of kilobase length, but Engler et al.  use 10 DThere are three main reasons for failure in the Golden Gate reaction in our experience. These points should be considered when the reaction does not work well.BsaI. BsaI needs to be fully active for efficient Golden Gate reaction. We advise to consider using a new lot when in trouble. To test whether the current lot retains sufficient enzymatic activity, we recommend digestion of an existing module plasmid with BsaI in the conditions for the Golden Gate reaction using a thermal cycler (b), rather than doing it simply at 37°C.First, decreased activity of l cycler b, rather−1. Unequal concentration among modules significantly deteriorates the efficiency to produce circularized plasmids.Second, concentration of each module was not equal, or was too high or too low. We strongly recommend to adjust the concentration of all modules to 20 fmol µlBsaI sites existed in the GOI module. Examine whether the GOI (and other modules) contains unintended BsaI sites inside. Existence of unintended BsaI sites in modules blocks circularization of the integration plasmid. For this reason, we removed two internal BsaI sites from pFA6a to create the vector module . Each portion can be amplified through PCR separately with oligonucleotide primers with a particular combination of cohesive ends, to yield three GOI fragments: b-P-x, x-Q-y and y-R-c. Note that ‘b’ and ‘c’ are cohesive ends connectable to the promoter and FPtag modules , hygromycin B , clonNAT  or blasticidin S , for selection of transformants by kan, hph, nat or bsd, respectively [Strains used in this study are listed in genetics ,40. For was used . Accordiectively ,24.TablFor confirmation of proper integration at the co2 site of chromosome I, colony PCR was performed with a pair of oligonucleotide primers: co2CP1F: 5′-AAGCCTCGTCTAAGCGAATC-3′ and Ttef-NtoC: 5′-CGACATCATCTGCCCAGATGCG-3′.4.2.c; tables Our Golden Gate DNA shuffling method is based on the original report of the method ,10. Detafigure 24.3.BsaI recognition site and a designed cohesive end was added to each side of module using a pair of oligonucleotide primers to amplify the module fragments.Modules except for GOI (N) and GOI (C) were cloned into the plasmid pCR-Blunt II-TOPO after PCR amplification, according to instruction given in the Zero Blunt TOPO PCR cloning kit . The sequence of a nmt1 promoter Pnmt1 [S. pombe genomic DNA (chromosome III: 1 837 263–1 838 542) in pombase [nmt41 and Pnmt81 were made through site-directed mutagenesis using the PrimeSTAR mutagenesis kit . The adh1 promoter Padh1 and its derivatives Padh41 and Padh81 were prepared similarly (chromosome III: 1 590 560–1 591 304).Origin of module fragments is as follows: for Promoter modules, the er Pnmt1  was ampl pombase . Its derAequorea victoria GFP (S65 T) and its variant CFP (ECFP) were amplified, using pFA6a-GFP(S65 T)-kanMX [Discosoma sp. was amplified similarly using pFA6a-mCherry-hphMX [For fluorescent protein modules, coding sequences for T)-kanMX  and pFA6T)-kanMX  as a temry-hphMX  as a temadh1 terminator (with or without a selection marker) were amplified from pFA6a-based plasmids containing antibiotics resistance genes kan [hph and nat [bsd [ura4+ and LEU2, respectively.Modules containing the enes kan , hph and and nat ,25, bsd nat [bsd , as wellBsaI sites, which hampers its use as an entry plasmid for the Golden Gate system. Therefore, we removed the sites as follows: a short fragment comprising 137 bases (5′-CAGCTGAAGC-· · ·-TGCCGGTCTC-3′), which contains a BsaI recognition site, was removed from the original pFA6a, and the other BsaI recognition site in the ampicillin resistance gene was eliminated through site-directed mutagenesis.The vector module was derived from the plasmid pFA6a . The oriFseI site was inserted at the border, which yields the fragment ‘co2R-FseI-co2F’.Target modules were created as follows. Regions corresponding to 2 939 891–2 940 410 (co2F) and 2 939 404–2 939 887 (co2R) of chromosome I were amplified through PCR. These two were connected in the inverted order and an additional FseI-z2F’. The arg1 module (arg1R-FseI-arg1F) is composed of arg1R (1 614 151–1 614 549 of chromosome III) and arg1F (1 613 754–1 614 150) connected with an FseI site. The lys1 module (lys1R-FseI-lys1F) is composed of lys1R (3 740 972–3 741 481 of chromosome I) and lys1F (3 740 472–3 740 971) connected with an FseI site.Similarly, z2R (3 485 793–3 486 320 of chromosome II) and z2F (3 485 246–3 485 782) were connected to make the fragment ‘z2R-4.4.ATGGGTGTCGCTGACCTTATCAAGAAGTTCGAGTCTATTTCTAAGGAAGAAGGGATCCCCGGGTTAATTAAGG-3′ and GFP-R: 5′-tttGGTCTCaAGACTTAACTAGTTTTGTATAGTTCATCCATGCC-3′. The underlined sequence encodes the lifeact protein [Fusion genes of lifeact-GFP and lifeact-mCherry were made as follows. The gene encoding GFP(S65T) with the linker sequence was amplified using pBMod-GFP(C) (#8) as a template with a pair of oligonucleotide primers: LA-linker-GG-F-2: 5′-tttGGTCTCaT protein . For lif4.5.oftWoRx system was used for conventional microscopy, as described previously [z-axis, with 0.4 µm intervals.Cells were grown for 12 h in YE5S liquid medium and then subjected to microscopy. The DeltaVision Seviously . BrieflyoftWoRx.Methods for head-on cell imaging were performed as described previously . In brie


Knowledge of factors that contribute to non-persistence with disease modifying anti-rheumatic drugs (NP) is essential to improve rheumatoid arthritis (RA) outcomes. Aims of the study were to investigate patient’s motivations and risk factors for NP in a cohort of early RA patients.Up to September 2012, data from 149 patients, who had at least 1 year of follow-up, at least one drug indication, and at least 2 consecutive six-months-apart rheumatic evaluations that included assessment of compliance were reviewed. NP and patient’s motivations of NP were evaluated according to a questionnaire. NP was defined when patients referred that they had completely stop RA medication, “Sometimes”, “Almost always” or “Always”. Patients had to pay for their medication.Descriptive statistics and logistic regression models were used. Statistical significance was set at a p value of less than 0.05. The study was approved by the internal review board.Up to cut-off, 715 questionnaires were applied to 149 patients, who had follow-up of 58.7 ± 27.9 months and were indicated 2.4 ± 0.7 DMARDs/patient/follow-up.Patients were most frequently female (88.6%), middle-aged ([mean ± SD] age of 38.5 ± 12.8 years) with lower-middle/lower socio-economic status (87.9%) and scholarship of 11 ± 3.9 years.Ninety-nine (66.4%) patients were NP and filled 330 questionnaires. Multivariate analysis showed that years of formal education , perception of at least some difficulty to find arthritis medication  and perception that arthritis medication is expensive  at the first evaluation of patient’s compliance were all predictors of NP.Among the 99 NP patients, 25 (25.3%) were recurrent-NP and accumulated more disease activity. The combination of both reasons of NP (“Because it was not available at the drugstore” and “Because the medication is very expensive”) when selected at the first evaluation of compliance was the only variable to predict recurrent NP, OR: 4.8, 95% CI: 1.1-20.8, p = 0.04.Health systems should provide (first line) treatment for RA as a strategy to improve compliance with therapy and clinical outcomes, particularly in vulnerable populations. Rheumatoid arthritis (RA) is a chronic inflammatory disease that may result in significant disability, morbidity and increased mortality -3. PatieCurrent RA treatment guidelines recommend early aggressive management with disease modifying anti-rheumatic drugs (DMARDs) in order to improve patient’s outcomes. However, poor compliance with therapy is a substantial problem that affects 20% to 70% of the patients during follow-up -16. LocaMany factors have been related to poor compliance with drugs in patients with RA  and inclthe Compliance Questionnaire (CQ), that evaluates both constructs, adherence with and persistence (P) on therapy and investigates patient’s motivations of inadequate persistence with therapy [In 2004, we establish an Early Arthritis Clinic in a referral Centre for Rheumatic Diseases in México City. Since the beginning, patient’s medication behavior has been assessed, initially through a structured interview and since November 2008 through an instrument locally designed,  therapy .In addition to the distinctive features above mentioned, RA patients from Latin-America are frequently uninsured, had a low socio-economical status and are less educated than RA patients from developed countries that may additionally impact compliance and outcomes. From the perspective of achieving desirable clinical outcomes, the negative effect of poor compliance with DMARDs needs to be minimized. However, in order to formulate effective strategies to contain the problem there is a need to investigate the factors that contribute to poor compliance. The aims of the study were:1. To investigate most frequent patient’s motivations for non-persistence with disease modifying anti-rheumatic drugs (NP) in early RA Mexican Mestizo patients.2. To identify risk factors of both, NP and recurrent NP.The early arthritis Clinic at the Instituto Nacional de Ciencias Médicas y Nutrición was initiated in 2004. Patients entering the Clinic had disease duration of less than a year when first evaluated and no specific rheumatic diagnosis but with RA. Patients are evaluated every two months during the first 2 years of follow-up and thereafter every 2, 4 or 6  months depending on patients and disease characteristics. Treatment is prescribed by the rheumatologist in charge of the Clinic and is “treat to target” oriented.Our Center belongs to the National Institutes of Health from México. Patients had health expenditures government coverage depending on their incomes; coverage varies from 0 to 100%. Most of the patients (70%) had 70% of their expenditures covered. Costs that are partially covered include: medical consultations, hospitalizations, emergency room and critical care unit, laboratory and all the diagnostic procedures available. Patients need to pay for their medication and these are not provided by the local pharmacy .At study entry a complete medical history and demographic data are recorded along with disease specific autoantibodies. Medical evaluations are standardized and included swollen and tender joint counts, acute reactant-phase determinations, patient- and physician- reported outcomes, comorbidity, and treatment’s assessment  along with the evaluation of compliance with therapy.CQ is a 22-items questionnaire  to determine NP; patients who scored item 10 as 1, 2, 3 or 4 are directed to answer item 11 mean while those who score it as 0 are directed to proceed to item 12; item 11 investigates patient’s reasons of inadequate medication taking behavior and includes 15 predefined answers (most of them obtained from literature review) and one open answer.Performance of the CQ has shown high sensitivity and satisfactory specificity to detect persistence on DMARDs . CQ is fDisease activity was defined according to cut-offs of the Disease Activity Score, when 28 joints are examined (DAS28), ,22.Disability was evaluated according to the HAQ .ersistent if in item 10 “In the past 6 months, how often did you completely stop taking your medication? “ boxes 0 (“Never”) or 1 (“Almost never”) were filled.A patient was considered to be PNP if in tem 10 boxes 2 (“Sometimes”), 3  and 4 (“Always”) were filled.A patient was considered to be be recurrent-NP if he/she was defined as NP at every time the CQ was applied.A patient was considered to Comité de Ética en Investigación del Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán). Written informed consent was obtained in order to have patient’s charts reviewed and data presented in scientific forums or published.The study was approved by the internal review board . Statistical significance was set at a p value of less than 0.05.From the present report, data from patients with at least 1 year of follow-up, at least one DMARD indication over follow-up, and at least 2 consecutive six-months-apart evaluations for compliance were reviewed. Up to September 2012, 149 patients met the inclusion criteria, which corresponded to 93% of the population of the Early Arthritis Clinic.Up to cut-off, 715 CQ were applied to 149 early RA patients who had (mean ± SD) follow-up of 58.7 ± 27.9 months and were indicated (mean ± SD) 2.4 ± 0.7 number of DMARDs/patient/follow-up. Main characteristics of the patients at cohort inclusion are summarized in Table Among the 715 CQ applied, 330 46.2%) were classified as NP. Table % were clEvery evaluation of compliance was performed as part of a complete rheumatic assessment which included disease activity evaluation as per DAS28. Accordingly, patients were classified as in remission (DAS28 ≤ 2.6) in 109 out of 330 evaluations and with disease activity in the remaining 221 evaluations. There were subtle differences in patient’s motivations of NP between active patients and patients who achieved remission: formers referred most frequently “I was not at home when I had to take my medication” and “nobody reminded me to take my medication” than their counterparts, as shown in Table In the past six months, how much difficulty did you had to find your arthritis medication at the drugstore?” and to the question (Number 9)” In the past 6 months, how much expensive did you consider was you arthritis medication?” (Table Ninety-nine (66.4%) patients were classified as NP at some point during their follow-up and filled 330 CQs, meanwhile 50 were classified as persistent (33.6%) and filled 385 CQs. P and NP patients were compared. No differences were found between groups regarding age, percentage of females, residence, occupation, marital status, health coverage and baseline percentage of patients with RF, baseline -disease activity,- comorbidity and –treatment, and cumulative-comorbidity and -treatment (data not shown). Nonetheless, NP patients had more years of formal education, longer follow-up, had more antibodies to cyclic citrullinated peptides (ACCP) and higher cumulative disease activity and disability than their counterparts, Table Multivariate analysis showed that years of formal education , perception of at least some difficulty to find arthritis medication  and perception that arthritis medication is expensive  at the first evaluation of patient’s therapy behavior were all predictors of NP (age and gender correction was done).As it has been demonstrated that persistence impacts outcomes, we ought to define if there was a particular number of times a patient had to be NP in order to have greater disease activity and disability. Recurrent-NP patients had greater disease activity and disability than their counterparts, (mean ± SD) cumulative DAS28: 3.7 ± 2 vs. 2.9 ± 1.7, p = 0.04 and cumulative HAQ: 0.3 ± 0.2 vs. 0.1 ± 0.1, p = 0.05.Among the 99 NP patients, 25 (25.3%) were recurrent-NP and their characteristics were compared to their counterparts (74 patients); no differences were found between groups besides those related to disease activity and disability. The number of patients with DAS28 remission was lower among recurrent-NP patients  as was the number of patients with disease activity improvement according to European League Against Rheumatism (EULAR) categories .Logistic regression models were constructed in order to investigate predictors on recurrent-NP; in all of them, any possible combination of the 15 patient’s motivations of NP when referred at first evaluation of compliance were included. Models yield similar results: the combination of both reasons of NP (“Because it was not available at the drugstore” and “Because the medication is very expensive”) when mentioned at the first time the CQ is applied was the only variable to predict recurrent NP, OR: 4.8, 95%CI: 1.1-20.8, p = 0.04. Results were similar after correction for disease duration, gender and age at disease onset.From the perspective of healthcare providers, therapeutic compliance is a major clinical issue in RA patients as it impacts disease’s outcomes ,17,18. IWe found that 2 out of 3 patients were classified as NP. In the literature, different studies have targeted adherence to DMARDs ,15,16,24Factors identified from studies and reviews with poor compliance may be grouped into several categories, and divided into 5 domains according to the World Health Organization: namely patient-centered factors, therapy-related factors, healthcare systems factors, social and economic factors, and disease factors ,26. In oPatient’s perception of at least “some difficulty to find arthritis medication” (previously reported as lack of availability at the drug store) and that “arthritis medication is expensive”  were among the most frequent motivations for NP; when both of them were selected at the first evaluation of patient’s compliance, they predicted NP (in addition to a higher education level) and also recurrent- NP. Cost is a crucial issue in patient’s compliance especially for patients with chronic diseases ,30. A nuIn our study, most frequent patient’s motivations for NP were “unintentional” motivations. As opposed to intentional motivations, they reflect a person’s ability and skill at medicine taking, including forgetting, poor manual dexterity, losing medicines or not being able to afford them. Meanwhile, intentional non-adherence is a behavior driven by the decision not to take medicines ,25,38; dSome limitations of the study need to be addressed. We did not use a well-validated questionnaire scale to assess persistence; we applied a short-patient-oriented questionnaire, locally designed which has shown adequate internal consistency, high sensitivity and satisfactory specificity to detect persistence on DMARDs . We did Compliance with medication is a dynamic process and fluctuates over time; with a more extended length of follow-up, patients formerly classified as persistent with therapy may become non-persistent. We limited the study of factors associated to medication persistence, to patient’s motivations; ultimately, it is the patient who decides whether or not to take his/her medication as prescribed, and non adherent patient’s opinion are essential in order to design effective interventions. Finally, we investigate a limited number of patient’s motivations for non-persistence with medication although they were selected based on the existing literature.Almost half of Mexican Mestizo patients with early RA and partial health coverage do not take their disease modifying anti-rheumatic drugs as directed. Most frequent patient’s motivations for non persistence were non-intentional motivations as forgetfulness, lack of financial resources and lack of availability at the drug store. When patients identified concomitantly “some difficulty to find arthritis medication and that arthritis medication is expensive” at their first evaluation of compliance with therapy they may be at risk of non-persistence during their follow-up and of deleterious outcomes. Health systems should provide (first line) treatment for RA as a strategy to improve compliance with therapy and clinical outcomes.RA: Rheumatoid arthritis; DMARDs: Disease modifying drugs; CQ: Compliance questionnaire; P: Persistence; NP: Non-persistence with disease modifying anti-rheumatic drugs; DAS28: Disease Activity Score ; HAQ: Health assessment questionnaire; ACCP: Antibodies to cyclic citrullinated peptides.The authors declare that they have no competing interests.VPR participated in the design and conception of the study, performed clinical evaluations and acquired data, drove statistical analysis and drafted the manuscript. ICY participated in the study design and conception, performed statistical analysis and helped to draft the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/336/prepubCompliance Questionnaire.Click here for file


Primary hyperuricemia, an excess of uric acid in the blood, is a major public health problem. In addition to the morbidity that is attributable to gout, hyperuricemia is also associated with metabolic syndrome, hypertension, and cardiovascular disease. This study aims to assess the genetic associations between Apolipoprotein E (APOE) polymorphisms and hyperuricemia in a Chinese population.A total of 770 subjects (356 hyperuricemic cases and 414 normouricemic controls) were recruited from the Ningxia Hui Autonomous Region, China. A physical examination was performed and fasting blood was collected for biochemical tests, including determination of the levels of serum lipid, creatinine, and uric acid. Multi-ARMS PCR was applied to determine the APOE genotypes, followed by an investigation of the distribution of APOE genotypes and alleles frequencies in the controls and cases.P = 0.003) and the APOE-ε2 allele  were significantly higher in the hyperuricemic group than in the normouricemic group. Furthermore, male cases were more likely to have the APOE-ε2ε3 genotype and APOE-ε2 allele, compared with male controls. In both Han and Hui subjects, cases were more likely to have the APOE-ε2ε3 genotype and the APOE-ε2 allele compared with controls. Furthermore, multivariate logistic regression showed that carriers of the APOE-ε2ε3 genotype  and the ε2 allele  were significantly more likely to experience hyperuricemia than carriers of the ε3/ε3 genotype and the ε3 allele after adjustment for sex, body mass index (BMI), diastolic blood pressure (DBP), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), creatinine (Cr) and fasting blood glucose(FBG).The frequencies of the APOE-ε2ε3 genotype (17.70% vs. 10.39%, The APOE-ε2ε3 genotype and the APOE-ε2 allele are associated with serum uric acid levels in Chinese subjects, indicating that individuals carrying the APOE-ε2 allele have a higher risk of hyperuricemia than non-carriers. Serum uric acid (SUA) is the final product of purine metabolism in humans. Hyperuricemia(HUA), which is caused by the overproduction of urate or, more commonly, by the reduced renal excretion of urate, contributes to the development of gout The apolipoprotein E (APOE) gene, located in the 19q13 chromosomal region, encodes the ApoE protein, which plays an important role in transporting cholesterol and other lipoproteins. The APOE gene has three common alleles, designated as ε2, ε3, ε4, and six different genotypes, ε2/2, ε2/3, ε2/4, ε3/3, ε3/4, ε4/4. These three common alleles result from two single-base polymorphisms within exon 4, at codons 112 and 158In addition to the impact on the serum lipid profile, blood pressure levels Therefore, the objective of this study was to assess the genetic associations of APOE polymorphisms and hyperuricemia in a Chinese population.The subjects were selected from a population-based cross-sectional survey, the Chinese Physiological Constant and Health Condition (CPCHC), in the Ningxia Hui Autonomous Region (2011). Briefly, this survey included 6056 participants, aged 10 to 80 years, who underwent physical examination and assays of serum biochemical indicators, including the serum urate levels. Of these 6056 subjects, 356 participants were initially screened as hyperuricemic cases in the present study. One or two region-, gender- and age-matched (the age difference did not exceed 2 years) normouricemic controls for each case were randomly selected at the time of the case diagnosis. Hyperuricemia was defined as SUA≥7 mg/dl  or SUA≥6 mg/dl , which is a widely accepted diagnostic criteria All procedures were performed following a 9–12 hour overnight fast and all subjects were told to consume a bland diet before blood testing. Blood pressure (BP) was measured using an OMRON HEM-7000 electronic sphygmomanometer  after the participant had rested for ≥10 min. Blood was drawn from the antecubital vein of the arm. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured with a Beckman AU Series Automatic Biochemical Analyzer (Japan), using Sekisui Medical (Japan) reagents. Fasting blood glucose (FBG), uric acid (UA), creatine (Cr), and blood urea nitrogen (BUN) were measured with the same instrument, using Beckman AU reagents. HbA1c was examined by high-performance liquid chromatography (HPLC) on a Bio-Rad Diamat automated glycosylated hemoglobin analyzer (USA).5′-ATGCCGATGACCTGCAGAATT-3′)/(5′ATGCCGATGACCTGCAGAATC-3′), Cys112/Arg112(5′-CGCGGACATGGAGGACGTTT-3′)/(5′-CGCGGACATGGAGGACGTTC-3′), and a common reverse primer (5′-GTTCAGTGATTGTCGCTGGGCA-3′). The common primer was paired with Cys158/Arg158 or Cys112/Arg112 and produced an amplicon of 451 and 588 base pairs (bp), respectively. A 218-bp fragment of the LDLR gene was co-amplified to function as an internal positive control, and the PCR primer sequences were 5′- GTGGCACCGAGACCAAACTC-3′ and 5′- AGTGCAAGGAGACCACGGGA-3′. The PCR conditions were denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 63°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 5 min. At the end of the PCR cycles, the products were resolved by electrophoresis on 2% agarose gels to validate the amplification of the specific PCR product expected.Genomic DNA for APOE genotyping was extracted from peripheral blood mononuclear cells with the Genomic DNA Purification System . The DNA samples were then analyzed using the amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) assay P<0.05, and all statistical analyses were conducted using SPSS.Continuous variables with a normal distribution are given as the mean ± standard deviation (SD) and were analyzed by t-test. Variables with a non-normal distribution are given as the median (interquartile range) and were compared by the Wilcoxon rank sum test. Categorical variables  were analyzed as percentages using the Chi-squared test. The extremely rare genotype groups  were excluded from the genotype analyses, but they were included in the allele analyses. ANOVA or the Wilcoxon rank sum test were used to compare the data related to the clinical parameters among the different APOE genotypes. Genotype and alleles frequencies for APOE were calculated and compared between the hyperuricemia cases and the control subjects using the Chi-squared and Fisher’s exact test. The associations between the ApoE genotypes and alleles and hyperuricemia were measured by calculating odd ratios (ORs) and 95% confidence intervals (CIs) using logistic regression. Univariate logistic analysis was applied to examine association between each potential risk factor and HUA. In multivariate analyses, all factors that were statistically significant were adjusted as potential confounding variables and the adjusted ORs and 95% CIs were calculated. Hardy-Weinberg equilibrium was assessed using the chi-squared test. In each model, the homozygous ε3/ε3 genotypes formed the reference group. Statistical significance was assumed at 1C), uric acid (UA), creatine (Cr) and blood urea nitrogen (BUN), but they had lower high-density lipoprotein cholesterol (HDL-C) levels compared with the normouricemic group (P<0.05). The levels of Total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were significantly higher among women in the hyperuricemic group than in the normouricemic group, while the diastolic blood pressure (DBP) was significantly higher for men in the hyperuricemic group than in the normouricemic group.As shown in P>0.05). The frequencies of the ε2ε3 genotype and the ε2 allele in the hyperuricemic group were significantly higher than those in the normouricemic group . However, the distributions of the ε3ε3 genotype and the ε3 allele were significantly lower in the hyperuricemic individuals .A total of 356 hyperuricemic cases and 414 normouricemic subjects were evaluated, and the frequencies of the APOE genotypes and alleles are shown in P = 0.039). Furthermore, the APOE ε2 allele frequency was significantly higher in the hyperuricemic group compared with the normouricemic group both in males (P = 0.014) and females (P = 0.049). In addition, the frequencies of the APOE-ε3ε3 genotype and the ε3 allele in the hyperuricemic group were significantly lower than those in the normouricemic group in males but not in females. Meanwhile, we observed that the prevalence of the APOE ε2 allele in males was higher than that in females in both the hyperuricemic (11.3% vs. 8.8%) and the normouricemic (6.7% vs. 4.8%) groups, although the difference was not statistically significant. Similar results were obtained from different ethnic groups  and ε2 allele  were significantly more likely to present hyperuricemia when the ε3/ε3 genotypes was set as the reference group. Furthermore, sex, BMI, WC, SBP, DBP, TC, TG, HDL-C, LDL-C, BUN, Cr, FBG and HbA1C were found significantly associated with hyperuricemia. Considering the high degree of correlation between some variables , in the next multivariate logistic regression model, only key factors sex, BMI, DBP, TG, LDL-C, Cr and FBG were included and adjusted as potential confounding variables. As shown in P = 0.001, OR = 2.194, 95% CI 1.362–3.537) and ε2 allele  were also significantly associated with hyperuricemia after adjusting for sex, BMI, DBP, TG, LDL-C, Cr and FBG.In the present study we observed that APOE gene polymorphism is associated with hyperuricemia in Chinese subjects. The frequencies of both the APOE-ε2ε3 genotype and ε2 allele were higher in the hyperuricemic group than in the normouricemic group (p<0.01), and individuals with the ε2ε3 genotype or ε2 allele had significantly higher SUA levels than ε3ε3 genotype or ε3 allele carriers. To the best of our knowledge, this is the first study to examine the possible associations between APOE polymorphism and hyperuricemia in China.APOE gene polymorphism is known to be closely related to cardiovascular disease, hypertension, dyslipidemia, diabetes, and metabolic syndrome In this Chinese population study, the frequencies of the ε2ε3 genotype and the ε2 allele in hyperuricemic individuals were significantly higher than those in normouricemic subjects, but no difference in the frequency of the ε4 allele was found between the two groups. We further analyzed the associations of gender and ethnicity with APOE polymorphism, and we found that the APOE ε2 allele frequency was also significantly higher in the hyperuricemic group compared with the normouricemic group, not only in males and females but also in the Han and Hui populations. InteresA logistic regression model demonstrated that the ε2ε3 genotype and the ε2 allele were significantly positively associated with hyperuricemia, and the association remained significant after adjustment for gender, BMI, DBP, TG, LDL-C, FBG and Cr. The ε2ε3 genotype or ε2 allele could be considered as independent risk factors of hyperuricemia. The underlying mechanisms for the increased risk of hyperuricemia in individuals with the ε2ε3 genotype or the ε2 allele are largely unknown, but they are most likely related to increased purine metabolism and uric acid production or decreased renal clearance of uric acid. The presence of the APOE-ε2 allele in patients with gout is associated with reduced renal excretion of urates Dietary habits, the intensity of physical activity, drinking and smoking may also be associated with the serum uric acid level. However, we did not include these factors in the multivariable analysis, which is a limitation of our study. In addition, due to the small sample size for subjects with the ε2ε2, ε2ε4 and ε4ε4 genotypes, these genotypes were not included in the logistic regression analysis.In conclusion, this study is the first to report that the APOE ε2 allele is positively associated with SUA and could be an independent risk factor for hyperuricemia in a Chinese population. Larger studies in the future will further clarify the correlation between APOE polymorphisms and uric acid metabolism.


Bovine and human plasma albumin were used. We observed that low albumin concentration decreases [Ca2+]c, while higher concentration, induces increase in [Ca2+]c. It was shown that increase in [Ca2+]c was mediated by inositol 1,4,5-trisphosphate and released from internal stores. Increase in [Ca2+]c was reduced to 19% by blocking the transforming growth factor-beta (TGF-βR) receptor. Albumin induces DNA synthesis in a dose-response manner. Finally, induction of DNA synthesis can be partially blocked by heparin and block of TGF-β; however, the combination of both incompletely inhibits DNA synthesis. Therefore, results suggest that mechanisms other than Ca2+ signals and TGF-β receptor activation might induce DNA synthesis in a lesser degree. These results may be important to further understand the mechanisms involved in the transition from normal to focal epileptic brain.So far, a little is known about transition from normal to focal epileptic brain, although disruption in blood-brain barrier and albumin had recently involved. The main objective of this work is to characterize the response of cultured human astrocytes to plasma albumin, including induction of DNA synthesis. Cortical tissue was obtained from 9 patients operated from temporal lobe epilepsy. Astrocytes were cultured for 3-4 weeks and cytosolic calcium concentration ([Ca Epilepsy is a major neurological disorder, affecting about 0.5–2% of the global population and approximately 20–30% of these patients are resistant to drug therapy –3. DespiβR) c through an Alb receptor, while higher p-Alb concentrations increased the [Ca2+]c in a dose-dependent manner, eliciting calcium-waves that travelled by gap-junctions, probably involving inositol 1,4,5-trisphosphate , could activate several types of cells, including xenopus oocytes, PC12, and fibroblasts microglia , 16–19. d slices . Indeed,te . These te  and operated to control their seizures. The patient's consent was obtained in all cases according to the Declaration of Helsinki and all protocols were approved by the Institutional Ethical Committee . A mass of brain tissue (approximately 3 g) containing grey and white mater, obtained from a nonspiking region of temporal lateral cortex, was obtained from 9 patients  suffering from drug-resistant temporal lobe epilepsy, all with a well-documented medical history.99mTc-HmPAO, magnetic resonance imaging (MRI) 1.5 T, and video-electroencephalography (v-EEG) using 19 scalp electrodes according to the international 10–20 system. In some patients, six-contact platinum foramen ovale electrodes (FO), with 1 cm center-to-center spacing , were inserted bilaterally under general anesthesia, as previously published c) was measured at room temperature from several regions of interest . Cells R) and basal values (R0) were subtracted (ΔR = R − R0 c in cortical rat astrocytes in a dose-dependent manner [2+]c decreased to low dose of BPA (0.1 mg/mL) but the maximum amplitude for [Ca2+]c increased with BPA concentration between 1 mg/mL and 80 mg/mL.It has been shown that bovine plasma albumin (BPA) induces oscillations in the c, as we can observe from Consecutive applications of BPA failed to induce any significant decrease in the maximum amplitude in [Ca2+]c, decreasing at low BPA concentration and increasing at higher concentrations. These responses do not show adaptation.Taken together, these results show that plasma albumin obtained from patients, such as commercially available BPA, induced modifications in c increase even in a calcium free-medium containing 0 Ca2+ + 10−3 mol/L EGTA. However, a second trial failed to induce any change in [Ca2+]c, likely because the internal stores were emptied by the previous pulse.In fact, as we can observe from −6 mol/L   did not modify the maximum peak of cytosolic calcium concentration   at a concentration high enough to block the receptor , decreased a 19.0% .The TGF-6 mol/L, , 34). As2+]c in human astrocytes are due to release from internal stores and are mediated through IP3 as second messenger, but not by calcium-induced-calcium release. Probably, heparin acts extracellularly, blocking the interaction between Alb and its receptor. The rise in [Ca2+]c, nevertheless, depended in a little degree (<20%) on TGF-β receptor. Therefore, we can rule out that the main effect of BPA onto [Ca2+]c is through the TGF-β receptor.These results suggest that increases in [Ca−4 mol/L) and albumin (20 mg/mL). Both glutamate and Alb are able to release calcium from internal stores. As we can observe from 2+]c increase. Then, we perfused heparin (1 mg/mL) during 24 hours. Eight minutes after heparin, glutamate was able to induce a response similar to the control one, but albumin failed to modify the [Ca2+]c. At 12 hours and 23 hours and 45 minutes in heparin, we obtained the same result. Nevertheless, at 24 hours and 10 minutes, that is, 10 minutes after having washed out heparin, both agents induced a [Ca2+]c increase similar to the control , a dose-response relationship between DNA synthesis and BPA concentration was observed .2+]c and DNA synthesis induced by BPA. As we can observe from 2+]c and DNA synthesis with BPA are rather different. In fact, the half maximal effective concentration (EC50) of albumin for [Ca2+]c is 14.5 mg/mL and 10.3 mg/mL for DNA synthesis. Furthermore, both curves are clearly different.We assessed the relationship between increases in c increase are involved in DNA synthesis.So, both mechanisms are responsible approximately for the same amount of DNA synthesis induced by 20 mg/mL BPA, but is there any other mechanisms? To answer this question, astrocytes were incubated with BPA (20 mg/mL) + heparin (1 mg/mL) + LY-364947  it seems unlikely that this effect would be due to an intracellular mechanism and, on other hand, there is no notice to authors about membrane mechanisms able to cross the membrane for a so big molecule. In fact, heparin acting in a selective way through IP3 receptors has to be introduced inside the cell [We have found that heparin can block the rise in [Cathe cell . PrelimiIn summary, it would be extremely important to assess what of the mechanisms effectively applies in humans, in order to design pharmacological strategies. On the other hand, it is conceivable that more than one mechanism acts synergistically.Any case, there is a real and exciting possibility to envisage a pathway running from the normal human brain to focal epilepsy after BBB disruption induced by a local insult . This po


In 2007, particulate organic carbon (POC) fluxes, measured below the euphotic zone at the edge of warm eddy were 136–194 mg-C m−2 d−1 which was greatly elevated over that (POC flux = 26–35 mg-C m−2 d−1) determined in the nutrient-depleted oligotrophic waters in the Western North Pacific (WNP). In 2010, higher POC fluxes (83–115 mg-C m−2 d−1) were also observed at the boundary of mesoscale eddies in the WNP. The enhanced POC flux at the edge of eddies was mainly attributed to both large denuded diatom frustules and zooplankton fecal pellets based on scanning electron microscopy (SEM) examination. The result suggests that mesoscale eddies in the oligotrophic waters in the subtropical WNP can efficiently increase the oceanic carbon export flux and the eddy edge is a crucial conduit in carbon sequestration to deep waters.Mesoscale eddies in the subtropical oligotrophic ocean are ubiquitous and play an important role in nutrient supply and oceanic primary production. However, it is still unclear whether these mesoscale eddies can efficiently transfer CO PP), but it is still unclear whether mesoscale eddies can efficiently carry organic carbon into the ocean interior; (2) elevated organic carbon export seems to appear at the eddy edge rather than inside of eddy. As mentioned above, there are numerous mesoscale eddies in the WNP and these eddy edges serve as natural laboratories for better understanding if sinking particles can efficiently transport organic carbon to the euphotic zone in an oligotrophic ocean. In this study, we investigated POC export fluxes at the eddy edge in the WNP in 2007 and 2010 and compared with POC fluxes from different eddy regions.http://eddy.colorado.edu/ccar/data_viewer/index) of the CCAR (Colorado Center for Astrodynamics Research). Temperature, salinity and density were recorded by a SeaBird CTD (SBE 9/11). Surface currents at 5 m along transect in 2007 were also obtained by an Acoustic Doppler Current Profiler (ADCP). Concentrations of the nitrate+nitrite (N), phosphate (P), silicate (Si), Chl a, POC and PP were measured according to Gong et al. (1999) [−3 h−1 and 2–5%, respectively. The 8-days mean PP values was derived from an empirical algorithm by Behrenfeld and Falkowski (1997) [http://www.science.oregonstate.edu/ocean.productivity/index.php).Seawater samples were collected by a rosette sampler aboard the R/V OR-II in the vicinity of cold and warm eddies in the WNP  during M. (1999)  and Hung. (1999) . The deti (1997)   with honeycomb baffles covering the trap mouths  at S3, S234Th/238U disequilibrium method and floating sediment traps collecting, suggesting that the values of POC export flux in this study are acceptable. Additionally, Hung et al. (2010b) [Floating sediment traps are directly used to measure POC fluxes in sinking particles, but the results may possibly be biases by hydrodynamic and biological effects –34. Howe (2010b)  reportedImages of some selected sinking particles were collected from subsamples of bulk sinking particles in the study area. In brief, sinking particles were collected on polycarbonate filters  and the membrane was washed by Milli-Q water. The membrane was dried and cut into 1-cm circles, and mounted on aluminum stubs. The stubs were coated with gold before scanning electron microscopy (SEM) imaging. The specimens were examined using a Hitachi (S-2400) SEM and clear images of bulk sinking particles were taken directly by SEM .In 2007, the average sea surface temperature (SST) ranged from 27.4 to 27.9°C at S2~S5 (including reference station S3) was higher than that on the edge of the eddy  [−2) than at S6–S7   concentrations were close to the detection limit among all the stations . A subst layers) –40 at S2mol m−2) . Higher at S2–S5 ,41.a concentrations were found in the surface layer (a maximum (SCM) at most stations, while two SCM peaks existed at the eddy edge (i.e. S7), the upper SCM was around 50 m and the lower was around 100 m  did not display significant variation  of POC were shown at S7 and low values at S3 (43–69 mg m−3). In-situ incubation of PP at S3 and S7 showed a similar trend, with higher values near the surface and lower values close to the bottom of the euphotic zone (−2 d−1), however, was lower than at S7 (373 mg-C m−2 d−1) (−2 d−1) in the main stream of the Kuroshio reported by Gong et al. (1999) [Low Chl ce layer  and gradnd 100 m  and the tic zone . The eupm−2 d−1) . The I-P−2 d−1, and 43, 61 and 39 mg-C m−2 d−1  at depths of 90, 120 and 140 m at S3 were 35, 26 and 35 mg-C m m−2 d−1 , Fig 4,  m−2 d−1 , respect m−2 d−1 , respect−2 d−1 at 120 m) is similar to the previously reported data (28 mg-C m−2 d−1 at 120 m) in the Kuroshio water by Hung and Gong (2007) [−2 d−1 at 120 m) in the WNP without extreme atmospheric events [−2 d−1 at S7, 115 mg-C m−2 d−1 at S8, 83 mg-C m−2 d−1 at S9, 94 mg-C m−2 d−1 at S10, In comparison, the POC flux at S3 (26 mg-C mg (2007)  and in tc events ,8. Remar234Th/238U disequilibrium method are comparable in the subtropical oligotrophic ocean [th in warm eddy’s ~135 days life cycle which was based on the successive images of SSHA from CCAR. The sampling period was associated with the warm eddy’s stage from maturing to decaying. During that stage, the dominance of entrained nutrients, supporting phytoplankton growth, might have shifted to predominance in the biological response, which would have resulted in a flux of dead phytoplankton cells sinking out of the euphotic zone [Since the POC export fluxes evaluated both by sediment trap-collected and ic ocean ,35,36, atic zone .a peaks were observed at S6 and S7. Usually, one SCM is observed in the oligotrophic WNP, and the SCM might be controlled by the input of nutrients from below, supplied by turbulent mixing or upwelling [ni  [Trichodesmium spp. can release other nitrogen sources  as nutrients for other phytoplankton, such as diatoms in the upper SCM. While we did not examine the main phytoplankton species composition in the water column or in sinking particles in 2007, we did examine images of sinking particles at S9 and S10 in 2010. The main composition of sinking particles included diatoms , fecal pellets and aggregates of small particles   also reparticles . Chen etearchers ,42,44.a at S3 and S7, one can see that the variation of Chl a concentrations and I-Chl a only showed slight difference , respectively, suggesting that the anticycling eddy can carry particle down to depth.From the vertical distributions and standing stocks of Chl plankton ,46. Addi−1), which is not suitable for zooplankton grazing, compared to the prevailing slow currents (5 m ADCP records: 0.4–0.6 m s−1) at S7. Additionally, our in situ incubation PP (228–430 mg-C m−2 d−1) is significantly higher than the modeled PP (159–275 mg-C m−2 d−1) suggesting that some of PP might be underestimated in this study. As a consequence, the e-ratio should be lower than about 59% if we use in situ-PP. Alternatively, fecal pellets generated by zooplankton, appear to be important at S7; we found an abundance of fecal pellets when we picked and removed small swimmers under a microscope. Furthermore, SEM images of bulk sinking particles at S9 and S10 contained many fecal pellets  of the reference station (S3) and edges of eddies (S7–S10) were 0.09–0.11 and 0.43–0.88, respectively . The for pellets , dead phin-situ incubation method and the modeled result, it could be attributed to the bias of adoptive optimal specific primary productivity [Even though the discrepancy of I-PP differed from the uctivity ,48, resu−2 d−1) at the eddy edges are significantly higher (a 2~7 fold) than fluxes determined by sediment traps from the reference location (i.e. S3: 26–35 mg-C m−2 d−1) and oligotrophic water in the WNP without and within extreme weather events (EWEs) (37 ± 6 and 52–71 mg-C m−2 d−1) [234Th /238U disequilibrium methods, as well as model calculation [−2 d−1, based on 234Th/238U method) are still lower than the measured values in this study. Besides, the reported POC fluxes (12–33 mg-C m−2 d−1) in the lee of eddy near Hawaii in the subtropical Pacific Ocean are also lower than our observed values. The possible reasons might be due to following factors: 1) different methods  [−2 d−1, respectively. It is noted that the lowest POC flux was obtained by Th-234 method. If we assume the ratio of two methods is 1.5 (trap/Th-234 method = 1.5), POC fluxes measured by traps in the SCS will be 63~68 mg-C m−2 d−1, which is slightly lower than our values (83–115 mg-C m−2 d−1) in 2010; 2) phytoplankton species shifts may be an important factor, as mentioned earlier, the main phytoplankton in sinking particle in this study composed of large denuded diatom frustules such as nitzschia, navicula, bacteriastrum, coscinodiscus, which could be different from other studies [The sinking POC export fluxes (77–194 mg-C mm−2 d−1) , but arem−2 d−1) . Even weculation ), one wiculation  estimate. 2013)  used Th-. (2013)  reported studies ,25; also studies ; 3) temp studies , total o studies . It sugg studies  have hig13  used  studies , the Sar studies ,45,49. O studies ,25. Howe2 partial pressure in the atmosphere. Mesoscale eddies have a notable impact on POC flux, but how important are they to the POC flux in the WNP. We briefly estimate the POC flux based on the eddy distribution in the WNP from 21°N to 26°N and 121°E to 125°E if we assume most eddies in the WNP have similar carbon sequestration rate. POC fluxes at the eddy edge and reference site are assumed 110 and 30 mg-C m−2 d−1, respectively. If 3 mesoscale eddies are formed each year, and the total high POC flux period is 30 days (10 days x 3), the eddy induced POC flux will be 3300 mg-C m−2 y−1 (= 110 mg-C m−2 d−1 x 30 d y−1), and the reference POC flux will be 10050 mg-C m−2 y−1 (= 30 mg-C m−2 d−1 x 335 d y−1). This suggests that the contribution of eddy in the WNP can account for at least 20~30% of annual carbon export flux. As mentioned earlier, an eddy’s life cycle, nutrient supply and phytoplankton physiology are difficult to predict or model. This study thus provides important information on the POC flux along the eddy’s edge that is relatively high as compared to the reference region. More intensive observations are needed to further validate and document the natural episodes in the oligotrophic oceans.The biogenic carbon flux to the deep ocean is one of the main factors affecting COBased on three sea-going expeditions in 2007 and 2010, elevated organic carbon fluxes, a 2~4 fold higher than POC fluxes in similar oligotrophic waters, were observed at eddy edges in the oligotrophic waters of the Western North Pacific Ocean. Although we are unable to exactly elucidate which factors  mainly drive organic carbon flux in those eddies, it is clear that enhanced POC flux appearing at eddy edges reshape the picture that “inefficient POC export” did not always happen in the warm waters in the subtropical. Most importantly, POC flux in the oligotrophic waters in the oligotropical open ocean could be underestimated due to limited sampling at mesoscale eddy edges. Hence, the large scale measurement of POC flux in the oligotrophic waters should be conducted in the further study.S1 File−3), temperature (°C), salinity and concentrations of nutrients  and Chl a (mg m−3) are within the supporting information file.Hydrographic data of density (kg m(XLSX)Click here for additional data file.


The presence of good collaterals on CT angiography (CTA) is a well-known predictor for favorable outcome in acute ischemic stroke. Recently, multiphase CT has been introduced as a more accurate method in assessing collaterals. The aim of this study was to assess the ability of dual-phase CT to evaluate collateral status and predict clinical outcome.Forty-three patients who underwent both dual-phase CT and transfemoral cerebral angiography (TFCA) for occluded intracranial internal carotid artery (ICA) and/or middle cerebral artery (M1 segment) were recruited from a prospectively collected database. The collateral status on dual-phase CT was graded by using a 4-point scale: grade 0 = no collaterals; 1 = some collaterals with persistence of some defects; 2 = slow but complete collaterals; and 3 = fast and complete collaterals. Univariate and multivariate analysis were performed to define the independent predictors for favorable outcome at 3 months.κ = 0.776). In the univariate analysis, age, history of hypertension, collateral scores on CTA, dual-phase CT, and TFCA, occlusion in intracranial ICA, final infarct volume, and symptomatic hemorrhage were significantly associated with outcome. Among them, only the dual-phase CT collateral score was an independent predictor for favorable outcome (OR = 26.342 (2.788–248.864); P = 0.004) in the multivariate analysis.Dual-phase CT collateral status (ρ = 0.744) showed higher correlation with TFCA collateral status than CTA collateral status (ρ = 0.596) and substantial interobserver agreement (weighted The collateral status on dual-phase CT can be a useful predictor for clinical outcome in acute stroke patients, especially when advanced CT techniques are not available in emergent situations. The presence of good collateral circulation to the ischemic territory is a notable predictor for favorable long term clinical outcome as well as small infarct size and good response to treatment in patients with proximal intracranial arterial occlusion Transfemoral cerebral angiography (TFCA) has been considered as a gold standard to assess collateral status owing to its temporal and spatial resolution. However, global evaluation of pial collateral flow requires catheterization of multiple vessels and, as an invasive investigation with recognized complication rate, is usually only performed when IA treatment is indicated. To aid selection of treatment strategy or to prognosticate, a method of collateral assessment for all stroke patients, not just for those undergoing intra-arterial therapy is warranted. Furthermore, accurate non-invasive assessment of collateral status prior to endovascular therapy would also be advantageous. Therefore, noninvasive assessment of collateral status before IA treatment or in patients who are not candidates for IA treatment may be helpful to select the treatment strategy and predict long term outcome.Because of short scan times and easy accessibility, computed tomography (CT) is used as the first imaging modality in most patients with acute ischemic stroke. Diverse collateral scoring methods have been suggested in the last decades using CT angiography source images (CTA-SI) The patients were retrospectively selected from a prospectively collected neurointerventional database in a single institution. From February 2011 to June 2013, we recruited consecutive patients who underwent TFCA for possible thrombolysis/mechanical thrombectomy for acute ischemic stroke and met the following inclusion criteria: (1) occlusion of the intracranial ICA or M1 segment; (2) time from symptom onset to admission <6 hours; and (3) presence of a standard local CT protocol including NCCT, CTA, and delayed CECT. Exclusion criteria included bilateral anterior circulation occlusion, posterior circulation occlusion, significant stenosis in contralateral anterior circulation, and past history of stroke.Clinical data were collected from a prospectively recorded stroke registry database. Type of stroke etiology was classified using Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria CT images were obtained on a 64 multi-detector row CT system . The sequential axial 3-mm NCCT was performed first with the following parameters from the skull base to the vertex: 120 kVp, 300 mAs, field of view (FOV) of 25 cm, feed/rotation 18 mm, 30×0.6 mm collimation and a H30s medium reconstruction kernel. Each of the conventional axial CT images was subsequently reconstructed into 1.2 mm slices and 1.2 mm increments. A nonionic contrast agent was administered followed by a 40 ml saline chaser at a rate of 4 ml/s by using a power injector. A bolus tracking technique was used with a marker placed at the ascending aorta, triggering at 100 Hounsfield Units (HU) above baseline with a 7 s delay prior to image acquisition. CTA from the aortic arch to the vertex was performed with the following parameters: 120 kVp, 160 mA, FOV of 18–20 cm, 0.33 s per rotation, 0.8 pitch, and a 0.6-mm section thickness. Delayed CECT was performed with the same parameters as NCCT 40 s after contrast injection. The effective dose which was calculated by the CTDI volume x the scan range x the conversion factor was 1.8 mSv for NCCT and CECT, respectively, 0.9 mSv for the cranial portion of CTA, and 3.0 mSv for the cervical portion of CTA. Therefore the total effective dose was 5.7mSv.TFCA was performed using biplane digital subtraction angiograms  in all patients for the purpose of IA thrombolysis/thrombectomy via bilateral ICAs and the dominant vertebral artery.Routine follow-up MRI was obtained 1 day after the onset of symptoms with the 3-Tesla MR system . DWI was performed with single shot echo planar imaging and parameters were as follows: b-value 1000 s/mm2, TR/TE 6500–7000/65–78 ms, slice thickness 3 mm, FOV 230 mm, matrix 128×128, and NEX 1.CT: The extent of ischemic lesions on NCCT scan images at admission was rated according to the Alberta Stroke Programme Early CT Score (ASPECTS) Collaterals on dual-phase CT were graded on CTA and delayed CECT axial MIP images by using a 4-point scale Occluded segments were recorded based on the location of the non-enhanced segment on delayed CECT as follows TFCA: The collateral flow grading system suggested by the Technology Assessment Committees of the American Society of Interventional and Therapeutic Neuroradiology and the Society of Interventional Radiology TICI score was recorded to assess recanalization using a 5-point scale The final infarct volume was measured on diffusion weighted images of 1-day follow-up MRI t-test, Mann-Whitney U test, chi-square test, or Fisher's exact test where appropriate with correction for multiple comparisons.Nonparametric tests were used for non-normally distributed data. Baseline characteristics were compared according to collateral status on dual-phase CT. Continuous variables are reported as mean ± SD or as the median (interquartile range (IQR)) and were compared using one-way analysis of variance or the Kruskal-Wallis test. Categorical variables are reported as numbers  and were compared using the Fisher's exact test. Post hoc analysis was also performed using the Students' κ) statistics κ≤0.2, poor; 0.2<κ≤0.4, fair; 0.4<κ≤0.6, moderate; 0.6<κ≤0.8, substantial, and 0.8<κ, good.Spearman's rank correlation was used to assess the correlation between collateral status on dual-phase CT or CTA and collateral status on TFCA. Interobserver agreement for assessment of collateral status on CT was determined using weighted Kappa (P<0.05) were included in the multivariable model. To assess independent predictors before IA treatment, multivariate analysis with clinical and CT variables was also performed. Receiver operating curve (ROC) comparison was performed to compare the prediction ability of models containing either dual-phase CT collateral (model 1), CTA collateral (model 2), or TFCA collateral (model 3) scores.To test the association between various clinical and imaging characteristics including collateral status on dual-phase CT and clinical outcome, univariate logistic regression was performed. Multivariate logistic regression with backward elimination  was used to identify independent factors for favorable outcome. Variables significantly associated with a favorable outcome in the univariate analysis  and SPSS, version 19 software . ROC curve comparison was performed by using MedCalc, Version 9.3.9.0 software . Among 166 patients who underwent TFCA for possible thrombolysis/mechanical thrombectomy for acute ischemic stroke, 123 patients were excluded due to posterior circulation occlusion (n = 34), anterior cerebral arterial occlusion (n = 4), occlusion distal to the M1 segment (n = 41), proximal ICA occlusion only (n = 12), old territorial infarcts (n = 8), multiple territorial infarcts due to cardioembolism (n = 2), significant stenosis in the contralateral M1 segment (n = 1), moyamoya disease (n = 1), and only NCCT acquisition (n = 20). Therefore, a total of 43 patients were recruited for this study. The patients underwent IA treatment with stent-assisted thrombectomy , Penumbra system (n = 1), both SAT and Penumbra system (n = 4), stent placement after mechanical thrombectomy (n = 4), Penumbra system with urokinase infusion (n = 2), and urokinase infusion only (n = 1).P = 0.415). Patients with incomplete collaterals were older than those with rapid-complete collaterals (P = 0.008). Patients with less collaterals had hypertension more often, although other cardiovascular risk factors did not show differences in the prevalence across different collateral patterns. The pattern of leptomeningeal collaterals on dual-phase CT was also associated with the TOAST classification of stroke: cardioembolic infarcts were observed more often in patients with less collaterals than those with large arterial infarcts. Less collaterals were associated with lower ASPECTS on NCCT (P = 0.002) and the presence of intracranial ICA occlusion (P = 0.018). Final infarct volume was significantly larger in patients with incomplete collaterals  ml) than that in patients with slow-complete  ml) and rapid-complete  ml) collaterals on dual-phase CT . Clinical outcome was also more favorable in patients with complete collateral patterns  than that of the CTA collateral scores . Interobserver agreement for the collateral scoring on dual-phase CT [weighted κ = 0.776 (0.619–0.933)] was substantial and better than that on CTA [weighted κ = 0.475 (0.285–0.665)].The dual-phase collateral scores showed higher correlation with collateral status on TFCA , absence of history of hypertension (P = 0.022), higher collateral scores on CTA (P = 0.002), dual-phase CT (P = 0.004), and TFCA (P = 0.002), absence of occlusion in intracranial ICA (P = 0.048), smaller final infarct volume (P = 0.003), and absence of symptomatic hemorrhage (P = 0.048) were all significantly associated with favorable clinical outcome. Multivariate analysis of all significant variables using backward elimination showed that only the collateral score on dual-phase CT  was an independent predictor for favorable outcome with adjustment for NIHSS scores at admission . Moreover, multivariate analysis with significant clinical and imaging variables before IA treatment also showed the dual-phase CT collateral score  as an independent predictor for clinical outcome even after adjustment of age, presence of hypertension, NIHSS score at admission, ASPECTS, and occlusion of intracranial ICA. Meanwhile, the CTA collateral score in model 2 was not an independent predictor (P = 0.062) unlike dual-phase CT collateral score in model 1. Although the TFCA collateral score was also an independent predictor , the area under the curve (AUC) of model 3 (0.874) was lower than that of model 1 (0.922). However, it failed to achieve a level of statistical significance ; P = 0.031]. On the other hand, the CTA collateral score could not independently predict clinical outcome in model 2. Also, although there was no statistical significance, the prediction model with the dual-phase CT collateral score was better than that with TFCA collateral scores.The present study suggests collateral status on dual-phase CT may be an independent predictor [OR = 26.342 (2.788–248.864); Leptomeningeal collaterals have been considered as having a beneficial role in patients with acute stroke despite various scoring scales and different imaging modalities A previous study using tri-phase CT In this study, collateral scores on dual-phase CT showed better correlation with collateral status on TFCA and higher interobserver agreement than did collateral scores on single phase CTA. Also, as mentioned above, collateral status on dual-phase CT was an independent predictor for clinical outcome; however, collateral scores on single phase CTA and even TFCA were not. On dual-phase CT, detection of collateral defects on CTA and delayed CECT was needed. On the other hand, additional detection on the degree  of collateral extent was required on single phase CTA. Therefore, the observers might grade collateral status more consistently on dual-phase CT. Both dual-phase CTA and TFCA provide dynamic information of collaterals; however, it is difficult to evaluate pial collaterals from whole cerebral circulation by TFCA, so the prediction power for clinical outcome might be lower than that of dual-phase CT.Among the patients with complete collaterals on dual-phase CT, patients with rapid-complete collaterals had higher ASPECT scores at admission (7.6 vs. 8.5), smaller final infarct volume (25.0 ml vs. 12.3 ml), and more frequent favorable clinical outcome (60.9% vs. 90%) than patients with slow-complete collaterals. Although there was no significant differences between the two groups as reported in a previous study There were several limitations in our study. First, this study was a retrospective study although the patients were recruited from a prospectively collected database. Also a small number of patients included in the analysis might cause a wide range of 95% CI of OR of dual-phase CT collateral status and its overestimation. Therefore, caution is needed in generalizing the results of this study. Second, more than 50% of patients were treated with IV thrombolysis. As a result of the IV treatment, potential confounding factors may have influenced the assessment of collateral status on TFCA and prediction of clinical outcome. There might be difference in thrombus extent and the collateral status between CT and TFCA. Third, there is the possibility that selection bias may exist. Patients who achieved recanalization only after IV treatment or had substantial ischemic change larger than 1/3 of the middle cerebral artery (MCA) territory were not subjected to IA treatment and excluded from this study. We also excluded considerable number of patients according to the exclusion criteria. However, our results could be applied to a relatively homogenous patient group with occlusion of distal ICA or M1 segments who are indicated for IA treatment. Fourth, we did not perform multiphase CT or perfusion CT, so direct comparison between these advanced techniques and dual-phase CT in predicting clinical outcome was not performed. Finally, our routine CT protocol was not optimized to assess collateral status in patients with acute ischemic stroke. According to a previous study using time-resolved CTA In conclusion, dual-phase CT is feasible for assessing collateral status in patients with acute ischemic stroke, showing substantial interobserver agreement. Collateral status on dual-phase CT can also be a useful predictor for clinical outcome in acute stroke patients subjected to IA treatment, especially when advanced CT techniques are not available in emergency situations.