ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels. P2Y receptors are a group of G protein coupled receptors (GPCRs) that are expressed in almost every tissue of the vertebrate body, including the nervous system . They arAccording to the fluid mosaic model of the membrane, proteins embedded therein may diffuse in a two-dimensional manner and randomly collide with each other . This alNevertheless, the tight regulation of ion channels via P2Y receptors has been observed repeatedly in neurons  and has The cell line tsA 201, a subclone of HEK293 cells stably expressing the SV40 large T-antigen, was cultured in antibiotic free Dulbecco’s modified Eagle’s medium  supplemented with 10 % fetal bovine serum . For patch-clamp experiments, cells were seeded onto conventional 35 mm culture dishes , whereas for fluorescence microscopy the transfected cells were plated on poly-D-lysine-coated glass cover slips or glass bottom dishes . For the transfection of calcium channels into tsA 201 cells prior to electrophysiological recordings, JetPrime®  was used according to the manufacturer’s recommendations. For all other transfections, the Turbofect® reagent  was used as follows: per 35 mm dish, a total of 1 μg DNA was mixed with 200 μl of 150 mM NaCl and 5 μl of transfection reagent, and after 10–15 min preincubation, this mixture was added to the cell cultures.Most of the plasmids as used here have been described before in more detail : P2X2-CFP/-YFP, P2Y1-CFP/-YFP, P2Y12-CFP/-YFP, and NTPDase1-YFP. Plasmids for fluorescently tagged human Kv7.2 and rat Kv7.3 as well as human M1 muscarinic receptors were provided by Mark Shapiro . A construct coding for human Cav2.2-GFP was obtained from Gerald Obermair , and the GFP tag was exchanged for EYFP. Cav2.2 was always coexpressed together with accessory rat β3 and rabbit α2δ1 subunits (from G. Obermair). The identities of all plasmids were verified by sequence analysis. When non-fluorescent proteins were expressed, EGFP was used to identify successfully transfected cells.2 (2), HEPES (10), Cs-EGTA (0.1), and Mg-ATP (2), adjusted to pH 7.4 with Cs-OH. For Ca2+ currents, the external bath solution consisted of the following (in mM): CaCl2 (10), TEA-Cl (145), and HEPES (10), adjusted to pH 7.4 with TEA-OH. With these solutions, the liquid junction potential is close to zero. Ca2+ currents were elicited once every 15 s by 30 ms depolarizations from a holding potential of −80 to +20 mV. The currents were quantified by measuring peak current amplitudes unless indicated otherwise.Patch pipettes were fabricated from borosilicate glass capillaries  with a Sutter P97 puller . Electrodes had tip resistances between 2 and 5 MΩ when filled with recording solution (see below). After establishing the recording conditions, series resistance values were between 10 and 20 MΩ and were routinely compensated for by 60–70 %. Electrophysiological recordings were carried out at room temperature (20–24 °C) from tsA 201 cells approximately 48 h after transfection. Currents through Cav2.2 channels were recorded in whole-cell mode with a pipette solution consisting of the following (in mM): CsOH (145), L-aspartic acid (145), MgCl2 (1), MgCl2 (0.7), HEPES (10), EGTA (10) adjusted to pH 7.4 with KOH. Thereafter, the electrodes were backfilled with the same solution containing 200 μg/ml amphotericin B (in 0.8 % DMSO). In these experiments, the external bath solution consisted of (mM): NaCl (140), CaCl2 (2.5), MgCl2 (2), KOH (3), glucose (20) and HEPES (10), adjusted to pH 7.4 with NaOH. These solutions result in a liquid junction potential of about 15 mV which was corrected for during experimentation. To activate these currents, cells were held at a potential of −30 mV, and once every 15 s currents were deactivated by 1 s hyperpolarizations to −55 mV. Currents through Kv7 channels were quantified by determination of deactivation amplitudes as described before [Currents through the Kv7.2/7.3 channels were recorded in perforated-patch mode to minimize their rundown. Pipettes were first front-filled with a solution containing (mM): KGluconate (133), NaCl (5.9), CaCld before .2 (2.5), MgCl2 (2), KOH (3), glucose (20), and HEPES (10), adjusted to pH 7.4 with NaOH. Fluorescent proteins were exited using the emission of a 100-W mercury burner (Zeiss) passed through a dielectric filter  mounted in a filter wheel  allowing for fast change of excitation filters. Resulting fluorescence was passed through a fixed double band dichroic mirror  and a dielectric emission filter , again mounted in a filter wheel. Images were captured using a cooled CCD camera . For measurement of FRET, three images of each cell were captured: one donor , one acceptor , and one FRET image . Normalized FRET (NFRET) was calculated according to the formula NFRET = (IDA − αID − βIA) / sqrt(ID × IA), with α and β being spectral bleedthrough factors measured in cells expressing donor or acceptor only [Epifluorescence three filter Förster resonance energy transfer (FRET) experiments were performed as described previously . In brietor only .To visualize membrane expression of receptors and ion channels, tsA 201 cells were transfected as described above. Images were captured on a Zeiss LSM510 using a ×63 NA 1.4 objective employing the 458 nm (ECFP) and 514 nm (EYFP) laser line of an argon gas laser. The pinhole was set to give a 1-μm optical section. To evaluate colocalization, images were background corrected, intensities for each channel were normalized, and channels were overlaid thereafter.To visualize fluorescence emission from CFP or YFP-tagged proteins in the immediate vicinity of the membrane, total internal reflection microscopy (TIRF) was employed . All TIR2 (2.5), MgCl2 (2), KOH (3), glucose (20), and HEPES (10), adjusted to pH 7.4 with NaOH. For measuring FRET efficiency images of both, ECFP and EYFP images were captured, followed by a 240-s bleaching step of the entire field of view using a 150-W Xenon lamp  passed through an EYFP excitation filter . This reduced the intensity of EYFP by 97.9 ± 0.4 %. Images were analyzed using ImageJ  and FRET efficiency was calculated as the percentage increase in CFP emission after YFP photobleaching [Donor recovery after acceptor photobleaching (DRAP) was used to measure FRET efficiency directly in TIRF mode. To this end, tsA 201 cells on poly-D-lysine-coated glass bottom dishes were used 48 h after transfection. Immediately before the experiment, the culture medium was exchanged by a buffer containing the following (in mM): NaCl (140), CaClleaching .corr = (FRAPnorm + (1 − CTRLnorm)).For fluorescence recovery after photobleaching (FRAP), a region of interest (ROI) was selected and five images were taken before and up to 100 after bleaching, both in TIRF mode at a frequency of 0.5 Hz. For bleaching, the illumination was switched to epifluorescence mode, and the preselected ROI was bleached using the 488-nm laser line. Analysis was performed using ImageJ. After subtracting the background, the fluorescence intensity of the bleached ROI was measured at each time point. To correct for bleaching caused by image acquisition, a control ROI in an adjacent, non-bleached cell was followed for its fluorescence intensity over time. All values were normalized to the mean value of the five pre-bleach images and afterwards corrected for acquisition-induced bleaching according to the following formula: FRAPTo investigate whether the modulation of neuronal ion currents by ADP is paralleled by a certain spatial arrangement of P2Y receptors and ion channels, P2Y1 and P2Y12 receptors, respectively, were expressed in tsA 201 cells together with either Kv7.2/Kv7.3 heteromers or Cav2.2 channels. First, electrophysiological experiments were carried out to reveal which of these two ion channels can be controlled via either of these two ADP receptors. Thereafter, the spatial relation between receptors and channels was investigated using FRET and a potential physical interaction was tested for via FRAP.n = 6). Likewise, in cells coexpressing P2Y12 receptors, this ADP concentration failed to cause alterations in current amplitudes  and maximal inhibition by 45.7 ± 7.2 %  for 8 min and were then exposed to 30 μM ADP, either alone or in combination with MRS 2179 . However, when either P2Y1  for P2Y1 and 165 nM  for P2Y12. In both cases, the maximal inhibition was about 80 %. Nevertheless, the inhibition via these two types of P2Y receptor was not identical: activation of P2Y1 receptors reduced current amplitudes to the same extent after 5 (42.5 ± 5.2 % of control) and 25 ms  of the depolarization, whereas activation of P2Y12 caused significantly more inhibition after 5 (35.0 ± 6.8 % of control) than after 25 ms . This latter difference reflects a slowing of channel gating and is characteristic of the voltage-dependent inhibition of Cav2 channels via G protein βγ subunits [Currents through Cav2.2 channels elicited by a 30-ms depolarization to 20 mV were not influenced by ADP (30 μM) in cells that had not been transfected with P2Y receptor plasmids, and current amplitudes in the presence of the nucleotide amounted to 99.7 ± 0.1 % of control . However, the nucleotide did not cause any change Fig. .To reveal whether the P2Y receptors, in addition to being in juxtaposition, might get in some physical contact with the ion channels, FRAP assays were performed. In this experimental paradigm, the mobility of ion channels can be altered by interactions with other membrane proteins such as accessory subunits , 41. To When fluorescent channel proteins were coexpressed with either P2Y1 or P2Y12, this maximum of recovery remained unaltered. For Kv7.2-YFP/7.3, the rates of recovery were also the same whether the P2Y receptors were present or not Fig. . In contAlmost all P2Y receptor subtypes have been found to control the gating of various neuronal ion channels, whether they were endogenously or heterologously expressed in different types of neuron. The ion channels that have been reported to be modulated by the largest number of different P2Y receptors are Kv7 and Cav2.2 channels . Here, t2+ channels [2+ channels only [2+ channels [2+ currents [Previously, endogenous P2Y1 receptors of rat superior cervical ganglion neurons have been shown to control the gating of Kv7  and Ca2+channels , while Pels only . Likewisels only  and Ca2+channels  channelscurrents . The pre2+ currents through P2Y12 receptors included a slowing of activation kinetics that is characteristic of the voltage-dependent interaction between G protein βγ subunits and channel proteins and was attenuated by pertussis toxin, but not by a phospholipase C inhibitor. Vice versa, Ca2+ current reduction via P2Y1 was pertussis toxin-insensitive and blunted by a phospholipase C inhibitor. Likewise, the closure of Kv7 channels via P2Y1 was abolished by the phospholipase C inhibitor and most probably involves depletion of phosphatidylinositol 4,5-bisphosphate [In neurons, the inhibition of channels of the Cav2 family via P2Y receptors can involve two pathways: (i) activation pertussis toxin-sensitive G proteins and subsequent binding of βγ subunits to the channel proteins; (ii) depletion of membrane phosphatidylinositol 4,5-bisphosphate via phospholipase C . The verhosphate . Hence, NFRET values derived from epifluorescence images can only be used for comparing the efficiency of fluorescence energy transfer between pairs of chosen FRET partners [NFRET values comparable to those of positive controls, and the distances calculated from the TIRF-DRAP measurements were similar and amounted to 7 nm (P2Y1) and 6.8 nm (P2Y12). These differences in distance go in parallel with the finding that P2Y1 receptors mediated inhibition of both types of channel, whereas P2Y12 receptors only did so with Cav2.2. This confirms previous reports demonstrating that GPCRs are unable to control the gating of ion channels over micrometer distances [To reveal whether a certain spatial arrangement of receptors and channels might parallel the functional data reported above, various microscopic techniques were employed. P2Y1 receptors showed robust FRET signals with Kv7 and Cav2.2 in both epifluorescence widefield as well as TIRF microscopy. While partners , the DRAistances , 20.D) and thus a slower recovery from bleaching. There might be several reasons to account for this apparent discrepancy. One comes from the fact that it is known that the scaling of D with size (R) is best described by an ln(1/R) relationship [D, which lies within the experimental variability and thus might not be detected. An alternative explanation would be offered by dynamic transient interactions between P2Y1 and the effector channels that could not suffice to form a co-diffusing complex.In addition to analyzing the juxtaposition of P2Y receptors and ion channels via FRET, FRAP experiments were carried out in order to gain insight into potential physical interactions. The FRAP recovery time constants obtained with Kv7.2/7.3 channels were in good agreement with those of the closely related Kv7.1 channels whose mobility can be altered by the presence of KCNE subunits , 46. In tionship . This meIn contrast to the results obtained with P2Y1, coexpression of P2Y12 with Cav2.2 led to pronounced changes in the FRAP recovery kinetics. The more than 50 % increase in recovery time constant might be attributed to the formation of large complexes. A candidate protein potentially participating in such a larger complexes would be, for instance, NHERF1, which is known to interact with P2Y12 receptors , 48. NHEThe present results regarding juxtaposition of and physical interactions between P2Y receptors and ion channels appear to be related to the interposed signaling cascades. Physical interaction was only detected between an ion channel and a GPCR which are functionally connected by the direct interaction of G protein βγ subunits with the pore forming protein . Previously, juxtaposition has been reported for Kir3.1/3.2 and activating GPCRs , 31 whicIn summary, the ADP receptors P2Y1 and P2Y12 control the gating of Kv7 and Cav2.2 channels in a selective manner, and the regulation of each of these neuronal ion channels is paralleled by juxtaposition of channel and P2Y receptor proteins within appropriate membrane microdomains.


Trametes pubescens FBCC735, isolated from Finland. The 39.67-Mb genome containing 14,451 gene models is typical among saprobic wood-rotting species.Here, we report the genome sequence of the basidiomycete white-rot fungus  Trametes spp. are known as efficient lignin degraders  was maintained on 2% (wt/vol) malt extract 2% (wt/vol) agar (MEA) plates. Four mycelium-covered plugs (Ø 7 mm) from MEA plates were used to inoculate 100 mL 2% (wt/vol) malt extract liquid cultures that were incubated stationary at 25°C for 21 days. Genomic DNA was extracted using cetyltrimethylammonium bromide–based buffer (Picea abies) wood sticks or 2-cm wheat (Triticum aestivum) straw pieces on 1% (wt/vol) water agar at 25°C for 21 days. Moisture content of the cultures was adjusted to 60% with sterile H2O. The cultures were inoculated with 4 mL of homogenized T. pubescens mycelium (The gDNA was fragmented using a Focused-ultrasonicator (Covaris). The NEBNext Ultra DNA library prep kit and NEBNext Ultra Directional RNA library prep kit for Illumina  were used for library preparation. Lab-on-a-Chip analysis and Fragment Analyzer were used for quality and yield checks of the library with fragments of approximately 300 to 500 bp and 500 to 800 bp.Illumina cBot and HiSeq 2500 standard Illumina primers and HiSeq control software HCS version 2.2.58 were used for clustering and DNA sequencing with concentrations of 8.0 pM of DNA and 16.0 pM of cDNA. The Illumina data analysis pipeline RTA version 1.18.64 and Bcl2fastq version 1.8.4 were used for image analysis, base calling, and quality checking.FASTQFilter version 2.05, a GenomeScan in-house pipeline, was used for adapter removing and quality checking: bases with Phred scores above Q22 and reads longer than 36 bp passed the filtering.k-mer length of 64 was used. Scaffolds shorter than 500 bp were removed. A total of 2,173 contigs was used for the assembly of the 39.67-Mb genome. The GC content was 57.50% as assessed by QUAST    softwareMNAD00000000. The version described in this paper is the first version and is also available through MycoCosm (This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under accession number MycoCosm .


Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally diverse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization. There has been a resurgence in group A streptococcal (GAS) invasive disease, which has been paralleled by the emergence of the highly virulent M1T1 GAS clone. Intensive research has focused on mechanisms that contribute to the invasive nature of this serotype, while the mechanisms that contribute to host susceptibility to disease and bacterial colonization and persistence are still poorly understood. The M1T1 GAS clone is frequently isolated from the throat, an environment highly abundant in blood group antigen structures. This work examined the interaction of the M1 protein, the preeminent GAS surface protein, against a wide range of host-expressed glycan structures. Our data suggest that susceptibility to infection by GAS in the oral tract may correlate with phenotypic differences in host blood group antigen expression. Thus, variations in host blood group antigen expression may serve as a selective pressure contributing to the dissemination and overrepresentation of M1T1 GAS. N-tetraose , lactosamine type 1 , and lactosamine type 2  forming the major backbone of all three antigens  gene encoding fucosyl-transferase 2, which facilitates secretion of the corresponding blood group antigens by mucin-secreting goblet cells  blood group antigen system. In addition to expression on red blood cells, ABO(H) glycan structures are expressed in extracellular fluids, such as saliva, tears, and breast milk, and at the surface of oral epithelial cells –3. The h–ectively , 6. Expret cells . Furtheret cells . The gloet cells . Frequenet cells –11.Helicobacter pylori, Campylobacter jejuni, Streptococcus pneumoniae, Salmonella enterica serovar Typhimurium and Staphylococcus aureus (––ABO(H) antigens are expressed by multiple host tissue types. Primarily localized at the surface of epithelial cells, these glycans are thought to mediate contact with multiple viral and bacterial pathogens. Phenotypic differences in host glycan expression have been correlated to host disease susceptibility for several human bacterial pathogens, including s aureus –18. The  aureus –, 14, 19. aureus ––22. As taureus ––.In the present work, we show that the globally disseminated M1T1 GAS clone binds multiple host blood group antigen structures via the surface-expressed M protein. Furthermore, we demonstrate that differences in M protein specificity for blood group antigens correlate with attachment to human oral epithelial cells. Blood group antigen structures and their related precursors are heavily represented on oral epithelia and in mucosal fluid, suggesting a role in GAS pharyngeal infection. These mechanisms of glycan interaction may contribute to the global dissemination of M1T1 GAS, particularly in Western populations.N-acetylglucosamine, mannose-containing, Neu5Ac-containing, fucosylated, and glycosaminoglycan structures  M1T1 GAS clone 5448, isogenic mutant 5448ΔM1, and recombinant M1 protein. Of the 358 glycan structures present on the array, GAS strain 5448 was observed to bind 204 distinct structures. In particular, binding was observed between strain 5448 and multiple terminal galactose, terminal ures see . We therures see . In addiures see . As bloo10.1128/mBio.02237-16.1TABLE S1 t test with a confidence level of 99.99% (P ≤ 0.0001), and only glycans that met these criteria for three biologically independent samples (n = 12 glycan spot replicates) were interpreted as positive binding interactions (highlighted green). Download Table S1, PDF file, 0.6 MB.Recombinant M1 protein, GAS strain 5448, and 5448ΔM1 glycan binding profile. Glycan binding was analyzed using the ProScanArray imaging software ScanArray Express , and the data were exported to Microsoft Excel for further analysis. M1 protein binding to a glycan was defined as a value representing a ≥1-fold increase above mean background RFU. The mean background was calculated from the average RFU of all empty spots on the array + 3 standard deviations. Furthermore, statistical analysis of the data was performed by a Student’s Copyright © 2017 De Oliveira et al.2017De Oliveira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the KD] = 114.4 ± 13.3 nM) compared with B antigen (KD = 518.5 ± 51.3 nM) and A antigen (KD = 2.5 ± 0.4 µM) structures   analysis was undertaken using a Biacore T200. In addition, the interaction between M1 and blood group A  and B  antigen trisaccharides was assessed . SPR revructures  and d. I no. 48)  and b. FLNT is a major precursor of H antigen type I synthesis. Both glycan species are highly similar in structure, differing only in α1-2-linked fucosylation at the terminal galactose residue . To asseKD = 6.8 to 14.1 µM) and H antigen type I (Kd = 238.3 to 609.6 nM)  . These d09.6 nM)  and b, sKD = 989.2 ± 153.4 nM; M1 B2, KD = 1.5 ± 0.2 μM) and H antigen type I  with various affinities (HHHHHHHHHH). SPR demonstrated that the M1 B1 motif peptide exhibited binding to LNT (KD = 3.8 ± 0.7 μM) and H antigen type I (KD = 90.7 ± 29.3 nM). A scrambled adaptation of the M1 B1 motif sequence  did not recognize LNT or H antigen type I , and only glycans that met these criteria for three biologically independent samples (n = 12 glycan spot replicates) were interpreted as positive binding interactions. Glycan index: 226, Galα1-3(Fucα1-2)Galβ; 359, Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ; 360, Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ; 362, Galα1-3(Fucα1-2)Galβ1-3GalNAcα; 363, Galα1-3(Fucα1-2)Galβ1-3GalNAcβ; 368, GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ; 7K, GalNAcα1-3(Fucα1-2)Gal. Download Fig. S1, PDF file, 0.2 MB.Glycan binding profile of M1T1 clone GAS strains 5448 and 5448ΔM1 to blood group antigen A- and B-related structures. Glycan binding was analyzed using the ProScanArray imaging software ScanArray Express , and the data were exported to Microsoft Excel for further analysis. Bacterial binding to a glycan was defined as a value representing a ≥1-fold increase above mean background RFU. The mean background was calculated from the average RFU of all empty spots on the array + 3 standard deviations. Statistical analysis of the data was performed by a Student’s Copyright © 2017 De Oliveira et al.2017De Oliveira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02237-16.2FIG. S2 O-glycans released from salivary glycoprotein proteins. Shown are collision-induced dissociation-tandem MS (MS/MS) fragment spectra of singly charged negative ions of three O-linked glycan structures from saliva at (a) m/z 530.31−, (b) m/z 733.31− and (c) m/z 895.31−. The fragmentation spectra of these unique glycan species represented here were used to identify secretor and ABO(H) blood group status. For example, the structure with m/z 530.31− is indicative of an H antigen and was observed to be present only in secretor individuals. Similarly, the fragmentation spectra of m/z 733.31− show an O-glycan structure with blood group A epitope and m/z 895.31− with blood group B epitope. Download Fig. S2, PDF file, 0.2 MB.Representative mass spectra of Copyright © 2017 De Oliveira et al.2017De Oliveira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.05), AB (P < 0.001), or B antigen structures (P < 0.001)  .10.1128/mBio.02237-16.3FIG. S3 Fig. S3, PDF file, 0.2 MB.Flow cytometry assay of WT 5448 binding to human buccal epithelial cells. Dot plots at the top of each panel show the forward scatter (FSC-A) and the side scatter (SSC-A) distribution, while the corresponding histograms of buccal epithelial cell count versus fluorescence intensity at 515 to 520 nm are shown below. (a) Autofluorescence of buccal epithelial cells alone. (b) WT 5448eGFP incubated with buccal epithelial cells. Download Copyright © 2017 De Oliveira et al.2017De Oliveira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assess the importance of M1 B repeat domains and M1 B repeat coiled-coil irregularities in blood group antigen recognition, quantitative HBE adherence and internalization assays were undertaken using the 5448 WT and 5448M1* . Replacement of alanine amino acids responsible for the irregularities of the coiled-coil structure in the B repeat domains has been used to generate the “idealized” coiled-coil version of the M1 protein in 5448M1*. Stabilization of the M1 B repeat domains has been previously shown to attenuate binding function , 30.P < 0.05), B (P < 0.05), or both A and B moieties (P < 0.05) (P < 0.05), demonstrating comparative levels of adherence to that of 5448M1* (P < 0.05) (GAS strain 5448 was observed to adhere to the surface of H-antigen-expressing HBE cells at a significantly higher level than HBE cells expressing A ( < 0.05) . In the  < 0.05) . No sign < 0.05) . These dN-neotetraose on red blood cells facilitates hemolytic activity  M28 and emm pattern A-C (throat-tropic) M6 GAS suggest that M protein interaction with fucose and galactose, essential components of blood group antigens, plays an important role in GAS adherence to the host pharyngeal mucosa (emm pattern D (skin-tropic) M98 was unable to bind blood group antigen structures. Therefore, we propose that interaction with blood group antigen structures may be a function of throat-tropic GAS. Here, we found that GAS strain 5448 interacts with the blood group antigen precursor LNT and H antigen type I in an M1 protein-dependent manner. Several blood group A and B antigen structures were also found to bind purified recombinant M1 and M1T1 GAS strain 5448 and also interacted with 5448ΔM1, suggesting that in addition to M1, GAS express other lectins with blood group antigen specificity. In the present study, LNT and H antigen type I interactions were shown to be restricted to a 12-amino-acid sequence (LEKELEEKKE/KAL) present at the start of both the B1 and B2 repeat domains of M1. In the reported crystal structure of the dimeric AB fragment of M1, the B repeat regions are splayed apart  Crystal structure of the dimeric AB fragment of M1 (Protein Data Bank ID 2OTO ) (b). AnCopyright © 2017 De Oliveira et al.2017De Oliveira et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Fucosylation of LNT, which results in blood group H antigen type I, significantly increased the affinity of M1 for LNT. Similarly, M1 protein had a significantly higher affinity for blood group A and B trisaccharides, which both contain α2-linked fucose residues, than for LNT. Studies analyzing the blood group recognition properties of rotaviruses show that fucose moieties do not play a direct role in blood group antigen binding . Rather,The interaction of M1 protein with fibrinogen both protects GAS against phagocytosis and stimulates neutrophil activation via activation of a proinflammatory cascade , 42, 46.M1T1 GAS are commonly isolated from the upper respiratory tract during the course of pharyngeal infection . Here, wThe globally disseminated M1T1 clone of GAS is a significant cause of human disease. Here, we have identified a range of novel M1 protein-dependent glycan interactions, suggesting a role for galactose-containing structures in GAS colonization of the upper respiratory tract. Furthermore, we have provided evidence to support a potential link between host blood group antigen expression and GAS colonization status. This study exemplifies the diverse nature of lectin-glycan binding in the human host-GAS pathogen interaction.Escherichia coli strains TOP10 and BL21(DE3), containing pGEX2T and pet-28b(+) expression plasmids, were cultured and used for expression of recombinant proteins at 37°C in lysogeny broth supplemented with ampicillin (100 µg/ml) or kanamycin (50 µg/ml), as previously described  of 0.5 by centrifugation , and 1 × 108 CFU were resuspended in array phosphate-buffered saline (PBS [PBS with 2 mM MgCl2 and 2 mM CaCl2]) containing 1 mM Cell Trace BODIPY TR methyl ester , to a final volume of 250 μl. Staining of bacteria was allowed to proceed in the dark at room temperature for 15 min. Following staining, bacterial cells were passed through a 5 μm-pore Millex-SV syringe filter unit  to remove clumps of aggregated bacteria. For glycan array analysis of recombinant M1 protein, purified M protein (1 μg) was preincubated with mouse anti-His tag antibody , rabbit anti-mouse Alexa Fluor 488 antibody conjugate  and goat anti-rabbit Alexa Fluor 488 conjugate , at a ratio of 4:2:1. For competitive glycan binding analysis, M protein-antibody complexes were preincubated with glycans of interest (600 nM to 60 μM) purchased from Dextra Laboratories (United Kingdom). Protein mixtures were made up to a final volume of 65 μl in array PBS and incubated at room temperature for 10 min to allow protein complex formation. Glycan microarray slides were blocked in array PBS containing 0.1% (wt/vol) bovine serum albumin  for 10 min at room temperature and dried by centrifugation . Following centrifugation, gene frames  were bonded onto each glycan microarray slide, and 65 μl of GAS or precomplexed protein mixture was applied. Mixtures were secured with the addition of a plastic coverslip, and samples were allowed to hybridize in the dark for 10 min at room temperature. Proceeding incubation, glycan microarray slides were washed in array PBS for 5 min with agitation, followed by a secondary wash in array PBS containing 0.001% Tween 20 for 2 min with agitation. After washing, glycan microarray slides were dried by centrifugation . Fluorescence intensity of array spots was measured using the ProScanArray microarray 4-Laser scanner  with the blue argon 488-nm excitation laser set to the fluorescein isothiocyanate (FITC) setting (494-nm excitation and 518-nm emission) and gain set to 40 to 60%. Image analysis was carried out using the ProScanArray imaging software ScanArray Express . Positive binding was defined as greater than a 1-fold increase of the mean background + 3 standard deviations. Statistical analysis of the data was performed using a Student’s t test with a confidence level of 99.99% (P ≤ 0.0001), and only glycans that met these criteria for three biologically independent samples (n = 12 glycan spot replicates) were interpreted as positive binding interactions.A glycan microarray was employed to identify novel GAS-glycan interactions . Whole-c2 for 1 min at 10 µl/min. Recombinant proteins were captured via the C-terminal histidine tag on flow cells 2, 3, and 4 for 120 s at 10 µl/min. Flow cell 1 served as a reference cell, whereby a negative-control his-tagged protein was immobilized. Glycan analytes were diluted in PBS, and binding assays were performed using various concentrations of analyte (0 to 100 μM) over a series of five 120 s injections at a flow rate of 30 μl/min. Regeneration of the flow cell surface was achieved with two separate injections of the EDTA regeneration buffer for 60 s at 30 μl/min. M protein-glycan interactions were analyzed via a steady-state affinity model using Biacore T200 evaluation software (Biacore AB).The interaction of M1 protein, M1 protein fragments and M1 peptides with specific glycan subsets was further investigated via single-cycle kinetic surface plasmon resonance (SPR) on a series S nitrilotriacetic acid (NTA) chip (Biacore AB) using a Biacore T200  at 25°C. The surface of the NTA chip was prepared by first stripping the surface with a regeneration buffer  for 1 min at 10 µl/min. The surface was then activated by an injection of 500 µM NiClHuman buccal epithelial (HBE) cells were used to assess the role of blood group antigens in GAS attachment to host cells. Blood group status of 38 donors was determined via mass spectrometry of saliva samples from each donor. Unstimulated saliva and HBE cells were collected and prepared as previously described  for 2 h at 37°C with 5% CO2. Postincubation, samples were resuspended in 750 μl DPBS, pelleted by centrifugation , and resuspended in a further 500 μl DPBS. Association of 5448eGFP with HBE cells was measured using an LSR II  flow cytometer. Gated HBE cells were analyzed with a 575/26-nm filter, with data being recorded for 10,000 events. Fluorescence intensity of uninfected HBE cells was also measured to allow for compensation of autofluorescence. The relative quantity of 5448eGFP associated with HBE cells was estimated by the mean fluorescence intensity (MFI) of eGFP-positive HBE cells. Data were analyzed and processed using FlowJo .To assess the association of GAS to HBE cells, 2 × 102. HBE cells were then pelleted by centrifugation, washed three times in DPBS, and lysed via resuspension in 0.025% (vol/vol) Triton X-100 . Internalized CFU were determined via enumeration on THY agar. To measure GAS adherence, HBE cells were incubated in the absence of gentamicin, and total adherence was determined by subtracting CFU of total internalized GAS. Adherence and internalization assays were undertaken in triplicate with HBE cells isolated from 3 different donors. Statistical analysis was determined via a one-way analysis of variance, with a Tukey’s post hoc test. Homogeneity of variances was confirmed using a Bartlett’s test for equal variances.To examine levels of bacterial internalization, HBE cells were incubated with either 5448 or 5448M1* as described above. Alternatively, 5448 was also incubated with HBE cells in the presence of M1 B fragment (100 µM). To kill nonadherent and HBE cell surface-adhered GAS, samples were treated with gentamicin (100 µg/ml) for 1 h at 37°C with 5% COCollection of HBE cells and saliva was performed with the approval of the University of Wollongong Human Ethics Committee (HE08/250). Volunteers provided informed consent before donating samples.


We measured kitchen PM2.5 and CO concentrations at one-minute intervals for an approximately 48-h period using the TSI DustTrak II 8530/SidePak AM510  or EL-USB-CO data logger  respectively. We also obtained information regarding fuel, stove and kitchen characteristics and cooking activity patterns. Household cooking was performed in two daily sessions  with diurnal variability in pollutant concentrations reflecting morning and evening cooking sessions and peak concentrations associated with fire-lighting. We observed a strong linear relationship between PM2.5 measurements obtained by co-located photometric and gravimetric monitoring devices, providing local calibration factors of 4.9 (DustTrak) and 2.7 (SidePak). Overall 48-h average CO and PM2.5 concentrations were 5.4 (SD 4.3) ppm (12 households) and 417.6 (SD 686.4) μg/m3 (8 households), respectively, with higher average concentrations associated with cooking and heating activities. Overall average PM2.5 concentrations and peak 1-h CO concentrations exceeded WHO Indoor Air Quality Guidelines. Average hourly PM2.5 and CO concentrations were moderately correlated (r = 0.52), suggesting that CO has limited utility as a proxy measure for PM2.5 exposure assessment in this setting. Domestic indoor air quality levels associated with biomass fuel combustion in this region exceed WHO Indoor Air Quality standards and are in the hazardous range for human health.Household Air Pollution (HAP) from biomass cooking fuels is a major cause of morbidity and mortality in low-income settings worldwide. In Nepal the use of open stoves with solid biomass fuels is the primary method of domestic cooking. To assess patterns of domestic air pollution we performed continuous measurement of carbon monoxide (CO) and particulate Matter  Indoor Air Quality Guidelines.Average PM•2.5 and CO were moderately correlated during cooking periods.Concentrations of PM The gre2.5 in large-scale epidemiological studies, due to influences of the indoor microenvironment  on the temporal and spatial relationship between pollutant concentrations , vegetables and warm buffalo milk. In the winter season the stove or fire in the central kitchen area may be lit overnight to provide warmth for kitchen livestock .This study was conducted in the Janakpur, the capital town of Dhanusha district in southern Nepal. Janakpur has a population of approximately 100,000 ), compri2.2The households in the study were a subset of homes of mothers recruited into a randomised controlled trial examining the effect of multiple micronutrient supplementation during pregnancy on child growth outcomes . At the 2.32.3.1The study fieldworkers observed and recorded information on kitchen construction  and characteristics  in addition to obtaining measurements of the stove (pan height) and kitchen floor, window and door areas. A ventilation index was calculated for each kitchen using a composite score for each open kitchen door (1) and window (0.5) . Informa2.3.22 (zero gas) and 100 ppm CO gas before transportation in airtight containers to Nepal with repeat calibration upon return to the UK. All monitoring commenced between 08:00 and 09:00 and was performed at one-minute intervals for a continuous period of approximately 48 h (range 45–50 h). All CO measurements below the LOD were assigned a value of one-half of the LOD (1.5 ppm). Data were downloaded using EasyLog USB software (Lascar Electronics Ltd).Real-time CO levels were measured at 1-min intervals using an electrochemical EL-USB-CO monitor  with lower Limit of Detection (LOD) 3 ppm. Monitoring was performed in a central location in each study kitchen at height 50 cm and distance 150 cm from the cooking stove. Monitors were calibrated in the UK using >99.9%N2.3.32.5 concentrations were estimated using the TSI DustTrak II 8530 (detection range 0.001–400 mg/m3) or AM510 SidePak ). The internal laser photometers of these devices measure airborne particle mass concentration using a 90° light-scattering laser diode. The monitors were co-located (within 10 cm distance) and time-synchronised with the Lascar EL-USB-CO device to measure PM2.5 at 1-min intervals. Sampling was performed using an upstream external size-selective PM2.5 inlet attachment, with a level greased well as the impaction surface. The monitors were calibrated to a zero filter before and after each sampling period. Continuous PM2.5 monitoring was performed in eight households for approximately 48 h (range 42–48 h) and for a shorter duration in three households (range 22.5–32.5 h), with reliance upon rechargeable batteries due to scheduled power cuts (load shedding) precipitated by electricity supply shortages in Nepal. Data were downloaded from the photometric devices using TSI Trakpro software .Kitchen PM2.4Particulate matter concentration measured by light scattering devices is subject to measurement error since standard dust calibration is performed using artificial aerosols with different physical parameters  to local cooking particles. To obtain a local ‘calibration factor’ as a reference to correct the photometric measurements, we performed simultaneous gravimetric sampling using the DustTrak filter facility (nine households) and using a co-located Casella Apex  gravimetric sampling device (four households). All gravimetric sampling was conducted in accordance with standard “Methods for Determination of Hazardous Substances (MDHS) no. 14/3 guidelines” . Samples2.5 cyclone head  using a portable flow meter with a flow rate 2.2 L/min . Flow rate calibration was performed with a Bios Dry Cal DC-Lite Primary flowmeter in the UK. The Apex monitor was co-located within 10 cm of the photometric device, with simultaneous gravimetric monitoring performed for up to 48 h (mean duration 20 h). We calculated a local calibration factor using the ratio of average PM2.5 concentrations obtained by each method (2 = 0.81 (n = 8), providing a calibration factor of 4.9 , providing a calibration factor of 2.7 , wall material (dirt/brick/mixed), kitchen size , ventilation index (high/low), eaves space  and stove type (single/multiple). We conducted multiple linear regression to determine a set of variables to best explain variability in log-transformed air quality measurements during cooking sessions, with likelihood ratio tests used to determine final multivariate models. Pearson's correlation coefficients were calculated evaluate the correlation between 1-h average PM2.5 and CO levels. All statistical analyses were performed in Stata .We calculated descriptive statistics for kitchen characteristics, cooking activity patterns and average  CO and PM2.6Ethical approval was granted by the Nepal Health Research Council and the UCL Research Ethics Committee. Written consent was obtained from participating mothers at the first point of contact and consent was obtained verbally at each sampling visit. Each participating family was reimbursed with 100 Nepalese Rupees and a packet of biscuits.33.12 (range 2.1–46.9 m2). There were no formal ventilation sources, but an open eaves gap provided informal ventilation in nine kitchens . Cooking stoves were all located against an external wall and consisted of single or multiple potholes. Meal preparation was performed twice daily by female family members, with children (age range 16 months to 8 years) present in the kitchen during cooking sessions in ten households. Morning cooking commenced between 07:00 and 08:00 with the stove lit for median duration 2.5 h (range 2–4 h), and a shorter evening session from 17:00 to 20:00 . An overnight fire was lit in five study households, to provide warmth for resident livestock.Detailed characteristics of the study kitchens are shown in 3.22.5 minute-level measurements respectively. Measured ranges of 48-h mean CO and PM2.5 levels by household were 3.0–6.8 ppm and 187.9–619.3 μg/m3, respectively. The continuous CO and PM2.5 concentration profile consistently showed diurnal peaks reflecting morning and evening cooking periods  and 15,900 μg/m3 (dung fuel), adjusted for calibration factor for CO and PM2.5 respectively.Descriptive statistics for pollutant measurements by household are presented in  periods . Extreme3.32.5 were 5.4 (4.3) ppm and 417.6 (686.4) μg/m3, respectively. Cooking activities were associated with elevated CO and PM2.5 concentrations of 8.3 (5.2) ppm and 966 (1384) μg/m3, respectively. Lighting of a kitchen fire to provide warmth for resident livestock was also associated with increased levels: mean CO concentration 6.3 (3.1) ppm and PM2.5 527.4 (409) μg/m3. Peak 1-h CO concentrations were in the range 8.1–31.9 ppm, with average peak 1-h concentration exceeding the 48-h mean by an average factor of 3.1. Corresponding values for peak 1-h PM2.5 concentrations were in the range 566.4–3805.0 μg/m3, exceeding the 48-h mean value by an average factor of 4.2.Summary statistics developed by averaging duration and stove activity are presented in 3.42.5 during cooking sessions, with the greatest variability in concentration magnitude associated with dung fuel. In ANOVA analyses, all kitchen and stove characteristics were significantly associated with average CO and PM2.5 concentrations during cooking periods  , than for wood fuel households .Hourly average CO and PM< 0.001) , more st42.5 concentration of 418 μg/m3, which greatly exceeds the WHO Air Quality Guidance (AQG) Interim-Target-1 recommendation of 35 μg/m3 for household fuel combustion (2.5. The highest recorded peak 1-h CO concentration (31.9 ppm) exceeded the WHO AQG 60-min exposure guideline (30 ppm)  , with ma2.5 of 9.1 ppm and 650 μg/m3 were measured respectively , which is lower than the correlation coefficient of 0.92 between CO and PM2.5 concentrations in wood fuel households in Guatemala, yak dung stoves in Tibet    and by D = 0.88) . Investi0 and CO . Such va0 and CO  and high0 and CO  suggesti0 and CO . Althoug0 and CO  our findoutcomes .2.5 and CO concentrations than those with a smaller floor surface area. These differences may be explained the location of stove which was frequently observed to be in a corner area close to the eaves space in small kitchens. We observed influences of stove activity patterns on CO and PM2.5, with peak pollutant concentrations associated with stove lighting, and return of concentrations to background values between cooking sessions. Previous studies have indicated that from a public health perspective increasing kitchen ventilation may be a low cost intervention to reduce HAP . The work of the UK Small Area Health Statistics Unit is funded by Public Health England as part of the MRC-PHE Centre for Environment and Health, funded also by the UK Medical Research Council (Grant number: MR/L01341X/1). The funders played no role in the conception, methodology, analysis or reporting of this study.The study was made possible by a The authors declare that they have no conflicts of interest.


GDC concentration increased with incubation time, as well as when incubated in the presence of H2O2 and/or Cu2+, which were effective even at the lowest concentrations. Dicarbonylic compounds did not increase the levels of GDC during incubations. 1H, 13C and FT-IR spectra from the purified fraction containing the chromophore (detected by UV/vis spectroscopy) showed oxidation products of glucose, including gluconic acid. Lens proteins solutions (10 mg/mL) incubated with glucose (30 mM) presented increased levels of carboxymethyl-lysine and hydrogen peroxide that were associated with GDC increase. Our results suggest a possible use of GDC as a marker of autoxidative reactions occurring during lens proteins glycation induced by glucose.Glucose autoxidation has been proposed as a key reaction associated with deleterious effects induced by hyperglycemia in the eye lens. Little is known about chromophores generated during glucose autoxidation. In this study, we analyzed the effect of oxidative and dicarbonyl stress in the generation of a major chromophore arising from glucose degradation (GDC) and its association with oxidative damage in lens proteins. Glucose (5 mM) was incubated with H Sustained hyperglycemia states constitute a risk factor for the development of numerous non-communicable chronic diseases, including cataract disease . Hypergl2+ (5–50 μM) or hydrogen peroxide (0.5–5 mM) during 10, 20 and 30 days at pH 7.4 and 37 °C. 2+ (5 µM) and H2O2 (5 mM), respectively. It can be observed that similar absorption spectra were obtained, independent of the pro-oxidant agent used in the incubations (Cu2+ and H2O2). 2+ (5–50 μM) or hydrogen peroxide (0.5–5 mM), respectively. It can be observed that both pro-oxidant agents induced an increase in the generation of the chromophores that possess an absorption maximum at 365 nm even at the lowest concentration used in these experiments. The increase in the absorbance was concomitant with the incubation time, which indicates an accumulation of the chromophores generated during the different times (2+ produced an increase in the levels of the glucose degradation chromophore (GDC) of 17.8 times when compared with glucose controls  with Cunt times C,D. Figucontrols E. This ixal/Cu2+ .2+ 5 μM during 30 days at pH 7.4 and the resulting solutions were then purified by means of column chromatography. −1 in agreement with the NIST Chemistry WebBook [−1  was stronger than in glucose. The absorption at 1630 (m) suggests the occurrence of a carbonyl function or C–O stretching vibration. The lower absorption wavelength of the C=O stretching vibration can be explained by the influence of hydrogen bonding. The absorbance at 1066 cm−1 can be related to the C–O stretching vibrations.Glucose 250 mM was incubated with Cu WebBook . In the 1H-NMR of the compound, a broad group of overlapping signals appeared in the range δ 3.4–4.2 ppm. No hemiacetal H were observed. Literature data for the 13C-NMR spectra of the α- and β-anomer of glucose show the typical hemiacetal C at δ 92.7 and 96.5 ppm, respectively, while C2, C3, C4, C5 and C6 appear at 72.14, 73.4, 70.4, 72.10 and 61.3 ppm for the α-anomer and 74.8, 76.4, 70.3, 76.6 and 61.5 for the β-anomer, respectively [13C-NMR spectrum of GDC showed a carbonyl function at δ 179.81 ppm, assignable to a carboxylic acid or ester (lactone) and doublet (d) at δ 73.29, 72.48, 71.16 and 70.92 ppm for H–C–OH atoms as well as a triplet (t) at δ 64.06 (minor) and 61.14 ppm (main) (–CH2OH) (2OH) indicates an equilibrium between the lactone (gluconolactone) and the open-chain form of gluconic acid. Furthermore, methylation of the product with diazomethane yielded a mixture with minor compounds showing two q at δ 54.99 and 55.05 ppm  and additional carbonyl C at δ 178.72 and 178.24 ppm, in agreement with possible isomers of the free carboxylic acid function. Hemiacetal signals and double bond sp2 carbons were absent, showing that the sugar is not a polyhydroxyaldehyde or an unsaturated sugar [In the (–CH2OH) . The preed sugar .2+. With the aim to determine the effect of the pH in the absorbance of GDC, solutions at different pH were prepared and a constant volume of GDC was added in order to achieve a constant concentration of 1.3 mg/mL. The results above suggest that GDC could be a coordination complex of a carboxylic acid arising from glucose oxidation and Cu2+ (5 μM) during 10, 20 and 30 days at pH 7.4 and 37 °C. The absorbance at 365 nm was measured after lens proteins precipitation with trichloroacetic acid, and increased linearly with the incubation time (p = 0.0076), hydrogen peroxide  and carboxymethyl-lysine levels  were calculated with incubation time, only correlations involving GDC and hydrogen peroxide were significant (p < 0.05). 2+during 10, 20 and 30 days. It can be observed that the levels of hydrogen peroxide and carboxymethyl lysine (CML) increased after 20 days of incubation  and CML  were statistically significant (p < 0.05).The association between the generation of the major chromophore arising from glucose degradation with oxidative stress and glycoxidative modifications in lens proteins was assessed incubating glucose (30 mM) with eye lens proteins (10 mg/mL) in the presence of Cuion time A. Pearsocubation B. We didNon-enzymatic reactions mediated by glucose have been proposed to play an important role in the etiology of nuclear cataract disease . The ana2+ or hydrogen peroxide  and H2O2 (0.5 mM)  . This fan of GDC . These rn of GDC .13C analysis of purified fractions containing GDC showed a signal at 179.8 ppm  and (2), followed by Fenton-type Reaction (3).The oxidation of glucose in the C1 position yielding d-1,4-gluconolactone or in C6 yielding glucuronic acid, has also been found in the glucose oxidation in aqueous colloidal MnO2 surface as well as in supported metal catalysts including platinum or palladium [d-glucoronic acid as the main products [d-glucoronic acid because of the absence of hemiacetal H signals observed in the 1H-NMR experiments. The generation of metal-gluconate complexes has been reported, and many of these complexes present absorption in the UVA-visible region [The synergism in the generation of GDC produced by incubations performed with the mixture glucose/Cualladium ,31. It hproducts . We can e region ,27.2+/eye lens proteins were incubated during 10, 20 and 30 days at 37 °C. The analysis of deproteinized solutions of these mixtures depicted an increase in the absorbance at 365 nm, showing a linear behavior with the incubation time. This fact is consistent with a previous report in which mixtures of glucose/Cu2+/eye lens proteins presented photosensitizing capacity after 10 days of incubation, which was attributed to GDC generation [2+/eye lens proteins also showed an increase of hydrogen peroxide concentration with the incubation time -Glucose (≥99.5%), Na2HPO4·7H2O, NaH2PO4·2H2O, Xylenol Orange tetrasodium salt and CuSO4·5H2O were obtained from Sigma-Aldrich . Pierce™ BCA Protein Assay Kit was obtained from Thermo Scientific .Ultrapure water obtained from a Barnstead EasyPure water filter system  was used for the preparation of all solutions. All solutions were filtered through a 0.2 μm sterile nitrocellulose filter.3. The suspension was homogenized and centrifuged at 15,000 rpm for 30 min at 4 °C . The supernatant was extensively dialyzed against deionized water at 4 °C. Protein concentration was determined by means of the BCA Protein Assay Kit, according to the manufacturer’s instructions. Potassium buffer 0.1 M pH 7.4 was added in order to achieve a protein concentration of 50 mg/mL.Bovine lenses were obtained from the slaughter-house. Twenty bovine lenses were decapsulated and stirred in a 50 mM Tris–HCl buffer pH 7.4 containing 0.2 mM KCl, 1 mM EDTA, 10 mM β-mercaptoethanol and 0.05% NaN4 (5–50 μM), H2O2 (0.5–5 mM), glyoxal (0.5–5 mM) or methylglyoxal (0.5–5 mM). Solutions were prepared in phosphate buffer 100 mM, pH 7.4, which was previously treated with Chelex in order to decrease the concentration of metals. Incubations were carried out during 10, 20 and 30 days in triplicate. Samples were prepared in order to analyze them the same day after the incubation period. Sterile solutions of glucose (5 mM) were incubated with and without CuSO4 5 µM. This solution was incubated during 30 days at 37 °C in the dark. After this period, glucose concentration was determined by photometric measurements by means of glucose assay kit , according to the manufacturer’s instructions. At the end of this period, the absorption spectra were recorded on a UV-visible photodiode array (PDA) spectrophotometer . The samples were partially lyophilized, distributed in aliquots and kept frozen at −20 °C until purification. Thawed samples were purified by column chromatography on a Sephadex LH-20 . Milli Q water was used as the mobile phase. The different fractions were recovered by means of a fraction collector . The flow rate was adjusted to 0.25 mL min−1, collecting in each tube a volume of 1 mL. The fractions were analyzed by means of absorption spectroscopy and determining the glucose concentration by means of a glucose assay kit. Purification was achieved selecting the fractions enriched in the chromophore (λmax = 365 nm) but low in glucose concentration.Glucose (250 mM) was prepared in phosphate buffer 100 mM, pH 7.4 in sterile conditions in the presence of CuSO1H and 100 MHz for 13C in D2O. Chemical shifts are given in ppm using residual water for calibration. The NMR spectra were recorded on a Bruker Avance 400  spectrometer at 400 MHz for w/w. The disk was prepared by applying a 40 mPa pressure during 2 min. The scan was performed in the range 700–4000 cm−1 at a resolution of 4 cm−1 and with 32 scans per spectrum. FT-IR spectra were obtained employing a Nicolet Nexus, 470-FT-IR Spectrophotometer . Glucose and GDC samples were scanned as KBr disks at a concentration of 1% w/v) SDS-PAGE gels. Electrophoresis was performed at 100 V for 1–2 h. Proteins were electrotransfered or stained with 0.1% (w/v) Coomassie Brilliant Blue and washed-out in a solution containing 10% (v/v) ethanol and 0.75% (v/v) acetic acid up to 24 h. SDS-PAGE analysis was carried out boiling 22.5 μg of protein per sample for 5 min in Laemmli sample buffer and loaded into 12% , which was then blocked with PBS supplemented with 1% BSA and 0.1% Tween 20. Membranes were incubated with an anti-carboxymethyl-lysine monoclonal primary antibody  at a dilution of 1/1000 and incubated overnight under agitation at 4 °C. Then membranes were washed with PBS, and incubated with HRP-conjugated secondary antibodies. Membranes were revealed with ECL Plus chemiluminescent detection system . Blots were scanned and signal intensities were quantified with ImageJ software . 4 (5 μM) and glucose (30 mM)/CuSO4 (5 μM) were incubated during 0, 10, 20 and 30 days at 37 °C, pH 7.4 in sterile conditions and peroxide concentrations were determined immediately after the incubation period was completed. Modified FOX-2 method was used for the determination of hydrogen peroxide and protein hydroperoxides. Total peroxides were determined by mixing 300 μL of incubated solutions (samples) with 50 μL of the working reagent and completed at 1 mL with milli-Q water. The working reagent contained 5 mM of ferrous sulfate and 4 mM of Xilenol Orange in 0.5 M H2SO4. An additional 15 min pre-incubation step with 20 μL of catalase in a concentration of 715 U mL−1 (instead of water) was carried out to eliminate H2O2 and measure only protein hydroperoxides. The mixtures were vortexed and left to react for 30 min in the dark at room temperature. The absorbance was quantified at 560 nm in an UV/vis spectrophotometer  and the concentrations were interpolated from a standard curve made with H2O2. To eliminate the interference of phosphate buffer, hydrogen peroxide standards were prepared considering a volume of 300 μL of 100 mM phosphate buffer, pH 7.4 and a good correlation was found.Mixtures of lens protein (10 mg/mL)/glucose (30 mM)/CuSOExperiments were performed in triplicate and results are expressed as mean values ± SD. Correlations between variables were analyzed by Pearson correlation with a 95% confidence limit.


The tyrosine kinase receptor (Trk) family comprises three members, namely TrkA,TrkB and TrkC. Various studies have confirmed that TrkA and TrkC expression is associated with a good prognosis in NB, while TrkB overexpression can lead to tumor cell growth and invasive metastasis. Previous studies have shown that As2O3 can inhibit the growth and proliferation of a human NB cell line and can also affect the N-Myc mRNA expression. It remains unclear whether As2O3 regulates Trks for the purposes of treating NB.Arsenic trioxide (As2O3 on Trk expression in NB cell lines and its potential therapeutic efficacy. SK-N-SH cells were grown with increasing doses of As2O3 at different time points. We cultured SK-N-SH cells, which were treated with increasing doses of As2O3 at different time points. Trk expression in the NB samples was quantified by immunohistochemistry, and the cell cycle was analyzed by flow cytometry. TrkA, TrkB and TrkC mRNA expression was evaluated by real-time PCR analysis.The aim of the present study was to investigate the effect of As2O3 significantly arrested SK-N-SH cells in the G2/M phase. In addition, TrkA, TrkB and TrkC expression levels were significantly upregulated by higher concentrations of As2O3 treatment, notably in the 48-h treatment period. Our findings suggested that to achieve the maximum effect and appropriate regulation of Trk expression in NB stages 1, 2 and 4S, As2O3 treatment should be at relatively higher concentrations for longer delivery times;however, for NB stages 3 and 4, an appropriate concentration and infusion time for As2O3 must be carefully determined.Immunohistochemical and real-time PCR analyses indicated that TrkA and TrkC were over-expressed in NB, and specifically during stages 1, 2 and 4S of the disease progression. TrkB expression was increased in stage 3 and 4 NB. As2O3 induced Trk expression in SK-N-SH cells to varying degrees and may be a promising adjuvant to current treatments for NB due to its apoptotic effects.The present findings suggested that As Neuroblastoma (NB) is an extracranial solid tumor that is frequently encountered in children and accounts for 8–10% of malignancies . The mai2O3) is a drug that has been used in China for approximately 2000 years  that was diluted to 0.5%. The probes were subsequently rinsed with PBS 3 times and staining was performed using diaminobenzidine (DAB) solution (DAKO). Following drying, the samples were counterstained with Meyer hematoxylin (Sigma-Aldrich). The immunostaining of all samples was conducted under the same conditions as the antibody reaction and DAB exposure. The evaluation of Trk expression was performed by observing 20 microscope fields at ×100 magnification .Consecutive 3-µm sections were cut from every block, and an immunoperoxidase technique was implemented following antigen retrieval by either microwave treatment (95 °C), pepsin  treatment for 20 min and/or citrate buffer (pH 6.0) treatment for 45 min. Following endogenous peroxidase blocking with 3% HThe staining intensity was scored on a scale of 1–3 as follows: 0 (absent), 1 (weak), 2 (moderate), or 3 (strong). The percentage of tumor cells that indicated positive staining was further assessed semi-quantitatively. The histochemical score (H-score) of immunoreactivity was obtained by multiplying the intensity and the percentage of positive cells, which yielded a final score of 0–300 . The med2 incubator.Human SK-N-SH cells were obtained from the Medical School of the Sun Yat-sen University. The cells were maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham   supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) at 37 °C in a 5% CO2O3 on the cell cycle and the expression levels of Trk, SK-N-SH cells were divided into two treatment groups, and both groups were exposed to 4 µM As2O3 for 48 h. A medium sample devoid of As2O3 was used as a control. To determine the effects on gene expression, the cells were cultured at three different concentrations, namely, 1, 2 and/or 4 µM of As2O3 ., along with a control group for 24 and 48 h [To examine the effects of As5 cells/well in 3 mL of medium and cultured overnight. Following serum starvation for 24 h, the cells were cultivated with half maximal inhibitory concentrations (IC50s) of As2O3 for 24 or 48 h in a 37 °C CO2 incubator. The cells were harvested by trypsinization, washed twice with PBS and fixed with cold 70% ethanol overnight. The samples were stained with propidium iodide (PI) solution containing 50 μg/mL RNase A and 0.1% Triton X-100. The cell cycle distribution was assessed using a flow cytometer .The cells were seeded into 6-well plates at a density of 2.5 × 102O3 treatment for 48 or 72 h, total RNA was isolated from SK-N-SH cell lines using the TRIzol reagent  according to the manufacturer’s protocol. The RNA was precipitated with isopropanol, washed with 75% ethanol, and dissolved in diethylpyrocarbonate (DEPC)-treated water. The integrity and purity of total RNA were assessed by DNA gel (1% agarose gel) electrophoresis and UV detection.Following AsFollowing cDNA synthesis, PCR amplification was performed in order to examine the number of Trk transcripts. The assay was carried out using a Biometra T-gradient Thermoblock thermal cycler ; a P value lower than 0.05 (P < 0.05) was considered statistically significant.Each experiment was performed at least three times. The data are presented as the mean ± standard deviation (SD). All data were analyzed by Student’s The pediatric Sun Yat-sen Memorial Hospital database resulted in the identification of 12 patients diagnosed with NB; nine were stage 4 and three were stage 2. Since the three children with stage 2 disease had lower N-myc amplification, they were classified as intermediate risk, whereas the remaining of the subjects was classified as high risk according to the INSS criteria.We performed immunohistochemical analyses of Trk expression in the 12 children with NB. The distribution of Trks was tissue-specific and correlated with the clinical heterogeneity of NB. TrkA expression was present in five (41.7%) tumors, with two (66.7%) stage 2 and three (33.3%) stage 4 tumors showing TrkA expression. A total of 11 (91.7%) tumors expressed TrkB, with two (66.7%) stage 2 and nine (100%) stage 4 tumors. Four (33.3%) tumors exhibited TrkC expression and were divided into two (66.7%) stage 2 and two (22.2%) stage 4 tumors. It is interesting to note that both TrkA (66.7%) and TrkC (66.7%) were strongly co-expressed in stage 2 samples, although they indicated low co-expression in stage 4 samples. Furthermore, TrkB (100%) was highly expressed in advanced-stage disease (stage 4), whereas it was expressed to a relatively lower extent (66.7%) in early-stage NB (stage 2)  was significantly higher (P < 0.01) than that noted in the control group (5.29%). These data suggested that As2O3 induced apoptosis of SK-N-SH cells following cell cycle arrest at the G2/M phase . In contrast to the 1 μM dose of As2O3, TrkA was downregulated following 2 and 4 µM As2O3 treatment. This trend was increased at the 48-h period compared with the 24-h period of treatment  compared with that of the control group. As2O3 increased TrkB mRNA expression in a dose-dependent manner at 24 and 48 h. In addition, RT-PCR analyses indicated that TrkC expression levels were significantly higher in SK-N-SH cells treated with 4 µM As2O3 for 48 h (5.22 ± 0.90) compared with untreated cells   induced rapid induction of apoptosis in NB cells at a rate of more than 50% in less than 16 h. Moreover, kinase activity significantly increased in those cells treated with 4 µM As2O3. The present study demonstrated that the treatment of SK-N-SH cells with 4 µM As2O3 for 48 h induced arrest at the G2/M phase. Our preliminary results (data not shown here) showed that the apoptosis rates of SK-N-SH cells during different time periods of As2O3 treatment were higher than those of the control cells. A similar study was performed by Woo et al. [2O3. Both cell lines were arrested at the S-G2/M phase and this effect was associated with increased cyclin B expression and CDK1 activity [2O3 on cell cycle progression in NB cells may be related to the p53 status [2O3 could arrest the cell cycle by modulating the expression and/or activity of several key G2/M regulatory proteins [2O3-induced mitotic cells only, as opposed to interphase cells [2O3 has a higher cytotoxicity against NB cells that are arrested at the G2/M phase. In addition, the capacity of enhancing treatment outcomes by arresting cells during the S phase of the cell cycle via the combination of As2O3 and chemotherapy should be considered.Aspoptosis . Suppresi et al. . reporteo et al. ,who usedactivity , 43. Oth3 status  and the proteins . Furtherse cells , suggest2O3 can influence TrkA, TrkB and TrkC expression in a time- and dose-dependent manner. We further demonstrated that increasing As2O3 concentrations caused significant upregulation of TrkA and TrkC expression levels. This observation was more obvious at the 48 h compared with the 24 h time point. In human NBs, TrkA is involved in the inhibition of cell growth, the induction of apoptosis, the inhibition of angiogenesis and the determination of either apoptosis and/or differentiation [2O3 treatment should be increased.Chemotherapeutics arrest the tumor cell cycle and further influence the expression of certain genes that closely correlate with cancer stage and prognosis . The prentiation . One repntiation . High le2O3, with the level of TrkB in the group treated with 4 µM for 48 h being significantly less than that in the control group. Increased TrkB expression is believed to be associated with aggressive NB. Zhang et al. [2O3 for the treatment of stage 3 and stage 4 NB, which indicate high levels of TrkB expression and poor prognoses; an appropriate concentration and treatment time for As2O3 remains to be determined.The receptor TrkB is preferentially expressed in NB patients who have a poor prognosis and confers invasive and metastatic potential to tumor cells in addition to enhancing therapeutic resistance . We furtg et al.  demonstrg et al.  reported


O-glucoside (PA1) and guaiacyl-pyranomalvidin-3-O-glucoside (PA2) are extensively revisited using ultraviolet (UV)-visible spectroscopy, dynamic light scattering (DLS) and quantum chemistry density functional theory (DFT) calculations. In mildly acidic aqueous solution, each cationic pigment undergoes regioselective deprotonation to form a single neutral quinonoid base and water addition appears negligible. Above pH = 4, both PA1 and PA2 become prone to aggregation, which is manifested by the slow build-up of broad absorption bands at longer wavelengths (λ ≥ 600 nm), followed in the case of PA2 by precipitation. Some phenolic copigments are able to inhibit aggregation of pyranoanthocyanins (PAs), although at large copigment/PA molar ratios. Thus, chlorogenic acid can dissociate PA1 aggregates while catechin is inactive. With PA2, both chlorogenic acid and catechin are able to prevent precipitation but not self-association. Calculations confirmed that the noncovalent dimerization of PAs is stronger with the neutral base than with the cation and also stronger than π–π stacking of PAs to chlorogenic acid (copigmentation). For each type of complex, the most stable conformation could be obtained. Finally, PA1 can also bind hard metal ions such as Al3+ and Fe3+ and the corresponding chelates are less prone to self-association.The physicochemical properties of the wine pigments catechyl-pyranomalvidin-3- Among flavonoids, anthocyanins are responsible for plant pigmentation and express a variety of colors from red to blue.+ is the only anthocyanin form. As pH increases, water addition at C2 (hydration) becomes thermodynamically favorable, leading to the colorless hemiketal . Around pH 4, fast deprotonation of AH+ competes with water addition, giving rise to the blue-purple quinonoid base A. Nevertheless, at equilibrium, A is in much lower concentration than the hemiketal, so that the solution is almost colorless. Around pH 4, deprotonation of AH+ also takes place, giving rise to blue-purple quinonoid bases , which are in low concentration at equilibrium. However, in their natural medium, the anthocyanin colored forms are prone to form noncovalent assemblies, mainly by π–π stacking interactions, either: (i) with themselves (self-association); or (ii) with the pool of colorless polyphenols especially, flavones, flavonols, and hydroxycinnamic acids (copigmentation) [In highly acidic aqueous solution pH < 2), the red flavylium ion AHntation) ,2,3. Cop, the redntation) . It is tp-hydroxyphenyl-pyranoanthocyanins result from reaction of anthocyanins with either vinylphenols [O-glucoside to form the catechyl-(PA1) and guaiacyl-(PA2) pyranomalvidin-3-O-glucoside derivatives  and accurate quantification of the major compounds by quantitative nuclear magnetic ressonance (qNMR) [During wine aging, anthocyanins are progressively converted to specific pigments, in particular pyranoanthocyanins with particular chromatic and physicochemical properties. Among them, lphenols  or hydrolphenols , the lativatives . A detaie (qNMR) .2H, Controversial results have been reported on the physicochemical properties of pyranoanthocyanins. Quijada-Morín et al.  investigmax values of PA1 and PA2 at pH 0.8 were reported at 4500 and 3060 M−1·cm−1, respectively [−1·cm−1 [There is also discrepancy about the values reported for the molar absorption coefficients of PAs. Namely, the εectively , whereas[−1·cm−1 .p-hydroxyphenyl-pyranoanthocyanins (PA1 and PA2) with a specific emphasis on: (a) proton transfer vs. hydration reactions; and (b) self-association vs. copigmentation. The experimental data by UV-visible spectroscopy and dynamic light scattering (DLS) are extensively analyzed and discussed with help of quantum chemical density functional theory (DFT) calculations.The aim of this work is to revisit the chromatic and physicochemical properties of max = 506 nm) and PA2 (λmax = 506 nm) were detected at m/z 625 and m/z 639, respectively. Full 1H and 13C NMR characterization confirmed the structures of both pigments  were measured at 18 × 103 and 42.5 × 103 M−1·cm−1 (λmax = 520 nm) for PA1 and PA2, respectively. Those εmax values are much higher than those previously reported in the literature, thereby attesting the high purity of the PA samples used in this work.The ε+ [cis–trans chalcone isomerization), i.e., from a few seconds (pH 2–3) to several minutes (pH 5–6). The apparent kinetics of hydration is largely governed by the fraction of AH+, the sole colored form to undergo water addition in solution. Unlike common anthocyanins, PA1 and PA2 do not clearly undergo such relatively fast color loss in agreement with previous investigations on various PAs [+ is the sole species. Above pH = 3.5, an instantaneous broadening and weakening of the visible band becomes observable with both PAs, which reflects proton loss. Therefore, a few seconds after mixing, the pH dependence of the visible absorbance at 505 nm  can be safely analyzed with the assumption of a single proton loss. In an acetate buffer containing 12% EtOH, the first pKa of PA1 (concentration. 50 µM) and PA2 (ca. 25 µM) was estimated at 4.30 (±0.09) and 4.35 (±0.11), respectively. In the citrate-phosphate buffer (no EtOH), the first pKa of PA1 was estimated at 4.69 (±0.06)  were added to buffered aqueous solutions in the pH range 1–6 and the spectral properties were monitored over 15–30 min. Under such conditions, common anthocyanins undergo a typical color loss, reflecting water addition onto AH+ ,18 durinious PAs ,19. Thusious PAs . By cont (±0.06) A. These  4.35 ±0., respectKa (50% flavylium + 50% base) and the spectrum of the pure AH+ form (pH ≈ 2). This deduced spectrum was used as the experimental control for calculations aimed at determining the distribution of the different tautomers  and their individual visible spectra (see below). Surprisingly, the experimental spectrum of A displays a main absorption band at λmax ≈ 480 nm, which means a hypsochromic shift of more than 20 nm with respect to AH+, and a shoulder in the range 530–540 nm. Its maximum molar absorption coefficient is circa half that of AH+. This uncommon hypsochromic shift is consistent with previous observations [The spectrum of the pure neutral base A B was dedrvations ,19. This+ form of PA1 exhibits a large band at 506 nm  being delocalized along an extended conjugated path A.+ and bases 1 and 2, deprotonation of C4E-OH or C4B-OH induces a destabilization of the HOMO  and is slightly bathochromically shifted (as for bases 1 and 2)  excited state theoretically lying at 446.4 nm A, which the HOMO A,B, ther1 and 2) D. Howeve1 and 2) B, thus i1 and 2) D. The agd 2)  represents only 1% of the total; their presence most probably contributes only slightly to the shoulder observed at 540 nm. The same rationalization also applies to PA2 see .max). Interestingly, investigations by 1H-NMR in acidic to mildly alkaline solutions [+: 600, A: 900, A−: 200 M−1. This means that, as expected, dimerization is stronger for the neutral form (pH 4–7), i.e., when electrostatic repulsion is minimized between the two chromophores.Self-association of anthocyanins has been investigated by UV-visible spectroscopy, circular dichroism and NMR ,22,23. Aolutions ,22 gave O-β-d-(6-O-p-coumaroyl)glucoside. At pH = 1.3, the AH+ form of this PA was shown to form a noncovalent dimer with a binding constant Kd of 3 × 103 M−1 [+ forms of PAs do not seem much more prone to self-association than the parent anthocyanins, under mildly acidic conditions, AH+···A or A···A self-association is likely to be stronger due to the decrease in electrostatic repulsion.By contrast, little data is available about the self-association of pyranoanthocyanins. One study was reported with the pyranoanthocyanin formed by addition of 8-vinylcatechin  and malvidin 3- 103 M−1 , which i 103 M−1 . AlthougIn this work, a surprising observation is the slow decay of the main visible band taking place in 50 µM solutions of PA1 at pH > 3.5 .+···A may form first, followed by aggregates of pure base A at higher pH, which is consistent with the spectral differences between pH 4.4 . To the best of our knowledge, such observations have never been reported before. A reasonable explanation for the slow changes observed in the visible spectra of PA1 at pH > 3.5 is that self-association involving the neutral quinonoid base occurs. Mixed aggregates AHn pH 4.4  and pH 5n pH 4.4 . The addn pH 4.4 .Ka value. Indeed, dilution by a factor of 2.5 shifted pKa value from 4.35 (±0.11) to 4.79 (±0.12) for PA2. This means that at high concentration, proton loss occurs at lower pH (acid-base equilibrium shifted toward the base) in agreement with A being more prone to self-association than AH+.The higher propensity of A for self-association is corroborated by the concentration dependence of the first pDFT calculations including dispersion correction confirmed the capacity of PA1 and PA2 to self-associate, both exhibiting negative energies of association  appeared likely to occur. For each, several conformers were obtained (see The three possible self-associations (AH+···AH+ complexes strongly distorted the geometries (compared to AH+···A and A···A), however without preventing the stabilizing π–π stacking forces.Three types of conformers could be distinguished: Group A, exhibiting interactions between A-, C-, and D-rings of both PA1 units; Group B, exhibiting interactions between A-, C-, and D-rings of one PA1 and the B-ring of the other PA1; Group C, exhibiting interactions between A-, C-, and D-rings of one PA1 with the E-ring of the other PA1. In each group, electrostatic and H-bonding interactions influence orientation of both units with respect to each other. In particular, the strong electrostatic repulsion in AH−1 than the most stable AH+···A and AH+···AH+ conformers. Moreover, stable A···A conformers appear more numerous, in particular when comparing to AH+···AH+ dimers. In this latter case indeed, only one AH+···AH+ dimer exhibits a stabilizing energy close to those of the AH+···A dimers. Hence, DFT calculations including dispersion forces confirm that AH+···AH+ (pH < pKa) forms to a lesser extent than AH+···A (pH ≈ pKa) and A···A (pH > pKa), in agreement with the experimental observations.Self-association is here studied in terms of electronic energy, not Gibbs energy, mainly because entropy related to self-association cannot be accurately calculated . Hence, −1, for PA2 and PA1, respectively  calculations, as the most stable A···A conformers exhibited association energies of −32.3 and −28.0 kcal·molectively . As expe0) was observed at pH 4.4 , which is in contrast to the slow spectral changes observed with PAs. It is thus very likely that PA self-association is not limited to the formation of noncovalent dimers or small-sized oligomers but leads to large aggregates. This was confirmed by DLS experiments. For PA1 (50 µM), an immediate increase in the relative scattering intensity (I/It pH 4.4 A and 5.2gregates A. At pH Adding a large excess of chlorogenic acid to PA1 aggregates (50 µM PA1 solution ca. 30 min after preparation) led to conversion of the visible spectrum back to that of a fresh solution  .max value by ca. 10 nm was observed, which is typical of copigmentation. Copigmentation is actually known to compete with anthocyanin self-association. Moreover, the time-dependent spectral changes that were observed due to PA1 self-association at pH value of 4.6 were markedly slowed down when the chlorogenic acid concentration was increased and totally abolished with 100 equiv. copigment (data not shown). DLS experiments confirmed that addition of chlorogenic acid (100 equiv.) inhibited self-association of PA1 and prevented the formation of large aggregates prone to precipitation (in acetate buffer containing 12% ethanol at pH = 4.4 and 5.2): the scattering intensity remained unchanged and only a slight Dh increase from 100 to 200 nm was observed , both chlorogenic acid and catechin (20–100 equiv.) were able to prevent precipitation at pH > 4 but not self-association. As said above, under more dilute conditions (10 µM), precipitation is not observed, even in the absence of copigment, and self-association is slow at pH = 4.4. However, at pH ≥ 5, self-association is much faster  and its + appeared less favorable than with A (deprotonation of C7A-OH). For instance, the stabilizing energies of the most stable PA1···chlorogenic acid conformers were −18.0 and −24.5 kcal·mol−1 for AH+ and A, respectively (−1) with PA2 , the B-ring (Group B), the E-ring (Group C) or the sugar moiety (Group D) of PA .In Group D, H-bonding would mainly drive copigmentation; as expected, the corresponding conformers are less stable than those of Groups A, B, and C. In Groups A, B, and C, π–π stacking is the driving force, intermolecular electrostatic interactions and H-bonding driving only the respective orientation of pigment and copigment. Copigmentation of PAs with chlorogenic acid appears less favorable than self-association. Indeed, when comparing geometries of noncovalent dimers and copigmentation complexes, π–π stacking in noncovalent dimers is much more favored due to more extended π-conjugated systems facing each other. This is consistent with the observation that chlorogenic acid has to be used in large excess (vs. pyranoanthocyanins) to prevent aggregation. Aggregation involves the formation of dimers and higher oligomers of high conformational variability. At high concentration, copigments can intercalate in small aggregates, thus slowing down or even inhibiting the formation of larger aggregates. Copigments can also intercalate in large aggregates and promote their dissociation.3+ and Fe3+. Under mildly acidic conditions, Al3+ binding and concomitant loss of the two catechol protons result in a strong bathochromic shift and a decrease in visible absorbance at λmax  as the spectral changes observed over 20–30 min are less intense than with metal-free PA1.max). Only the spectral characteristics of the AH+···chlorogenic acid and A···A complexes will be detailed. In the former case, λmax is bathochromically shifted in agreement with the experiment. The low-energy band is assigned to the first excited state (S0 → S1), which is mainly described by HOMO → LUMO electronic transition. As already observed [The spectral shifts characteristic of self-association and copigmentation were studied by TD-DFT calculations and analyzed according to the π → π* electronic transitions constituting the first excited states . Purified deionized water  was used for the preparation of all solutions.All solvents were of HPLC quality and all chemicals of p.a. grade. Vinylguaiacol and glycerol were purchased from Sigma . Malvidin-3-Saccharomyces cerevisiae MTF3686 . A two liters cell culture was prepared as described earlier [p-coumaric acid was added to induce the decarboxylase activity. Cells were recovered in exponential growth phase, washed twice with 50 mM citrate-phosphate buffer (pH = 3.5). Highly dense suspensions of resting cells were prepared in 1 L of citrate-phosphate buffer (pH = 3.5) containing 2% glucose and supplemented with 0.5 mM of caffeic acid as a bioconversion substrate. The medium was agitated at 27 °C during 4 days, then centrifuged (1000× g) for 10 min. The supernatant was passed through a Sep-Pak® tC18 cartridge , eluted successively with H2O and MeOH. The MeOH fraction containing vinylcatechol was mixed with water and concentrated under vacuum to remove MeOH, analysed by HPLC-DAD as described earlier [Vinylcatechol was obtained from caffeic acid by bioconversion using the yeast strain  earlier  with somO-glucoside (2 mM) and vinylguaiacol (2 mM) or vinylcatechol (2 mM equiv. vinylguaiacol) were prepared in MeOH/H2O mixture (70/30). The pH of the mixture was adjusted with concentrated HCl to pH 3. The reactions were carried out during 5 days at 35 °C [2O.Malvidin-3-at 35 °C . The reaSemi-preparative purification of PA1 and PA2 was performed using a Varian LC 940 system  with a UV detector and fraction collector. After filtration on a 0.45 µm HVLP Millipore filter, the solution was injected onto a Microsorb 100—3 µm, C18 column . Elution was achieved at a flow rate of 10 mL/min with solvents A (0.1% aqueous HCl) and B (0.1% HCl in MeCN-H2O (4:1)) with the following gradient: 0–6 min, linear 39%–41% B, 6–8 min, linear 41%–80% B, 8–9 min, isocratic 80% B, 9–10 min, linear 80%–39% B. The collected fractions were freeze-dried and stored at −80 °C until analysis.1H and 13C, respectively, and equipped with a 5 mm indirect detection Z-gradient probe. The chemical shifts were expressed with MeOH as the internal reference . All data were collected with active sample temperature regulation. Assignments of proton and carbon signals were deduced from homonuclear 1H correlation spectroscopy, rotating-frame overhauser spectroscopy, diffusion-ordered spectroscopy and heteronuclear 1H/13C heteronuclear single quantum coherence and heteronuclear multiple bond correlation experiments. Data were processed and analyzed using the VNMRJ software .All experiments were performed on an Agilent 500 MHz DD2 NMR spectrometer  operating at 500.05 MHz and 125.75 MHz for Spectra were recorded with an Agilent 8453 diode array spectrophotometer fitted with a quartz cell  equipped with a stirring magnet. A constant temperature of 25 °C in the cell was obtained by use of a water-thermostated bath.3 or Fe(NO3)3) in acetate buffer + 12% EtOH.Small volumes of concentrated PA solutions in acidified MeOH  were added to a 0.2 M acetate buffer containing 12% EtOH or to a citrate-phosphate buffer (pH range 1–6). In the cell, the pigment concentration was ca. 50 µM for PA1 and 25 or 10 µM for PA2. The spectral monitoring was carried out over 15–30 min. Then, the samples were collected and kept overnight at room temperature to repeat the spectroscopic analysis. The experiments were also repeated with buffers containing a copigment (CP: chlorogenic acid or catechin) in large excess (20–100 equiv.) or a metal ion  and the UV-visible spectra were recorded.DLS experiments were conducted with a Malvern Autosizer 4800 HS  equipped with a 30 mW He-Ne laser (λ = 633 nm) and APD detection. Measurements were carried out at an angle of 90° from the incident beam. Prior to experiments, all solvents were filtered on 0.2 μm microfiltration units . Both PA1 and PA2 were prepared in 0.1 M HCl in MeOH. PA solutions (10 µL) were diluted in acetate buffer  and introduced in the measurement cell at 25 °C. Each experiment was carried out in duplicate. Control measurements were performed on solvents. Addition of chlorogenic acid (100 equiv.) to PA1 and PA2 was performed in acetate buffer containing 12% ethanol at pH = 3.5, 4.4 and 5.2.Sample evolution was followed by measuring the scattered intensity and the autocorrelation function G(t) of the scattered light. Each measurement was the average of 10 subruns. A rise in scattered intensity above control values indicated the onset of aggregation.0/λ)·sin(θ/2) is the scattering vector, n0 the refractive index of the solvent, λ the laser wavelength and θ the scattering angle. The average hydrodynamic diameter of the particles Dh was then derived from D using the Stokes-Einstein equation and assuming spherical shapes:The autocorrelation function of the scattered light was first analyzed using the cumulant method, which gives an average value of the aggregate size in the dispersion. The logarithm of the normalized correlation function was fitted to a polynomial:Finally, the polydispersity index PI (0 < PI < 1), defined as c/2b, was estimated as a measure of the variance (squared standard deviation) of the particle size distribution.Geometry optimization were achieved using density functional theory (DFT) calculations with the hybrid functional B3P86, which has appeared particularly adapted to assess both the thermodynamic and UV-visible absorption properties of polyphenols ,25. The The TD-DFT calculations were performed both in the gas phase and in solvent, using the Polarizable Continuum Model (PCM) scheme. The integral equation formalism PCM (IEFPCM) method coupled to UA0 radii was used. In the PCM formalism, the solute is embedded in a shape-adapted cavity surrounded by the solvent implicitly described as a dielectric continuum characterized by its dielectric constant ε (ε = 32.6 for MeOH). All calculations were performed with the Gaussian software package .Self-association and copigmentation complexes were obtained from pure quantum chemistry calculations, using dispersion corrected DFT , which is a successful approach to evaluate noncovalent (mainly π–π stacking) interactions in copigmentation complexes. However, pure quantum chemistry methods do not allow the explicit description of solvent, especially water, for such quite large molecular systems. As a consequence, the contribution of hydrogen bonding to stabilizing energies is overestimated. Moreover, only the stabilizing electronic energies , not the free enthalpy changes, were evaluated as calculation of entropic contributions in self-association and copigmentation is still an unsolved theoretical challenge . In otheAll geometries of the dimerization and copigmentation assemblies were obtained with the COSMO B3P86-D2/def2-SVP formalism in the ORCA package [O-glucoside (PA1) and guaiacyl-pyranomalvidin-3-O-glucoside (PA2) are stable wine pigments that do not undergo water addition and thus simply behave as monoacids in mildly acidic solution. However, in the pH range 3.5–6 that is most important for application as food colorants, their intense color tends to evolve due to self-association of the neutral quinonoid base and eventual precipitation. If controlled by appropriate matrices, possibly including some copigments to avoid precipitation, this phenomenon, reported in this work for the first time, is very interesting as natural colors from red (monomer) to blue (aggregates) can thus be produced.In conclusion, catechyl-pyranomalvidin-3-