O-GlcNAcylation are widespread post-translational modifications (PTMs), often sharing protein targets. Numerous studies have reported the phosphorylation of plant viral proteins. In plants, research on O-GlcNAcylation lags behind that of other eukaryotes, and information about O-GlcNAcylated plant viral proteins is extremely scarce. The potyvirus Plum pox virus (PPV) causes sharka disease in Prunus trees and also infects a wide range of experimental hosts. Capsid protein (CP) from virions of PPV-R isolate purified from herbaceous plants can be extensively modified by O-GlcNAcylation and phosphorylation. In this study, a combination of proteomics and biochemical approaches was employed to broaden knowledge of PPV CP PTMs. CP proved to be modified regardless of whether or not it was assembled into mature particles. PTMs of CP occurred in the natural host Prunus persica, similarly to what happens in herbaceous plants. Additionally, we observed that O-GlcNAcylation and phosphorylation were general features of different PPV strains, suggesting that these modifications contribute to general strategies deployed during plant-virus interactions. Interestingly, phosphorylation at a casein kinase II motif conserved among potyviral CPs exhibited strain specificity in PPV; however, it did not display the critical role attributed to the same modification in the CP of another potyvirus, Potato virus A.Phosphorylation and  Numerous viral proteins can be post-translationally modified at different stages of infection, probably a result of co-evolutionary strategies, whereby viruses acquire the ability to hijack host enzymes . There aPotyvirus is one of the largest and more economically relevant groups of plant viruses [Potato virus A (PVA) has been thoroughly studied. PVA CP is phosphorylated at the C terminus of the protein by casein kinase II (CK2) in the motif (S/T)XX(D/E) conserved in most potyviruses [Plum pox virus (PPV), is also phosphorylated [O-GlcNAcylation [The genus  viruses . Potyvir viruses ,20. A fr viruses ,22,23. Pyviruses ,25,26. Iyviruses . The CP orylated ,29. Intecylation ,30, whiccylation  and has cylation ,33,34,35O-GlcNAcylated by the O-GlcNAc transferase (OGT) SECRET AGENT (SEC) along the N-terminal region of the protein [O-GlcNAcylation nor phosphorylation of CP is essential for virus viability, but both appear to contribute to infection efficiency [O-GlcNAcylation at N terminus of the protein regulate CP stability, thus providing appropriate CP amounts for each phase of viral infection, while phosphorylation at core region controls viral particle assembly/disassembly [CP from virions of the PPV R isolate, which belongs to strain D, has been shown to be extensively  protein ,36,37. P protein ,39. Neitficiency ,37,38. IPrunus trees causing sharka, the most devastating viral disease that affects stone fruit trees worldwide, but it is also able to infect a wide range of experimental herbaceous hosts, among them Nicotiana spp. [O-GlcNAcylation and phosphorylation affecting PPV CP have been narrowed to infections in herbaceous hosts, mainly in Nicotiana spp., and no data concerning PTMs of CP during infections in natural woody hosts are available. On the other hand, up to 10 different PPV strains have been described [O-GlcNAcylation and phosphorylation affecting CP may be a common attribute of PPV species [PPV naturally infects ana spp. ,41. All escribed ,43. Alth species ,29,44, tO-GlcNAcylation and phosphorylation of PPV R when infecting seedlings of the natural host of the pathogen Prunus persica, as well as PTMs of PPV CP from isolates of strains Rec and C in Nicotiana plants. Our results indicated that modification of CP by O-GlcNAcylation and phosphorylation took place in at least two evolutionarily distant hosts, Nicotiana and Prunus, and was a general feature of different PPV strains. Moreover, we showed that O-GlcNAcylation and phosphorylation were not exclusive to CP arranged in virions. Remarkably, although phosphorylation at conserved casein kinase II (CK2) motif of CP did not have in PPV the same pivotal role reported for PVA, it did appear to have some strain-specific contribution to PPV infection.In this work, we used a combination of proteomics and biochemical approaches to study P. persica and Nicotiana benthamiana plants by agroinfiltration. This plasmid was created by engineering the chimeric cDNA of PPV pICPPV-5′BD-GFP [Prunus plants but by biolistic method, into pSN-PPV [Plasmid pSN-PPV-5′BD-GFP  was use5′BD-GFP , also us pSN-PPV   in the CP of PPV-R and threonine 306 (T306) in that of SwCM were respectively engineered into plasmids pICPPV-NK-lGFP and pICPPV-CPSwCM-R . ReplaceMutations at T304 and T306 were created by site-directed mutagenesis using a three-step PCR approach, as previously described . PrimersP. persica and N. benthamiana plants agroinoculated with pSN-PPV-5′BD-GFP, which were grown in a climate chamber at 22 °C with the same photoperiod. For agroinoculation, young P. persica cv. GF305 and N. benthamiana plants (four-to-six-leaf stage) were infiltrated with cultures of Agrobacterium tumefaciens GV3101  transformed with plasmid pSN-PPV-5′BD-GFP, as previously described [Prunus plants, the agrobacterium pellet was suspended in inoculation buffer to reach an OD600 of 1, and leaves were infiltrated by pressing strongly and repeatedly on the syringe plunger, in overlapping patches, to cover most of the foliar area.Plants were cultured in a glasshouse at 19–23 °C and a 16 h/8 h (light/dark) photoperiod, except for escribed . In the N. benthamiana plants, pICPPV-NK-lGFP- or pICPPV-CPSwCM-R-derived plasmids were dispensed in three leaves per plant  and rubbed using Carborundum as an abrasive agent. Similarly, crude homogenates from tissue infected with PPV-BOR-3, an isolate belonging to strain Rec, were used to inoculate N. benthamiana and Nicotiana clevelandii plants (three leaves per plant), by rubbing 10 µL of extract (2 mL of 5 mM sodium phosphate pH 7.2 per g of tissue) per leaf. Serial passages of PPV-NK-lGFP- and PPV-CPSwCM-R-derived mutants through N. benthamiana plants were also carried out by hand-rubbing with crude homogenates of infected leaves, as explained above.For manual inoculation of P. persica cv. GF305 with microgold particles coated with DNA of pICPPV-5´BD-GFP was performed with a Helios gene gun device , as previously described [Biolistic inoculation of escribed .Viral infection was monitored by visual inspection of symptoms and by observing GFP fluorescence under a long-wavelength UV lamp or a stereomicroscope, as previously described . Viral ag). The supernatant was filtered through a Miracloth membrane , and Triton X-100 was added slowly to a final concentration of 5%. The stirring process was maintained during 1 h, and then the mixture was ultra-centrifuged through a layer of 20% sucrose in 0.02 M HEPES buffer, pH 6.8 . The resulting pellet was suspended overnight in 0.1 M sodium borate, 5 mM EDTA buffer, pH 8.2 (borate-EDTA buffer), with a magnetic stirrer. After removal of debris by centrifugation in a tabletop microfuge, the supernatant was subjected to a new step of ultracentrifugation through a continuous sucrose gradient (10%–40%) in a borate-EDTA buffer . Fractions in which virions are usually concentrated were diluted in the borate-EDTA buffer, adjusted to 0.005% of Triton X-100, and dialyzed during 3 h through a 10 kDa exclusion membrane SnakeSkin Dialysis Tubing  against borate-EDTA buffer with 0.005% Triton X-100. Dialyzed samples were centrifuged at 81,630× g for 180 min, and the pellet was resuspended for at least 2 h in 5 mM sodium borate buffer, pH 8.2 with a magnetic stirrer. All purification steps were carried out at 4 °C. The yield and quality of samples were checked by 12% SDS-PAGE. In cases when an excess of plant contaminants was detected, samples were subjected to an additional purification step, by repeating the sucrose gradient sedimentation and further concentration by ultracentrifugation.Virions were purified by the method previously described in Laín et al. , slightlN. benthamiana leaves, prepared as previously described [g. Aliquots were collected by gravity from the bottom with a capillary tube and analyzed by Western blot anti CP. For CP immunoprecipitation, pools of one of the fractions of 4 independent gradients  were mixed with an equal volume of 200 mM Tris-HCl, 600 mM NaCl, 10 mM DTT, 0.4% Triton X-100 buffer, pH 7.4 (BI buffer) supplemented with 0.2 % cOmplete EDTA-free Protease Inhibitor Cocktail . Then, 10 µL of anti-CP rabbit polyclonal serum was added to the mixture. After five hours of incubation at 4 °C in rotary motion, 50 μL of Protein A Sepharose Fast Flow  was added, and incubation continued overnight under the same conditions. Immuno-complexes were collected by centrifugation in a tabletop centrifuge and washed three times with BI buffer. Finally, immunoprecipitated CP was eluted from sepharose beads with 80 μL of disruption buffer .Crude extracts from systemically infected escribed , were suPPV virions (1–5 µg) were digested with 50 ng (10 ng/µL) of Pierce MS grade trypsin  in 50 mM ammonium bicarbonate for 20 min at approximately 22 °C, desalted with a Zip-Tip reverse-phase C18 column, and eluted in 70% aqueous acetonitrile and 0.1% trifluoroacetic acid (TFA), as previously described . EluatesPPV virions were precipitated with methanol–chloroform mix (4:1) and reconstituted in 8 M urea, 25 mM ammonium bicarbonate solution. After following reduction (10 mM dithiothreitol) and alkylation (50 mM iodoacetamide) in 25 mM ammonium bicarbonate, samples were digested at 37 °C with Lys-C (6 h) and trypsin (15 h) at an enzyme:substrate ratio of 1:50. Phospho-peptide enrichment procedure concatenated two in-house packed microcolumns—Immobilized Metal Affinity Chromatography (IMAC) and Oligo R3 polymeric reversed-phase—, which provided selective purification and sample desalting prior to LC-MS/MS analysis . Phosphohttp://www.matrixscience.com; Matrix Science, London, UK) against a PPV protein database, containing 13 protein sequences together with commonly occurring contaminants. In addition to Mascot, an in-house licensed Peaks Search engine 7.5  was also used. Search engines considered fixed carbamidomethyl modification of cysteine and the following variable modifications: oxidation of methionine, phosphorylation and glycosylation of serine and threonine, and phosphorylation of tyrosine. No enzyme restriction was considered.MS/MS spectra were exported to mgf format and searched using an in-house licensed Mascot Server 2.6.1 (The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner O-GlcNAcylated by the O-GlcNAcyltransferase (OGT) SEC [N. benthamiana plants were inoculated with a PPV-R cDNA clone, pSN-PPV, and systemically infected leaves were homogenized in a phosphate buffer. The resulting extract was fractionated by sucrose gradient centrifugation, and collected fractions were analyzed by Western blot anti-CP  from the PPV-R isolate (R-CP) assembled in virions was extensively OGT) SEC ,37 and pOGT) SEC . To dete anti-CP A.O-GlcNAcylation of peptide 1–39, which has been shown to contain up to two O-GlcNAcylations, at T19 and T24 [O-GlcNAcylation patterns of CP assembled and non-assembled in virions ran similarly, and the intensity ratios among non-modified, mono-, and di-O-GlcNAcylated forms for both samples were comparable (A virion-containing fraction (fraction 8) and the gradient-top fraction, where small complexes and free CP are retained (fraction 19), were separately immunoprecipitated using a specific anti PPV CP serum. Both samples were trypsinized, and  and T24 , was anamparable A.N. benthamiana plants systemically infected with PPV-BOR-3 was fractionated by sucrose gradient centrifugation. Western blot analysis of all gradient fractions detected the double band that is characteristic of phosphorylated BOR-3-CP, both in virion-hosting fraction 7 and in fraction 11, containing CP not assembled in virus particles  has the peculiarity of migrating as a doublet in SDS-PAGE, presumably because of its partial phosphorylation . To assearticles B. This rO-GlcNAcylation in Nicotiana spp. and Arabidopsis thaliana [Nicotiana spp. [PTMs of PPV CP had been shown to take place in experimental herbaceous hosts: thaliana  and phosana spp. ,39. HowePrunus persica seedlings were biolistically inoculated with the full-length chimeric clone pICPPV-5´BD-GFP, which expresses R-CP. Infection was verified by visual inspection for symptoms  and by monitoring of virus-derived GFP fluorescence. Virions were purified from systemically infected tissue, partially digested with trypsin, and subjected to MALDI-TOF analysis to inquire about the putative O-GlcNAcylation of CP. The N-terminal tryptic peptide 1–39 of CP was detected as both non-glycosylated and mono- and di-O-GlcNAcylated forms  [O-GlcNAcylation at these sites appeared to be somewhat higher in this experiment compared to that found in previous works. To confirm such a difference and assess whether it is caused by the use of the PPV-5′BD chimera or it is specifically linked to the host type, P. persica and N. benthamiana plants were inoculated with pSN-PPV-5′BD-GFP. This last plasmid is another PPV-5′BD-GFP chimeric clone but allowing inoculation of PPV by agroinfiltration. After agroinoculation, the O-GlcNAcylation pattern of peptide 1–39 was analyzed in two biological samples of virions purified from each of the infected host types.These and S65) ,38. HoweN. benthamina samples compared with those obtained from P. persica (average of 25.8% vs. 8.2%). Accordingly, the amount of di-O-GlcNAcylated peptide was almost twice in the Prunus sample compared to that of N. benthamina (average of 43.0% vs. 24.6%) (Estimates based on peak intensities showed a relative abundance of the non-modified peptide more than three times higher in . 24.6%) B.O-GlcNAcylation of R-CP was enhanced in the natural PPV host compared with that occurring in herbaceous plants.Although the sample size of this study was not large enough to perform a statistical analysis that would rigorously support the results, our data suggested that N. benthamiana at specific serines  and, more sporadically, at threonines  [P. persica seedlings infected by agroinfiltration with pSN-PPV-5′BD-GFP and analyzed by LC-MS/MS after trypsin/Lys-C digestion and phospho-peptide enrichment.Former results had shown that R-CP was targeted by phosphorylation in PPV-infected nd T313) ,39. To dPrunus-derived virions at three of the four phosphorylatable serines  and one of the four threonine targets (pT254) previously identified in R-CP produced in herbaceous infections  and PPV-SwCM (strain C) were analyzed. Rec was selected because signs of phosphorylation and O-GlcNAcylation had been previously reported for isolates of this strain [Former studies concerning s strain , and strs strain .N. benthamiana [N. benthamiana plants infected with PPV-BOR-3 and the chimeric plasmid expressing SwCM-CP were subjected to mild trypsin digestion and MALDI-TOF analysis. For SwCM-CP, no tryptic peptides covering the region between amino acids 17 and 72, where presumably O-GlcNAcylations would take place, could be recovered. For its part, the poor digestibility of the CP of PPV-BOR-3 (BOR-3-CP) did not allow to obtain useful results using this type of approach either.PPV-SwCM causes lethal systemic necrosis in thamiana . To overthamiana , thus alO-GlcNAcylation of CPs from these isolates, we took advantage of the small differences in electrophoretic mobility in SDS-PAGE previously observed when comparing R-CP produced in wild-type N. benthamiana plants with the same protein expressed in O-GlcNAcylation-deficient SEC-b2 plants [O-GlcNAcylation target) is a consequence of different O-GlcNAcylation levels of CP, and, although the difference is subtle, it allows determining whether the OGT SEC is modifying the protein. Extracts from systemically infected leaves of N. benthamiana plants, wild type, and SEC-b2, infected with PPV-BOR-3 and PPV-CPSwCM-R, were subjected to SDS-PAGE and Western blot anti-CP. Analysis of CP electrophoretic mobility indicated that—similar to R-CP—BOR-3-CP and SwCM-CP from SEC-b2 plants exhibited greater electrophoretic mobility compared to those from wild-type plants  A had nevTo explore this possibility, mutations were introduced in pICPPV-NK-lGFP and pICPPV-CPSwCM-R, to replace residues T304 of R-CP and T306 of SwCM-CP by alanine  or aspartic acid  to, respectively, prevent or emulate phosphorylation at those sites. Aspartic acid serves to mimic steric and electrostatic effects of phosphorylation without suffering such modification. Replacements by asparagine, a neutral amino acid with a bulky side chain similar to the negatively charged aspartic, were included as controls  .N. benthamiana plants by hand-rubbing, and symptoms of infection and GFP fluorescence expressed from recombinant viruses were monitored. Tracking viral infection for up to 21 dpi did not show differences in infectivity or progression of infection between the different mutants and the wild-type virus  or with mutant SwCM-T306A (7/8) showed signs of infection at an early time (12 dpi); while the number of plants showing symptoms after being inoculated with mutants SwCM-T306D or SwCM-T306N  was clearly smaller at that time. However, after 21 dpi, infection symptoms of all mutants tended to equalize  allowed a complete reversion of alanine to threonine at position 306, emulating wild-type SwCM-CP sequence. In turn, the two analyzed lineages of the SwCM-T306D mutant showed a mutation (ACA X AAA), causing a change from threonine to lysine at position 273. In one of these lineages, a second mutation (GCC X ACC) appeared, which resulted in a change from alanine to threonine at position 128. Finally, each of SwCM-T306N lineages took a different evolutionary path. In one of them, a point mutation (AAT X GAT) was introduced, causing a change from asparagine to aspartic acid at position 88; while in the other, mutations ATT X AGT and CTC X TTC, respectively, produced changes from isoleucine to serine at position 77 and from leucine to phenylalanine at position 121  has been shown to be essential for efficient viral movement, but functions of TEV CP in virion assembly and movement appeared to be somewhat distinct [O-GlcNAcylation, two very frequent and closely related PTMs [O-GlcNAcylations and phosphorylations, while that of PVA is only phosphorylated. Moreover, PPV and PVA CP phosphorylations have shown specific peculiarities [The function of the coat protein of potyviruses, which is a multitasking protein essential for viral infection, goes beyond forming stable virion structures to protect genomic RNA . For insdistinct . Moreovedistinct ,30, partdistinct . The supdistinct . On the distinct ,63,64,65ted PTMs ,66. Thusted PTMs ,29,67. Hiarities ,38,39. IO-GlcNAcylations of PPV, which have been exclusively studied on purified virus particles [O-GlcNAcylation are reversible PTMs, enzymatic removal of phosphate or O-GlcNAc groups from potyviral CPs has not been demonstrated. If these reverse reactions indeed take place, we do not know if they occur on free or assembled CP, or at both states of protein aggregation. The MALDI-TOF approach used here did not allow identification of which specific residues are modified by O-GlcNAcylation. However, analysis of peptide 1–39 seemed to indicate that the same amino acids previously mapped in CP of purified virions (T19 and T24) were modified in not-fully assembled CP. This would suggest that the O-GlcNAcylation of PPV CP does not depend on the assembly level of the protein. The scope of our phosphorylation analysis is also limited. It clearly demonstrates this PTM can take place in CP not assembled in mature virions, but it only provides information about a PPV-BOR-3-specific phosphorylation target (see below). This is especially important because very different functions have been suggested for phosphorylations at different locations of PPV CP [The widely studied phosphorylation of PVA CP has not been reported to occur in virions . This coarticles ,39. In tO-GlcNAcylation of PPV CP have been previously studied in herbaceous experimental hosts exclusively; thus, analyzing them in P. persica has the interest of extending our knowledge to a quite different plant species and bringing it closer to natural conditions. Phosphorylation mapping in PPV virions from P. persica confirmed this modification also comes about in this host  [O-GlcNAcylable threonines of PPV CP by alanines has comparable effects on infections of P. persica and herbaceous hosts [O-GlcNAcylation of CP appears to be important for PPV in both experimental herbaceous plants and natural woody hosts, it is not essential for infection in any of these plants.Here, we demonstrated that PPV CP  persica . MALDI-T persica A seemed ana spp. ,68. Thisfections B could bnd gT24) . Howeverus hosts . TherefoO-GlcNAcylation and most of the phosphorylation targets are focused. Thus, we wondered if PTMs widely analyzed in an isolate (PPV-R) of the strain D would similarly affect other PPV strains. Sequence alignment of CPs from isolates of different PPV strains showed that most potential targets of O-GlcNAcylation in PPV-R virions  were conserved in the other two isolates analyzed in this study, PPV-BOR-3 (strain Rec) and PPV-SwCM (strain C). While S65 was not conserved in SwCM-CP, T58 was not conserved in either SwCM-CP or BOR-3-CP  were conserved in PPV-BOR-3 and PPV-SwCM, while phospho-targets aligning with S25 and S106 were absent from the isolate of strain Rec . Some phO-GlcNAcylation role in protein stability suggested for the PTMs of the N-terminal protrusion of PPV CP, based on former results obtained with PPV R [Our LC-MS/MS analyses showed phosphorylations in the CP of PPV-BOR-3 and PPV-SwCM modifying residues not equivalent to those previously identified as phospho-targets in PPV-R CP . Phosphoth PPV R .Moreover, we detected the phosphorylated residue T306 in SwCM-CP . This amIt has been suggested that phosphorylation at CP residue T243 of PVA-CP by protein kinase CK2 plays a crucial role in RNA translation/replication transition. Phosphorylation of T306 at the CK2 motif-like of SwCM-CP could have a similar function, but in this case, the contribution would be much less significant. However, we could also envisage the possibility that phosphorylation of T306 is involved in a function related to that proposed for phosphorylation at the core domain of R-CP in the control of virion assembly/disassembly. Moreover, we could not rule out another possible role, still non-characterized, specific for the CP of this particular PPV strain.O-GlcNAcylation appears to be essential for PPV infection agrees with a recent acquisition of these PTMs in potyvirus evolution. The divergences on features and impact of phosphorylation of CPs of PPV and PVA CPs also support this assumption. On the other hand, conservation of these PTMs among different PPV strains suggests that they were acquired soon in PPV evolution, undergoing strain-specific adjustments throughout the diversification of the species. The preservation of phosphorylation and O-GlcNAcylation during the evolutionary history of PPV provides clear evidence of its usefulness for the virus. We presume that PTMs provide the chance to carry out varied improvements on the regulatory capacity of multifunctional proteins as potyviral CP, in a manner that they can be specifically adopted by different viruses at different evolutionary stages.Outcomes described in this paper, along with previously reported data, demonstrate that PTMs are general features of particular potyviruses, whose relevance varies between different members of this virus group. The fact that neither phosphorylation nor


Neuronal apoptosis plays a critical event in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). This study investigated the roles of Tauroursodeoxycholic acid (TUDCA) in attenuate neuronal apoptosis and underlying mechanisms after SAH.Sprague–Dawley rats were subjected to model of SAH and TUDCA was administered via the internal carotid injection. Small interfering RNA (siRNA) for TGR5 were administered through intracerebroventricular injection 48 h before SAH. Neurological scores, brain water content, Western blot, TUNEL staining and immunofluorescence staining were evaluated.TUDCA alleviated brain water content and improved neurological scores at 24 h and 72 h after SAH. TUDCA administration prevented the reduction of SIRT3 and BCL-2 expressions, as well as the increase of BAX and cleaved caspase-3.Endogenous TGR5 expression were upregulated after SAH and treatment with TGR5 siRNA exacerbated neurological outcomes after SAH and the protective effects of TUDCA at 24 h after SAH were also abolished by TGR5 siRNA.Our findings demonstrate that TUDCA could attenuated neuronal apoptosis and improve neurological functions through TGR5/ SIRT3 signaling pathway after SAH. TUDCA may be an attractive candidate for anti-apoptosis treatment in SAH. Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease with high mortality and morbidity . StudiesTauroursodeoxycholic acid (TUDCA) is an endogenous hydrophilic bile acid used clinically to treat certain liver diseases . PreviouTUDCA may activate TGR5-mediated signaling to reduce DNA damage and improve embryo development after ultraviolet light exposure . The effIn the current study, we hypothesized that (1) TUDCA provides neuroprotective effects after SAH; (2) TUDCA attenuates apoptosis by activating TGR5 signaling after SAH.A total of 254 rats were used and 224 rats underwent SAH induction. There were no deaths in sham group. For groups of SAH, the mortality rate was 11.6% (26 of 224)  ,     Fig. a. The 10P < 0.05 versus Sham).Double immunofluorescence staining showed that TGR5 was upregulated in neurons in the ipsilateral cortex at 24 h after SAH and was also localized in microglia  and TUDCA prevented the reductions (P < 0.05) Fig.  a, b. FuTo further assess the role of TUDCA in SAH, TGR5 siRNA was administered by ICV injection to knockdown endogenous TGR5. Western blot showed that TGR5 expression was partially prevented by TGR5 siRNA   , SAH + TUDCA (100 mg/kg), SAH + scramble siRNA, SAH + TGR5 siRNA, SAH + scramble siRNA + TUDCA (100 mg/kg), SAH + TGR5 siRNA + TUDCA (100 mg/kg) and sham + TUDCA (100 mg/kg). The rats were raised on a 12 h dark–light cycle circumstance with free access to food and water.The endovascular perforation was performed as previously described with modifcations . AnestheSAH grade was determined by the high-resolution images of the blood clots in the basal cisterns, as previously described . AccordiBrain water content was evaluated at 24 and 72 h after SAH. The brains were separated into cerebrum, cerebellum, and brainstem. Each part was weighed immediately after removal (wet weight) and then dried in an oven at 80 °C for 72 h (dry weight). After that, the percentage of brain water content was calculated as [(wet weight—dry weight)/wet weight] × 100% .Saline or TUDCA , dissolved in phosphate buffer, pH 7.4 at 400 mg/ml, was slowly injected into the internal carotid at 1 ml/kg of bodyweight (bw). TUDCA at 50 or 100 mg/kg of bw was administered 1 h after SAH model.TUNEL assay was conducted as previously described with modifications . TUNEL rIntracerebroventricular (i.c.v) drug administration was performed was performed as described previously . TGR5-siDouble fluorescence staining was performed as described previously . The ratWestern blot tests were performed as previously described . Brains Data were analyzed using SigmaPlot 11.0 and GraphPad Prism 6 , expressed as means ± standard deviation (SD). Data from different groups were compared using 1-way ANOVA followed by post hoc Tukey tests. Non-parametric data  were analyzed with the Kruskal–Wallis test followed by Dunn’s post-hoc. Significant differences were accepted when a value of p < 0.05.Additional file 1: Table S1. Animal number  in each group. Figure S1. (a) The effect of TUDCA in different administration time on neurological damage. n = 6 per group. *P<0.001 vs SAH+vehicle. (b–d) Representative Western blot images and quantitative analyses of SIRT3 and BAX in sham group and sham+TUDCA group. n = 6 per group.Below is the link to the electronic supplementary material.


MLCP contains a phosphatase subunit (PP1c) that is regulated through myosin phosphatase target subunit 1 (MYPT1) and other subunits, such as glycogen-associated regulatory subunit and myosin-binding subunit 85 kDa. Interestingly, MLCP inhibition may contribute to exacerbation of smooth muscle contraction by increasing MLC phosphorylation to induce hypercontractility. Many pathways inhibiting MLCP activity in airway smooth muscle have been proposed and are focused on inhibition of PP1c, inhibitory phosphorylation of MYPT1 and dissociation of the PP1c-MYPT1 complex.Smooth muscle is a central structure involved in the regulation of airway tone. In addition, it plays an important role in the development of some pathologies generated by alterations in contraction, such as hypercontractility and the airway hyperresponsiveness observed in asthma. The molecular processes associated with smooth muscle contraction are centered around myosin light chain (MLC) phosphorylation, which is controlled by a balance in the activity of myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP). MLCK activation depends on increasing concentrations of intracellular Ca Kinases and phosphatases are key regulatory components of many cellular processes. Mathematical analysis of kinase–phosphatase pathways suggests that kinases have an important role in modulating the signal amplitude, while phosphatases control the duration of responses . In addiSmooth muscle can be classified into two types according to its contractile properties: tonic and phasic. Although phasic and tonic smooth muscles differ in the time of contraction maintenance, both share the same signaling and regulatory pathways. In particular, airway smooth muscle becomes tonic after birth because it does not generate spontaneous contractions . The intFew studies have addressed the relationship between the role of MLCP and airway smooth muscle hypercontractility and airway hyperresponsiveness. MLCP-associated signaling is an interesting perspective from which to understand the possible dysfunctions in the contractile response of smooth muscle. Therefore, in this mini-review, we attempt to elucidate the possible role of MLCP in the development and regulation of airway smooth muscle contraction to generate hypercontraction.To expand knowledge on the regulation of smooth muscle contraction, specific reviews have covered this topic in detail . In gene19) residue allows interaction between actin and myosin filaments, which results in smooth muscle contraction. This phosphorylation occurs after the increase in intracellular Ca2+ (Ca2+i) levels induced by contractile agonist stimulation. The high Ca2+i levels saturate the binding sites on the calmodulin (CaM) protein and allow the formation of a complex between the C-terminal domain of CaM and the N-terminal domain of MLCK, causing its activation and subsequent phosphorylation of MLC20 , a regulatory subunit called myosin phosphatase target subunit 1 (MYPT1), and an accessory subunit of 20 kDa called M20 that is linked to MYPT1 (MLCP is a serine (Ser)-threonine (Thr) phosphatase expressed in smooth muscle whose function is independent of Cato MYPT1 . In addito MYPT1 . The regto MYPT1 .PP1CA, PP1CB, and PP1CC. In particular, the protein product of PP1CB is involved in muscle function and is controlled by various regulatory proteins, such as MYPT1, glycogen-associated regulatory subunit (GM), and myosin-binding subunit 85 kDa (MBS85), and is involved in sarcoplasmic reticulum Ca2+ loading and regulating several transcription factors in myocytes (The mechanisms of MLCP regulation in smooth muscle are mediated mainly by the PP1c and MYPT1 subunits. PP1c belongs to the phosphoprotein phosphatase (PP) family and is the main Ser-Thr phosphatase involved in several functions. The specificity of PP is achieved through its different catalytic isoforms, such as PP1, PP2, PP3, PP4, PP5, PP6, and PP7, some of which encode unique genes. PP1c is encoded by myocytes . Various507, Thr853/850, and Thr696/695 residues mediates the inhibition of MLCP activity and therefore the inhibition of airway smooth muscle relaxation. In addition, MYPT1 phosphorylation at Ser965, Ser696, and Ser852 is also observed in airway smooth muscle.A second pathway regulating MLCP involves phosphorylation of the phosphatase regulatory subunit MYPT1. This protein has multiple phosphorylation sites that negatively or positively regulate MLCP activity. The most important phosphorylation sites in MYPT1 are Ser and Thr residues . In partThe role of ROCK in airway smooth muscle contractility has been widely studied . RhoA isknockout mice in 2015, Kasahara and co-workers reported that ROCK expression is directly correlated with the increase in hyperresponsiveness caused by exposure to ozone. Similarly, they determined that reduced expression of either ROCK isoform – ROCK1 or ROCK2 – prevents the development of hyperresponsiveness. In fact, the degree of hyperresponsiveness and MLCP regulation are directly related, because the degree of hyperresponsiveness is associated with increased ROCK expression in the airway, which in turn results in an inhibitory effect via MLCP phosphorylation ; PP1c inhibition by PHI-1 results in ROCK-activated MLCP inhibition . However850 phosphorylation but had little effect on Thr695 phosphorylation. Another inhibitor examined was calyculin A, which acts directly on the catalytic subunit PP1c and also affects MYPT1 by increasing Thr850 dephosphorylation without noticeably affecting the phosphorylation level of Thr695. The lack of inhibitory phosphorylation at Thr695 may be due to phosphorylation by kinases other than ROCK, suggesting that each phosphorylation event can independently regulate MYPT1 and is mediated by different kinases. For example, p21-activated kinase (PAK-1), a protein activated by ROCK, can also induce the phosphorylation of MYPT1 at Thr696 in airway smooth muscle  was shown to induce this di-phosphorylation of MLC20 via a mechanism independent of ROCK and dependent on ZIPK (in vitro assay, ZIPK directly inhibited MLCP by phosphorylating MYPT1 at Thr696 (ZIPK has a zipper design similar to those of MYPT1 and M20 . In huma on ZIPK . In colo on ZIPK , althougt Thr696 . The subt Thr696  because t Thr696 .695, Ser697 and Ser852 – and at Thr855 without affecting MLCP (in vivo data suggest that phosphorylation at Ser695 attenuates inhibitory phosphorylation of Thr696 in rabbit ileum smooth muscle via competition between these phosphorylation sites. The same study showed that thiophosphorylation of MYPT1 by PKA reduces phosphorylation catalyzed by ROCK, thereby preventing MLCP inhibition and inhibiting smooth muscle contraction (Smooth muscle relaxation can be generated by several mechanisms, including the formation of cyclic nucleotides such as cAMP that activate PKA or cGMP, which in turn activates PKG. MYPT1 can be phosphorylated by PKG and PKA at three serine residues – Sering MLCP . Additiotraction .2+ sensitization are increased in smooth muscle. After PKC activation, arachidonic acid enhances MLC20 phosphorylation in smooth muscle of the rabbit femoral artery due to inhibition of MLCP by dissociation of the enzymatic complex, demonstrating the participation of non-metabolized fatty acids in the regulation of smooth muscle contraction (On the other hand, the effects of PKC on Catraction . Therefotraction  and actitraction .2+i is necessary for MLCK activation, MLC20 can be phosphorylated by ILK without an increase in Ca2+i (696 or through the activation of endogenous inhibitors of PP1c, such as CPI-17 and PHI-1 (Although an increase in Cain Ca2+i . In addind PHI-1 . Anothernd PHI-1 . The myond PHI-1 . Howevernd PHI-1 .In conclusion, airway smooth muscle hypercontractility might result from inhibition of PP1c, inhibitory phosphorylation of MYPT1 and/or dissociation of the PP1c/MYPT1 complex. Many signaling pathways converge in the regulation of MLCP, a key regulator of contraction; however, other potential perspectives on the hypercontractile phenotype of airway smooth muscle might remain to be explored.MÁ-S and BB-P contributed to the conception and design of the work. MÁ-S and MÁ-G wrote the first draft of the manuscript. BB-P and SE-S revisited critically the intellectual content. All authors contributed to the manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, left bundle branch area pacing (LBBAP) has been shown to be feasible. However, the right ventricular (RV) implantation site for LBBAP remains elusive. We believe that the RV implantation site should be located at the posteromedial basal septum, and in this paper, we propose a new method to help guide lead implantation. The aim of this study is to demonstrate the feasibility of the proposed method.The RV implantation site was positioned by a combination of a nine-grid system on fluoroscopy and the use of intracardiac echocardiogram (ICE) and then verified by ICE.P > 0.05). The operation time was 133 ± 32.9 min. The time of X-ray fluoroscopy was 21.2 ± 5.9 min. The mean time for lead positioning during LBBAP was 33.8 ± 16.6 min. During a follow-up of 3 months, the LBB capture threshold remained stable in 12 patients, except for one patient who had an increase in the LBB capture threshold to 3.0 v/0.4 ms.Fifteen patients were enrolled for LBBAP using our method. The acute success rate was 86.7% (13/15), which demonstrated that our method is useful for assisting with lead implantation. According to ICE, the distance between the implantation site and apex (the front) and the distance between the implantation site and tricuspid annulus (the back) were 44.9 ± 10.7 and 33.2 ± 10.4 mm, respectively, and the ratio of the front and the back was 1.57 ± 0.80. The distance between the implantation site and the front junction point of the left-right ventricle (the upper) and the distance between the implantation site and the back junction point (the lower) were 33.4 ± 10.6 and 24.5 ± 10.2 mm, respectively. The ratio of the upper to the lower was 1.76 ± 1.36. These results suggest that the implantation site was at the posteromedial basal septum. The width of the QRS duration increased from 110.4 ± 33.1 ms at baseline to 114.1 ± 16.1 ms post LBBAP (Our preliminary results indicate that the posteromedial basal septum could be seen as the implantation site for LBBAP. As a technique for LBBAP, ICE is a useful method for assisting with lead implantation. It is feasible and safe to use a nine-grid system combined with ICE for LBBAP. His-Purkinje system pacing has been proposed as the most physiologic mode of ventricular pacing. Previous studies have demonstrated that bundle branch block could be corrected by pacing at the distal His bundle and validated the safety and clinical benefits in patients with various cardiac diseases –4.However, previous studies have shown that the His bundle pacing threshold significantly increased with time, even went over the capture threshold, to correct the left bundle branch (LBB) block , 5. OtheIt is known that after penetrating the membranous atrioventricular (AV) septum, conductive fibres of the LBB are spread beneath the endocardium of the interventricular septum (IVS) over a relatively large area, which offers an easier approach for pacing the LBB . RecentlHowever, the right ventricular (RV) implantation site for LBBAP remains elusive. We believe that the implantation site at the RV side of the IVS and the implantation direction of the lead tip in the septum are two essential elements to warrant successful LBBAP. The RV implantation site is usually identified on the basis of the His bundle potential signal, so the dual-lead method is helpful in finding the implantation site , but it LBB originates in the branching portion underneath the membranous septum ; as a reThis study was approved by the Ethics Committee of Xinhua Hospital Affiliated with Shanghai Jiaotong University School of Medicine  and performed in accordance with the Declaration of Helsinki. The RV implantation site was positioned by our proposed methods and then verified by ICE.Patients who had indications for pacing therapy according to the 2013 ESC/EHRA Guidelines and underwent ICE guided LBBAP in our centre from Nov 1, 2018 to Jun 31, 2019 were enrolled, and the clinical data were analysed retrospectively. All patients submitted written informed consent and demonstrated an understanding of LBBAP as a nonstandard approach to achieve physiologic pacing.As previously described –16, a SeAn ICE catheter  was advanced through the right femoral vein towards the mid-right atrium (RA). The basic rule to advance the ICE catheter in vascular or cardiac chambers is to maintain an echographic clear space (black) ahead of the catheter and avoid pushing when an echogenic space (white) is ahead of the catheter .The ICE catheter is initially in “home” view in the mid-RA, and the transducer in a neutral position is facing the tricuspid valve (TV). From the home view, clockwise rotation to the six o’ clock of the catheter brings into view the upper and lower right pulmonary veins; then, the catheter is “P” flexed and could be advanced gently into the RV. Once the catheter tip passes through the TV, the deflection is released gently until the tip points to the RV apex, and a view of the inferior RV is obtained. From this RV view, the catheter is gradually rotated clockwise and “L” flexed to image the long axis view of the left ventricle (LV) and IVS .When the catheter passes through the TV, the “P” flex is released gently to position the tip close to RVOT. Then, the catheter is gradually rotated counterclockwise to image a short axis view of the LV and IVS. The LV, RV and IVS should be visualized in both the short axis around the mitral plane and a longitudinal view of the LV Fig. .Fig. 1IThere is no true bifurcation of the bundle of His in the human heart . The sumSingle-plane right ventricle fluoroscopy in RAO 30° projection was performed, and the images of fluoroscopy were reviewed. The RV end-diastolic images were determined. Then, two parallel lines along the long axis plane and the short axis plane were drawn, which divide every plane into three parts, and the distance between two adjacent lines was equal. The RV in this projection was divided into nine grids, and the desired implantation site was proposed as the intersection region between the posterior part of the long axis plane and the middle part of the short axis   1 should display a “W” morphology with a mid-notch close to the bottom  referred for primary pacemaker implantation and underwent LBBAP procedures with our proposed method during the study period were included. Among 15 patients, LBBAP was successfully achieved and demonstrated an RBBB pattern during UTP in 13 patients. The acute success rate was 86.7% (13/15), which demonstrated that our method is a useful method for assisting with lead implantation. Of two unsuccessful patients, the RBBB pattern disappeared during C315 sheath removal in 1 patient, and another patient with baseline RBBB received LV septum pacing with a paced QRSd > 130 ms after failed LBBAP 5 times. All procedures were performed under the guidance of ICE. The operation duration was 133 ± 32.9 min. The time of X-ray fluoroscopy was 21.2 ± 5.9 min. The mean time for 3830 lead positioning during LBBAP was 33.8 ± 16.6 min.n = 3) or RBBB (n = 3) was present in 6 patients .Among 15 patients, three remained in sinus bradycardia. Sinus pause-dependent atrial fibrillation was noted in 1 patient. Atrioventricular block was present in the remaining 11 patients . The left ventricular ejection fraction (LVEF) was 62.6 ± 10.5%, and a reduction in LVEF was found in two patients (25 and 34%). Three patients  had histories of myocardial infarction or percutaneous coronary intervention, 66.7% (10/15) had hypertension, and 20.0% (3/15) had diabetes mellitus. Baseline left bundle branch block (LBBB) (ECG characteristics and pacing parameters.P > 0.05 vs. baseline). Among 13 LBBAP patients, LBB potential could be recorded in 10 patients from the LBB lead , and the mean interval of LBB potential to the beginning of the QRS complex was 29.5 ± 4.4 ms. The LVAT for all LBBAP patients was 76.2 ± 8.6 ms, and the R wave sensing amplitude, pacing impedance, and unipolar pacing capture threshold were 9.5 ± 2.7 V, 845 ± 106.4 Ω, and 1.08 ± 0.86 V/0.4 ms respectively after implantation  or RBBB (n = 3), LBBAP corrected both the LBBB (n = 3) and the RBBB (n = 2) with a successful bundle branch correction rate of 83.3%. The RBBB morphology could be corrected by bipolar LBBAP at a low output. In the case shown in Fig. For patients with complete LBBB  . During the 3-month follow-up period, pacing parameters, including the sensing amplitude, pacing threshold and impedance, remained stable in 12 patients  and LVEF  were similar between the 3-month follow-up and baseline.Transthoracic echocardiogram evaluation data at baseline and 3-month follow-up were available in 13 patients receiving successful LBBAP  [Using our methods, assisted by pacing localization, we could quickly locate the desired implantation site at the RV septum and avoid damaging the His bundle system. The two-way monitoring method, with the help of the notch in the paced QRS complex in lead V4.1 min) , which sTo date, the precise and ideal anatomical location of LBBAP, which should theoretically be located distal to the His bundle, trunk of the LBB, or proximal to the left anterior/posterior fascicle bundle, has not yet been well elucidated. The LBB potential, a determinant of LBB capture, confirms the pacing site at the left bundle branch or nearby the left bundle branch. The threshold differences between LBB capture and myocardial capture may be too close to discriminate . To avoiThe use of ICE allowed us to monitor all procedures. The procedure was successfully completed without acute complications, and LBBAP electrocardiogram post procedure was presented in the 13 patients. ICE showed that the ratio of the front to the back was 1.57 ± 0.80, and the ratio of the upper to the lower was 1.76 ± 1.36, suggesting that the implantation site was at the posteromedial basal septum. ICE was also helpful in defining the aetiology of failed LBBAP procedures. In our two failed patients, the lead all deviated from the original direction, even though the tip moved back in a patient.Our experience suggests that ICE guidance may have the following merits. ① The direct visualization of endocavitary structures, which is currently not provided by any other real-time mapping system. ② Monitoring of the lead orientation in the septum during fixing. ③ Direct visualization of the depth of the tip into the septum reduces the risk of perforation. However, unskillful handling of ICE could also interfere with the lead, even pulling it back.Considering both pacing efficiency and safety, a series of tips and tricks for lead implantation may be shared as follows. Heparinized saline flush pre-procedures are used to prevent thrombus formation within the C315 sheath lumen. A guide wire  is available and easily guides the C315 tip to the desired site. A suitable power is needed to keep the C315 tip perpendicular to the IVS. A combination of using the two-way monitoring method and ICE could identify the lead orientation in the septum and avoid deviating from the original direction. The 3830 lead depth should be carefully estimated during implantation to avoid passing through IVS.Some limitations need to be discussed. First, ICE is expensive, which is linked with increased medical cost. Second, the present study had a short follow-up interval. We expect long-term favourable clinical benefits, as observed in the case reported by Huang et al. . Third, Our preliminary results indicate that the posteromedial basal septum could be seen as the implantation target site for LBBAP. As a technique for LBBAP, ICE is a useful method for assisting with lead implantation. It is feasible and safe to use a nine-grid system combined with ICE for LBBAP.


Coreopsis tinctoria Nutt., arousing huge interest recently for its considerable biological characteristics including antioxidant, antineurotoxic, and antidiabetic activities. An ultrahigh performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS) was successfully used to determine okanin in rat plasma after oral administration of okanin. Bavachalcone acted as an internal standard (IS). By gradient elution, IS and analyte were separated on a C18 column for 7 min at a flow rate of 0.25 mL/min with acetonitrile-0.1% acetic acid mobile phase. The stability, matrix effect, extraction recovery, accuracy, precision, linearity, and selectivity of the method were firstly demonstrated. The major pharmacokinetic parameters of okanin in rat plasma were then measured using the developed UPLC-MS method. An UPLC-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was finally established to obtain the specific and accurate mass of okanin in rat plasma after oral administration, and its proposed fragmentation was further elaborated.Okanin is a major flavonoid found in  Coreopsis tinctoria Nutt.'s (C. tinctoria) capitula is consumed as herbal tea, and its extracts exhibit various biological activities including antioxidant [C. tinctoria [C. tinctoria. Okanin had the prominent effect on DPPH scavenging acts with EC50 as 6.2 μM, which was stronger than those of butylated hydroxytoluene and ascorbic acid with EC50 as 45.8 μM and 30.4 μM, separately [50 as 11.0 μM [α-glucosidase with IC50 values about 0.02 mM, suggesting that it is a potential drug for the treatment of diabetes [50 value at 15.54 μM, suggesting that it can treat neurodegenerative diseases [In China, ioxidant –4, antihioxidant , 6, antiioxidant , antidiaioxidant –11, hepaioxidant , and antioxidant . Okanin inctoria  having aparately . Meanwhi 11.0 μM . Kil et  11.0 μM . Furtherdiabetes . Besidesdiseases .C. tinctoria become necessary. The flavonoids including okanin from C. tinctoria had been successfully analyzed by UPLC, HPLC-MS, high-performance thin-layer chromatography, and capillary zone electrophoresis methods [C. tinctoria among these methods [C. tinctoria in order to better understand their pharmacological action. Only one report emphasized the characterization of the pharmacokinetics of okanin and isookanin, the two most prominent antioxidants, in rat plasma after oral administration of 100 mg/kg ethanol extract of C. tinctoria capitula; however, the pharmacokinetic parameters of two targets only measured the time to reach Cmax (Tmax) and the maximum plasma concentration (Cmax) [Smilacis glabrae extract [Due to the significant pharmacological effects, effective methods for the identification of flavonoids in  methods , 18–21.  methods , 19. Mean (Cmax) . Neverthn (Cmax) –25. Thern (Cmax) –24, but In this work, an effective UPLC–MS/MS method coupled with multichannel segmented selected reaction monitoring program was developed for measuring the levels of okanin in rat plasma after oral monomer administration. Bavachalcone served as an internal standard (IS). The structures of the okanin and IS are shown in 18, 6 cc/500 mg) were provided by Wan Cheng Bo Da . Triple deionized water was adopted during the study and then purified with a Millipore purification system .Okanin and bavachalcone with purities over 98% were provided by Shanghai Yuanye Bio-Technology Co., Ltd. . Heparin sodium was provided by Affandi . HPLC grade acetonitrile, acetic acid, and methanol were purchased from E. Merck . The SPE cartridges  with an electrospray ionization (ESI) interface. An ACQUITYTM BEH C18 column  combined with an ACQUITYTM UPLC C18 guard column was used for the separation. The column temperature was set at 35°C. The binary mobile phase system was composed by 0.1% acetic acid (A) and acetonitrile (B). The gradient conditions of the mobile phase included: 0–2 min, 95–20% (A); 2-3 min, 20–20% (A); 3–3.5 min, 20–95% (A); 3.5–7 min, 95–95% (A). The flow rate was kept at 0.25 mL/min for an overall 7-min run time. The volume of sample injection was 5 μL. Mass spectrometer was further improved as follows: capillary voltage, 3.50 kV (+); source temperature, 120°C; and desolvation temperature 300°C. Nitrogen was served as the desolvation and cone gas, and flow rates were set at 600 and 50 L/h, respectively. Argon acted as the collision gas at 2.89 × 10−3 mbar pressure. Multiple reaction monitoring (MRM) in the positive ion mode was selected in this study. The dwell time was automatically set by MassLynx NT 4.1 software. All data obtained from the centroid mode were further processed by MassLynx NT 4.1 software. The improved MRM transitions and energy parameters including cone voltage and collision energy of okanin and IS are listed in Supplementary An UPLC-MS/MS method was carried out using an ACQUITY2 was 35 V.The full-scan mass range was set at 50–1200 for obtaining accurate mass of okanin by UPLC-Q-TOF-MS analysis. The mass spectrometer conditions were listed as follows: gas temperature 550°C, spray gas 50 psi, auxiliary gas 50 psi, curtain gas 35 psi, ion spray voltage 5500 V, declustering potential 90 V, and collision energy for MSOne mg/mL of okanin was prepared with methanol as standard stock solution. Then, the standard stock solution was diluted 10 times as working solution. In the meantime, one mg/mL of bavachalcone was prepared with methanol as an IS solution. Calibration standards, low, intermediate, and high levels of quality control (QC) samples were prepared using spiking blank rat plasma with appropriate volumes of the working solutions.Six serial concentrations were 1.956, 10.95, 40.40, 200.20, 800.24, and 1390 ng/mL for calibration standard solution. The concentrations of the low, intermediate, and high levels of QC samples were 10.01, 500.10, and 1000.20 ng/mL for okanin. All samples were incubated at −20°C before application.μL blank plasma, and 50 μL of IS solution (22.06 ng/mL) was added to respective tube.Two hundred microlitres of calibration standards, QC samples, and plasma samples were added to a 1.5 mL Eppendorf tube containing 400 μL methanol and centrifuged at 16,000 ×g for 15 min. Five microlitres of aliquot was subsequently injected into the UPLC-MS for investigation.Before loading the sample, the SPE cartridges were washed with 3 mL of methanol and 5 mL of purified water separately. The mixture in respective tube was vortexed for 1.0 min and then loaded onto a SPE cartridges. The SPE cartridges were separately eluted with 2 mL purified water and 4.5 mL of methanol. The resulting eluate was dried with nitrogen. The residue was redissolved in 50 Rt) and MRM transitions were adopted for analyzing okanin and IS. Meanwhile, the accurate mass of okanin and its proposed fragmentation mechanism in rat plasma after oral administration were obtained using UPLC-Q-TOF-MS.In order to study the possible endogenous interference on okanin and IS, the blank plasma from six rats, the corresponding blank plasma added with okanin and IS, and the plasma of rats after oral administration of okanin were analyzed by UPLC-MS/MS, respectively. The retention times (x2-weighted liner least-square regression model. The lower limit of detection (LLOD) was defined as the lowest concentration with at least three signal-to-noise ratios. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the standard curve that can be quantitated with an accuracy of 80–120% and a precision value not exceeding 20%.Calibration curve for okanin was plotted for the peak area ratio of okanin/IS versus plasma concentrations by a 1/n = 6) of low, middle, and high concentrations of QC samples in the same day and over three consecutive days, respectively. The concentration of sample was calculated according to the standard curve equation prepared on the same day. The precision of the method was calculated as the relative standard deviation (RSD) and was less than 15%. Accuracy was expressed as the relative error (RE) and was within ±15%. The recovery and matrix effects were measured using low-, middle- and high-concentration QC samples with six replicates. The recovery was assessed by comparing the peak areas of the spiked samples to those of the untreated sample. The matrix effect was determined by comparing the peak areas of spiked blank plasma (A) to the peak areas of the solution standards in methanol at equal concentrations (B). The matrix factor (MF) was expressed as the ratio of (A/B × 100), and the MF values ranged from 80% to 120%. The stabilities of okanin in plasma were evaluated by analyzing the three concentrations of QC samples at different storage and processing procedures. The short-term stability was also assessed by analyzing the QC sample retained in the autosampler (4°C) for 36 h. The QC sample was stored at −20°C for 30 days in order to evaluate long-term stability. The freeze-thaw stability of QC samples was evaluated by transferring them from −20°C to room temperature for three complete freeze-thaw cycles. The stability was demonstrated if the percentage deviation was no more than 15% of the nominal concentration.Intraday and interday accuracy and precision were assessed from replicate analyses  rats (250 ± 20 g) from Animal Safety Evaluation Center of Heilongjiang University of Chinese Medicine (China). The rats were divided into control and treatment groups with six animals each. The rats were maintained under a light/dark cycle of 12 h, in the temperature range of 22°C ± 2°C to ambient temperature, and at a humidity of 50% ± 5%. Before the experiment, all rats were fasted for 12 h, but allowed to drink water freely. Each rat in the treatment and control group were treated with 1 mg/kg okanin and an equal volume of distilled water in an oral manner, separately. Blood samples (approximately 0.4 mL) were collected into 1.5 mL heparinized polythene tubes from the suborbital venous plexus before administration and at 0, 0.083, 0.167, 0.33, 0.50, 0.75, 1.0, 2.0, 4.0, 8.0, 12.0, and 24 h after administration. Then, the blood samples were immediately centrifuged at 13,000 rpm for 10 min, and samples were processed as described in Cmax) and the time to reach Cmax (Tmax) were directly determined. The elimination rate constant (Kel) was then obtained via linear regression analysis on the log-linear portion of the plasma concentration versus time curve. The other major pharmacokinetic parameters of okanin in rat plasma including half life (t1/2), the mean residence time from zero to the sampling time (MRTt0–) and to infinity (MRT∞0–), and area under the plasma concentration versus time curve from zero to the sampling time (AUCt0–) and to infinity (AUC0–∞) were determined using WinNonlin Professional Version 5.2.1 (http://www.pharsight.com). The results were expressed as arithmetic mean ± standard deviation .According to observing plasma concentration versus time curve, maximum plasma concentration  by the high-resolution Q-TOF analyses for structural confirmation. In UPLC-Q-TOF-MS/MS experiment, the ion at m/z 289.0717 yielded four fragment ions at m/z 271.0431, 163.0388, 153.0182, and 135.0439, respectively. As shown in m/z 163.0388 was produced by losing A's ring. The predominant fragmentation ion at m/z 135.0439 and 153.0182 was formed from the cleavage of position 3 in the three-carbon chain, and the charge is retained in ring A and ring B, respectively. Minor fragment m/z 271.0431 was produced directly from the parent ion of m/z 289.0717 due to the neutral loss of one molecule of water, indicating the existence of orthophenol hydroxyl in the structure. The proposed fragmentation mechanism was elaborated in Okanin in rat plasma eluted at 2.38 min showed a major molecular ion at y = 19.979x + 337.59 and r2 = 0.9980  of 1296.12 ± 60.31 ng/mL (mean ± SD), revealing quick absorption of okanin into blood circulatory system. An existing report suggested Tmax and Cmax values for okanin of 0.333 h and 10.5 μM after oral administration of 100 mg/kg ethanol extract of C. tinctoria capitula [Tmax values, okanin was absorbed into blood more quickly by orally administered monomer than the extract of C. tinctoria, indicating a possible pharmacokinetic interaction of multiple components in the extract of C. tinctoria. Our conclusion was consistent with that of literatures [As listed in Supplementary capitula . In conteratures –25.t1/2, Kel, AUCt0–, AUC 0–∞, MRTt0–, and MRT∞0– values were determined here. The concentrations of okanin in rat plasma were decreased quickly, and the shortest elimination half life (t1/2) at 0.89 ± 0.09 h was observed for okanin. The Kel values were 0.75 ± 0.08 for okanin. The AUCt0– and AUC ∞0– values of okanin were 1728.78 ± 146.64 and 1826.51 ± 149.59 ng·h/mL, respectively. MRTt0– and MRT0–∞ values for okanin were 1.14 ± 0.06 h and 1.36 ± 0.10 h, separately.To further study the pharmacokinetic behaviors of okanin, more pharmacokinetic parameters including Tmax, Cmax, t1/2, Kel, AUCt0–, AUC 0–∞, MRTt0–, and MRT∞0– values were obtained in this study. Meanwhile, target okanin had shorter Tmax by monomer administration than that of herb extract administration, suggesting a potential pharmacokinetic interaction among multiple components in herb extract. The pharmacokinetic behaviors for okanin provide the basis for further studies to elucidate the mechanisms of action and facilitate clinical application.An effective UPLC–MS/MS method was developed for analyzing okanin in rat plasma after oral administration okanin monomer for the first time. Compared with the literature report, more pharmacokinetic parameters of okanin including


Co-crystal structures of GLIC, a bacterial ligand-gated ion channel, in complex with monocarboxylate and dicarboxylate derivatives are reported. It is shown that binding occurs at two pharmacological sites in the extracellular domain, which is in agreement with the reported effect of some carboxylates as allosteric modulators of GLIC. GLIC is a bacterial homologue of the pentameric ligand-gated ion channels (pLGICs) that mediate the fast chemical neurotransmission of nerve signalling in eukaryotes. Because the activation and allosteric modulation features are conserved among prokaryotic and eukaryotic pLGICs, GLIC is commonly used as a model to study the allosteric transition and structural pharmacology of pLGICs. It has previously been shown that GLIC is inhibited by some carboxylic acid derivatives. Here, experimental evidence for carboxylate binding to GLIC is provided by solving its X-ray structures with a series of monocarboxylate and dicarboxylate derivatives, and two carboxylate-binding sites are described: (i) the ‘intersubunit’ site that partially overlaps the canonical pLGIC orthosteric site and (ii) the ‘intrasubunit’ vestibular site, which is only occupied by a subset of the described derivatives. While the intersubunit site is widely conserved in all pLGICs, the intrasubunit site is only conserved in cationic eukaryotic pLGICs. This study sheds light on the importance of these two extracellular modulation sites as potential drug targets in pLGICs. A variety of pharmacological molecules modulate pLGIC activity and thus modify their response towards their respective agonists. The unexpected occurrence of pLGIC orthologues in prokaryotes was discovered only a decade ago , was mixed in a 1:1 ratio with a reservoir solution consisting of 16% (2.2 M) glycerol, 12–14.5% (34–41 mM) polyethylene glycol 4000, 2% (0.28 M) dimethyl sulfoxide, 400 mM sodium thiocyanate with the buffering carboxylic acid adjusted to pH 4 using NaOH–HCl. The concentration of the monocarboxylic or dicarboxylic acid compound used as a buffer in the reservoir solution was 100 mM, except for fumarate (30 mM) owing to a solubility issue. The quality of the crystals was improved by the microseeding technique: a suspension of crushed crystals grown in the same crystallization condition but in the presence of acetic acid/acetate was added upon setting up the crystallization experiment. Crystals appeared overnight with a parallelepiped-like shape and grew for one week before reaching their final dimensions. All of the crystals were directly flash-cooled in liquid nitrogen prior to data collection.All crystals were obtained by vapour diffusion in hanging drops at 18°C. The concentrated protein solution  with one pentamer in the asymmetric unit, with the exception of the GLIC–succinate structure, in which two pentamers, oriented back to back, were found in the asymmetric unit .X-ray data sets were collected on the PROXIMA 1 and PROXIMA 2 beamlines at the SOLEIL synchrotron, Gif-sur-Yvette, France and on the ID23-1 and ID29 beamlines at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Reflections were integrated using 2.4.REFMAC5 . Here, we solved a 3.2 Å resolution structure of GLIC in complex with propionate obtained from crystals grown in a solution buffered at pH 4.0 using 100 mM propionate instead of acetate. Similarly to acetate, propionate molecules were found both in the intrasubunit site, where propionate interacts with the side chains of Tyr102 and Arg85, and in the intersubunit site at the bottom of the agonist site, where propionate interacts with Glu181 and Arg77 of one subunit and with Arg105 of the complementary subunit . The overall structure was similar to the acetate-bound structure , and no significant remodelling of the intrasubunit and intersubunit pockets was observed upon propionate binding.We previously identified ten acetate-binding sites (two per monomer) within the high-resolution GLIC X-ray structure obtained from crystals grown at pH 4 in acetate buffer . In this structure, two identical GLIC pentamers were found in the asymmetric unit, with a c unit-cell parameter that was twice as large as in the other structures described in this study . Strong Fo − Fc densities, reminiscent of succinate binding, were observed in the intersubunit and intrasubunit sites, and succinate was modelled in both sites previously described to bind acetate or propionate . The succinate molecule bound in the intersubunit site is modelled with one of its carboxylate moieties superimposed on the position of acetate below Loop C. This carboxylate moiety interacts with the side chains of residues from two adjacent subunits: Arg77 (Loop A) and Glu181 (Loop C) from one subunit and Arg105 from the neighbouring subunit. The other carboxylic moiety of succinate interacts with the side-chain amino group of Asn152 from the complementary subunit .So far, only monocarboxylic acids have been reported to bind GLIC [acetic .The succinate molecule in the intrasubunit site displayed a higher flexibility than the intersubunit succinate, as seen in the different subunits. Indeed, the first succinate carboxylic moiety binds in the same way  as acetate: through a hydrogen bond to the hydroxyl group of the well conserved Tyr102 and a salt bridge to the guanidinium group of Arg85, which itself makes a salt bridge with Glu104. However, the second carboxylic moiety of the intrasubunit succinate molecule only interacts with the main-chain carbonyl group of Pro74, leading to different binding poses of succinate in the intrasubunit site in the different subunits .This analysis thus reveals new residues that are involved in carboxylate binding, namely the side chain of Asn152 in the intersubunit pocket and the main chain of Pro74 in the intrasubunit pocket (3.3.b and 2c). The first carboxylic groups of malonate and glutarate occupied the position of acetate, with a similar interaction pattern with Arg77, Glu181 and Arg105. The second carboxylic group of glutarate forms a hydrogen bond to Asn152, as for succinate, whereas that of malonate interacts with the side chain of Arg105 In attempt to check whether other dicarboxylic acids with different lengths are also able to bind GLIC, we solved crystal structures of GLIC in complex with malonate (diC3) and glutarate (diC5) at 2.9 and 2.65 Å resolution, respectively. Surprisingly, densities corresponding to malonate and glutarate were only found in the intersubunit sites, whereas a globular density, modelled as a chloride ion, was found in the intrasubunit sites of both structures  at 0.1 M as a buffer produced co-crystals of GLIC that provided a 2.9 Å resolution structure in which a crotonate molecule occupied the intersubunit pocket, whereas the spherical density in the intrasubunit pocket was modelled as a chloride ion . The crotonate molecule was oriented along the axis between Arg77 and Asn152, as was observed for the succinate molecule present in the intersubunit pocket. This orientation partially overlaps with that previously described by molecular docking  was then used as a buffering agent at 0.03 M, producing a 2.9 Å resolution structure. A very similar orientation was adopted by fumarate in the intersubunit pocket, whereas a chloride ion again occupied the intrasubunit pocket . The first carboxylic acid moiety occupied the same position as acetate, near Arg77 and Arg105, and the C4 atom could be superimposed on the C4 atom of crotonate. The distances between the C4 atom and Glu181 or Asn152 were unchanged in the fumarate and crotonate structures, although Asn152 is involved in the coordination of fumarate but not crotonate through hydrogen bonding , the proton concentration thus might be too high to allow the equilibrium to shift towards channel closure, even at 100 mM crotonate. At the molecular level, the intersubunit site that partially overlaps with the common pLGIC orthosteric site appears to be the principal binding site for carboxylates. Indeed, all of the monocarboxylic and dicarboxylic acids were found to bind in this pocket with a similar binding mode for the first carboxylic group involving Arg77 and Glu181 from one subunit and Arg105 from the neighbouring sububnit. For dicarboxylates, except for malonate, Asn152 from the complementary subunit contributes to the coordination of the second carboxylic group. For malonate, the molecule is too short to reach the Asn152 side chain and the second carboxylate is thus coordinated by Arg105. In the crotonate structure, the hydrophobic tail is partially coordinated by weak hydrophobic interactions involving Ile131 and Phe41. These latter residues have been suggested to be involved in crotonate-mediated inhibition of GLIC currents  are actually allosteric inhibitors, all of the structures presented in this work show that they  bind the apparently open conformation of this ion channel. In particular, crotonic acid was described to be an inhibitor of GLIC, with an ICbSupplementary Fig. 2). Why these carboxylate derivatives bind in this intrasubunit pocket while others do not is not completely obvious, at least based on the co-structures. Indeed, simple docking experiments with fumarate show that it could also bind to the intrasubunit site. Crotonic acid, which is a monocarboxylic derivative of fumarate, might be unable to stably bind the intrasubunit site because of the absence of the second carboxylic group and be easily replaced by chloride. It is also noteworthy that succinate has higher B factors (mean intersubunit Bsuccinate of 85.1 Å2 versus a mean intrasubunit Bsuccinate of 106.5 Å2) in the intrasubunit binding site than in the intersubunit binding site, as well as a higher variability of binding poses within the intrasubunit site . Unlike the intersubunit succinate, where both carboxylic groups are tightly coordinated through hydrogen bonds to Arg85, Tyr102 and Glu104, the second carboxylic group of succinate in the intrasubunit site points almost freely towards the solvent and is only loosely coordinated through a hydrogen bond to the main chain of Pro74 in most of the modelled succinate poses. This flexibility might explain why fumarate, which is a cis-unsaturated (and thus more rigid) derivative of succinate, was not seen experimentally in the intrasubunit site. From a functional point of view, this intrasubunit site, which is also called the vestibular site, has already been described as a potent modulation site in other pLGICs. Indeed, along with the intersubunit site, the vestibular intrasubunit site has also been described as a benzodiazepine-binding site . The authors correlate this bimodal modulation with intrasubunit versus intersubunit site binding, where flurazepam would promote ELIC potentiation when bound to the ‘high-affinity’ intrasubunit site and inhibition, at higher concentrations, when bound to the ‘low-affinity’ intersubunit site. Besides ELIC, sTeLIC, a receptor that we have recently characterized, is also positively modulated by cinnamic acid derivatives that bind to the intrasubunit vestibular site with a similar potency as flurazepam in ELIC . Bromoethanol . The consensus emerging from the different functional/structural studies in ELIC and sTeLIC is that the intersubunit site would be a negative modulation site and the intrasubunit site a potentiating site. In the case of GLIC, a comprehensive functional analysis of the effect of these carboxylates is required to link binding to modulation. Furthermore, the crystal structures of the same complexes with the closed form of GLIC would be necessary in order to predict the overall activating or inhibiting properties of these compounds, as recently shown for barbiturates .At this point, it is tempting to speculate that the existence of two regulatory pockets that are rather close in space might be an evolutionary remnant of a primordial catalytic activity in the extracellular domain of pLGIC likely involving amino-acid metabolism. More specifically, the direct catalytic decarboxylation of an amino acid might have been possible at some point during evolution of the pLGIC family in the vestibular site, with the product diffusing rapidly to the other (orthosteric) binding site, resulting in the coupled opening of the pore. Indeed, several agonists of pLGICs are directly derived from the simple decarboxylation of amino acids or derivatives thereof (GABA, 5HT3, histamine 5.In conclusion, GLIC can accommodate monocarboxylate or dicarboxylate derivatives in two distinct sites of the extracellular domain. Some carboxylates are known to be allosteric inhibitors of GLIC, but others still need to be characterized from a functional point of view. At the molecular level, all of the carboxylates bind to the intersubunit site that partially overlaps the pLGIC orthosteric site, but only acetate, propionate and succinate bind in the intrasubunit vestibular site. This latter site is reported to be a potentiation site in other bacterial pLGICs and is conserved in eukaryotic cationic receptors. This vestibular site is likely to be a key pharmacological site and thus an important target for rational drug design against some neurological disorders involving human pLGICs.GLIC–glutarate, 6hjaPDB reference: GLIC–malonate, 6hjbPDB reference: GLIC–crotonate, 6hjiPDB reference: GLIC–succinate, 6hjzPDB reference: GLIC–fumarate, 6hj3PDB reference: GLIC–propionate, 6hppPDB reference: 10.1107/S205979832000772X/ag5034sup1.pdfSupplementary figures. DOI:


Drosophila melanogaster. In the task, a wing-shortened fruit fly freely moves on a round platform surrounded by a 360° white screen with two vertical black stripes placed at 0° and 180°. A normal fly will tend to approach the stripes one at a time and move back and forth between them. A variety of tasks developed based on the Buridan’s paradigm were designed to test other cognitive functions such as visual spatial memory. Although the movement patterns and the behavioral preferences of the flies in the Buridan’s or similar tasks have been extensively studies a few decades ago, the protocol and experimental settings are markedly different from what are used today. We revisited the Buridan’s paradigm and systematically investigated the approach behavior of fruit flies under different stimulus settings. While early studies revealed an edge-fixation behavior for a wide stripe in the initial visuomotor responses, we did not discover such tendency in the Buridan’s paradigm when observing a longer-term behavior up to minutes, a memory-task relevant time scale. Instead, we observed robust negative photoaxis in which the flies approached the central part of the dark stripes of all sizes. In addition, we found that stripes of 20°-30° width yielded the best performance of approach. We further varied the luminance of the stripes and the background screen, and discovered that the performance depended on the luminance ratio between the stripes and the screen. Our study provided useful information for designing and optimizing the Buridan’s paradigm and other behavioral tasks that utilize the approach behavior.The Buridan’s paradigm is a behavioral task designed for testing visuomotor responses or phototaxis in fruit fly  Drosophila melanogaster serves as a good animal model for behavioral studies because of its complex behavior and abundant genetic tools [The fruit fly ic tools . Among mic tools –7.In the Buridan’s paradigm, a single fly with shortened wings is placed into a round arena. Two black vertical stripes, serve as the visual cues, are attached on the wall of the arena and are separated by 180°. The fly is allowed to freely move in the arena. Without any training, a naïve fly tends to perform the approach behavior by walking back and forth between two black stripes persistently ,8,9. TheIn the present study, we systematically investigated the approach behavior with different stimulus settings at the time scale of minutes and revisited the question: what is the behavior preference of fruit flies in the Buridan’s paradigm? We tested the flies under three hypotheses for the behavioral preferences: (1) The flies prefer salient visual objects, (2) they are attracted by high-contrast edges, and (3) they like to approach dark regions. In the classical Buridan’s paradigm, which uses two narrow black stripes, all the three hypotheses lead to the same behavior preference, or the approach behavior toward the stripes. Therefore, the hypotheses are indistinguishable in this paradigm.To address this issue, we modified the experimental setup by using different stripe widths so that we could dissociate the factors correlated with each of the hypotheses. We also introduced several behavior metrics that are more accurate in quantifying the detailed approach behavior than the metrics proposed earlier . We furtw+, which was obtained from Brain Research Center (BRC) in National Tsing Hua University (NTHU), Taiwan.The fruit flies were incubated at 25°C with a 12:12 h light and dark cycle with a humidity level of ~50%. We used the wild-type strains, We established the behavioral tracking tool which followed the general design of previous researches . The areDrosophila melanogaster) were used in the present research and we shortened the wings one or two days before the experiments. The behavioral task was modified from the classic Buridan’s paradigm [Two or three days old wild-type flies , respectively. We also use the window films to adjust the luminance of the background screen, and the number of layers used for the background screen was always smaller than that used for the stripes, so that the stripes were always relatively darker than the screen. In the experiments, we tested 6 conditions of luminance as indicated by the layers of films for stripes vs. for screen: 3 vs. 0, 2 vs. 0, 1 vs. 0, 3 vs. 1, 2 vs. 1 and 3 vs. 2. The positions of the fly were recorded by a CCD camera at a speed of 15 frames per second for off-line analysis.To test the effect of luminance of the stripes and the background screen and luminance ratio (stripe luminance / background luminance) on the approach behavior, we replaced the cardboard by dark window films  which has a transparency of 5% in the visible and UV light ranges. The luminance of the stripes can be adjusted by stacking different layers of the films. We expect that stacking two layers of the films reduced the transparency to 5% × 5% = 2.5 × 10To quantify the approach behavior, we need to analyze the movement of each fly with respect to the stripes . IntuitiWe discovered that the approach behavior consisted of two distinct movement patterns. When away from the edge of the platform, a fly tended to fixate on a stripe by moving toward it. After reaching the edge, the fly tended to spend a significant amount of time moving along the edge of the platform near the stripe . This beTo calculate the deviation angle based on the movement direction, several different methods were implemented in various studies ,9,13. Mop(θ), of the data falling in each quantile. θ denotes the azimuth angle of the center of each quantile, i.e. θ = 0°, 10°, 20°, …. 350°.For better presentation and analysis, after calculating the deviation angle in each video frame, we binned the data into 36 quantiles with each quantile spanning 10° in azimuth. Next, we calculated the percentages, APD) and approach strength (AS). APD measures the difference between approach toward the dark regions approach toward the bright regions. Specifically, the APD was calculated by subtracting the normalized p(θ)’s of the quantiles in the bright regions from the normalized p(θ)’s of the quantiles in the dark regions:We determined the behavioral performance of the flies by calculating the approach probability density  toward the dark regions than the approach toward the bright regions, while a negative value indicates the opposite. Zero indicates non-approaching movement that is toward the dark and bright regions with equal frequency. In addition, in the all-bright or all-dark conditions, the APD cannot be defined and therefore was not calculated.A positive To visualize the approach behavior, we created the radar plot, which indicated the frequencies of the movement toward each quantile. Due to the symmetric nature of the arena, we merged the data in the pair of quantiles that are 180° apart, e.g. quantiles centered at 90° and 270°, and calculated their averaged percentage. This procedure made the radar plot point-asymmetric. If a fruit fly did not exhibit any approach behavior, it would move toward all directions with equal probability, and the expected value of each quantile was 1 / 18 (~5.56%). A value that was significantly different from 1 / 18 indicated some form of approach behavior.σf) and the approach strength (AS). The approach angle (σf) represents the direction of the strongest approach tendency and is defined by the angle σ of the axis that yields the smallest second moments M(σ) on the radar plot:θi is the angle of each quantile . The angle 0° is defined as the direction corresponds to the center of one of the dark stripes. σf can be obtained by taking the derivative of M(σ) with respect to σ.The radar plots allowed us to visually inspect the existence of approach behavior and its direction. We also defined a metric to quantify the approach behavior based on the radar plots. The radar plots usually form an ellipse and the longest axis, or the major axis indicates the direction of approach. The strength of approach can be measured by the ratio between the lengths of the major and the minor axes. To identify the major axis, one simply calculates the second moment of the ellipse with respect to any axis and the major axis is the one give rise to the smallest value. Based on this concept, we defined the approach angle (θi and p(θi) into this equation and solving for σ using trigonometric identities, we obtainϕ is a variable depending on p(θi) based on the following equations:Plugging M (denoted as Mmin) and correlates with the length of the major axis. The other gives rise to the maximum value of M (denoted as Mmax) and correlates with the length of the minor axis. Finally, the approach strength (AS) is defined as follows:The approach angle given in Eq  correspoMmin and Mmax indicates with the roundness of the ellipse, which correlates with the strength of approach. The approach strength AS is a value between 0 and 1. AS ≈ 1 for a long ellipse that is close to a line segment (perfect approach), and AS = 0 for an extremely round ellipse that is close to a circle (no approach). To determine the minimum level of AS that represents a statistically significant approach behavior, we performed simulations by generating random movement traces for 25 simulated flies and calculated their approach strength. The mean approach strength was 0.224 and the standard deviation was 0.160 for the simulated data. We took two standard deviations above the mean (AS = 0.544) as our statistical criterion for strong approach behavior and one standard deviations above the mean (AS = 0.384) for weak approach behavior. No significant approach behavior is considered when AS is smaller than 0.384.The ratio between APD and AS serve different purposes in quantifying the approach behavior. APD measures the tendency of approach toward the dark versus the bright regions. The value is normalized by the width of each region. By contrast, AS measures the tendency of approach toward any part of the screen and is independent of the presence of the dark and bright regions. The exact azimuth angle of the approach is specified by σf.We stress that To quantify whether different stripe widths led to different levels of locomotion, we analyzed the average movement speed and activity level of the fruit flies . The aveA total of 576 flies were used in the study but we removed 21 individuals which had significantly less locomotion than the others. The removal criterium was two standard deviations below the group mean of the activity level or the speed. Therefore, 555 flies were included in the analysis with 20–25 flies for each test condition.The statistical analyses were performed using Statistical Product and Service Solutions 22.0 (SPSS 22.0). A multi-factor within group analysis of variance was used for analyzing the probability density, and a mix variance analysis was used for evaluating differences between groups of different experimental conditions.APD, activity level and speed were observed between female and male flies. For different recovery periods, there were neither differences in APD nor activity level. However, flies with 5-day recovery period exhibited slight but statistically significant reduction in the speed comparing to the flies with 1-day recovery period.The present study used both genders and 1–2 days of recovery period . To evaluate the effect of gender and recovery period on the behavior performance, we conducted a small set of experiment  separately for the male and female flies, and also for 1, 3 and 5 days of recovery period . No signWe constructed the behavioral arena  based onThe approach behavior of fruit flies in the Buridan’s paradigm is known to be robust. However, the underlying behavioral mechanism was still unclear and not systematically tested. We proposed three hypotheses for the mechanism: (1) The saliency hypothesis. In this hypothesis, the flies tend to move toward any visual pattern that stands out of the background, whether it be a dark stripe in a bright background, or a bright stripe in a dark background . (2) Thew+) in different conditions of stripe widths in several example trials (APD) (see APD was large and positive for narrow dark stripes (20°-40°), but reduced when the widths of the stripes increased. Although the APD was low for wide dark stripes, visual inspection still revealed clear approach patterns that were toward the dark stripes  see  for indiAPD) see ; S5 Fig. stripes . No appr stripes .APD and AS  produced AS with magnitudes comparable to those by the narrow stripes (20°-40°). To further examine the movement patterns of flies in more detail, we plotted the radar plots, which indicate the frequency of movement toward all angles in the 36 quantiles  (FWHM) of each distribution and plotted FWHM as a function of the widths of the dark stripes (ter rim) . We also stripes .FWHM, stripes of 20° to 30° width induced the most concentrated approach behavior.For all stripe widths, the distributions always peaked at the center of the dark stripe. However, we discovered a couple interesting trends regarding the widths of the distributions. First, a wider stripe helped to reduce the width of the distribution. When the dark stripes were only 10° or 20° wide, the flies could not focus their approach in the azimuth range of the dark stripes and the distributions extended into the surrounding bright regions considerably . HoweverAPD against the luminance ratio and the luminance, we concluded that the approach behavior correlated with the luminance ratio between the stripes and the screen  the approach behavior depends on the luminance of the stripes, and 2) the approach behavior depends on the luminance ratio between the stripes and the background screen. To this end, we vary the luminance of the stripes and the background screen by using different layers of dark window films . By plote screen . AS alsot of APD .In the present study, we sought to provide a behavioral-level explanation of the visual approach in the fruit flies. We tested how widths and the luminance of the stripes affected the approach behavior. We discovered that among the different explanations of the approach behavior, our result supported the shade hypothesis, in which the flies prefer to approach dark objects. We introduced new metrics that worked better than previous metrics for evaluating the approach behavior of wide stripes. We found that the stripes with a width of 20°-30° were most effective in eliciting approach behavior, and the flies approached the entire azimuth range of the dark stripes. Wider stripes also elicited the approach behavior with comparable strengths, but the flies only approached the central part of a stripe with relatively weak approach toward the peripheral part. Further tests discovered that the approach is strongly correlated with the luminance ratio of the stripes and the background screen, but not the luminance of either the stripes or the screen.The approach toward the center of a wide stripe is a surprising observation and is worth discussion. Although the behavioral results of the present study support the shade hypothesis, it would predict that the approach is toward the entire azimuth range of a wide stripe. Therefore, the observation of approaching toward the central part of a wide stripe suggested that other behavioral drives might be involved. Could the bright regions (which were narrower than the stripes when they were larger than 90°) produce certain “repulsive” effect that drove the flies toward the central portion of the dark stripes? Do the specific compound eye structure or the visual system properties play roles here? Novel experimental settings need to be developed in order to study this phenomenon.Some early studies ,7 with dOne more interesting issue to discuss is the early studies of fruit fly choice experiments by Götz ,15, in wIn the present study, we only consider the explanation of approach at the behavioral level. The neural mechanism underlying such shade-seeking behavior, or negative phototaxis, is still not clear. In a previous study, photophobism has been observed in larva , but thew1118 flies are known to have poor vision, in another ongoing study we discovered that this types of flies actually perform decently in an arena that used dimmer green LEDs as the light source [It is interesting to note that, fruit flies with different genetic backgrounds may perform differently in Buridan’s paradigm. For example, although the white-eyed t source . TherefoS1 FigAPD based on two different methods for calculating the deviation angle. In Method 1, the deviation angle was calculated based on the movement vector regardless of the location on the platform. In Method 2, which was used in the present study, the deviation angle was calculated differently in the inner circle (< 0.85 radius) and in the outer rim (> 0.85 radius). See APD between the two methods. (C) Movement traces from five representative flies in this subgroup. (D)-(F) same as in (A)-(C) but for the subgroup of flies which spent majority of the time in the outer rim. Method 2 gave rise to a large APD but not Method 1, despite the visually significant approach patterns as seen in five representative traces shown in (F).Comparison between flies which exhibited higher density of movement trace in the inner circle than the outer rim (A)-(C) and flies which were opposite (D)-(F). (A) This subgroup of flies spent majority of the time in the inner circle. (B) (TIF)Click here for additional data file.S2 Fig(A) Using Columb’s method, the maximum possible deviation angle α depends on the position of the fly. α < 90° at the position shown here. (B) Using Columb’s method, depending on the position of the fly, the same deviation angle may or may not indicate approach toward the stripes. (C) The distribution of deviation angle in the all-bright condition, in which the flies performed non-approaching movement without any directional preference. Our method (blue) gave rise to a flat curve that covers all angles (0°-90°), while Colomb’s method gave rise to a curve with a dip at the large angle region due to the issue illustrated in (A). (D) The distribution of deviation angle in the 30°-stripes condition. In our method, angles that are below 15° (shaded area) indicate the approach behavior. However, in Colomb’s method, due to the issue illustrated in (B), it is difficult to define an angular value as the criterium for the approach behavior. (E) When a fly approaches a location on the screen and moves across the platform, our method produces a constant deviation angle indicating the azimuth angle of the approached position, while Colomb’s method produces a curve with a large change in angle.(TIF)Click here for additional data file.S3 FigAPD and AS from the deviation angles. (B) In Colomb’s method, in addition to calculating the median deviation angle, another metric called “number of walks” was also used. This metric measures the number of walks a fruit fly made between the shaded areas. However, when the stripes become very wide, some walks that still clearly approach the dark stripes may out count in (C) Due to the issue illustrated in (B), the number of walks declines as the width of stripes increases. (D) Our AS still gave rise to large values even for wide stripes.(A) In our method, we first calculate the deviation angles for each video frame and then derive (TIF)Click here for additional data file.S4 FigAPD, activity level and speed. (B) Comparison between different recovery periods .(A) Comparison between female and male for (TIF)Click here for additional data file.S5 FigAPD for different dark stripe widths. (K) The fly did not exhibit any approach behavior in the all-dark condition.(A) Without any stripe , the fly did not exhibit any approach behavior. (B)-(J) Example traces and the time-resolved (TIF)Click here for additional data file.S6 FigAPD separately calculated for the inner circle and the outer rim. Both regions contribute to the APD significantly. (B)-(J) Time-resolved APD for all tested stripe widths. APD is not defined for the all-bring or all-dark conditions. The shaded area indicates the standard error of the mean.(A) The (TIF)Click here for additional data file.S7 FigDark stripes of all sizes elicited significant approach strength with the approach direction toward the center of the dark stripes.(TIF)Click here for additional data file.S8 FigSimilar to (TIF)Click here for additional data file.S9 FigAPD for all three conditions. (C)-(D) Distributions of deviation angle for all three test conditions.(A) Example movement traces for one dark stripe, two dark stripes (Buridan’s paradigm) and one bright stripe. (B) The population mean (TIF)Click here for additional data file.S1 VideoThe dark stripes existed in the upper and bottom of the screen.(MOV)Click here for additional data file.S2 VideoThe dark stripes existed in the upper and bottom of the screen.(MOV)Click here for additional data file.S3 VideoOne dark stripe and one bright stripe. The dark or bright stripe existed in the upper of the screen.(MOV)Click here for additional data file.