These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography–multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man The molecular characterization of bioactive food components is essential for understanding the mechanisms of their beneficial or detrimental effects on human health.Widely-consumed legume seeds  have been studied with the specific aim of identifying and characterizing the proteins accounting for their health-promoting properties ,2 and/orin vitro [γ-Conglutin, a minor component of the mature lupin seed  having iin vitro ,11, was in vitro ,12. Puriin vitro , and hadin vitro .in vitro and/or in vivo settings [The allergenic properties of the lupin seed have been ascribed to the abundant components β- and α-conglutin , while fsettings ,18,19,20Lupinusalbus) seed is a basic 7S protein which is prevalently tetrameric or hexameric at neutral pH [131 within the canonical eukaryote N-glycosylation consensus sequence Asn–Xaa–Ser/Thr (where Xaa is not Pro), and Asn132 within the less common sequon (Asn–Xaa–Cys), recently also described in plant cells [γ-Conglutin from the white lupin . γ-Conglutin extracted from lupin flour was purified as described in the Supporting Information (ormation .Purified γ-conglutin (10 µg) was analyzed (n=4) under reducing or non-reducing conditions by SDS-PAGE  .In-gel trypsin digestion (with reduction and carbamidomethylation) was done on gel-separated γ-conglutin subunits or monomer bands according to Schiarea et al. . In soluDried trypsin digests of the large subunit band were treated with two N-glycosidase enzymes of different specificity, i.e. PNGase A and F , while dIn order to determine the structure(s), micro-heterogeneity profile, and attachment site of the N-glycan bound to γ-conglutin we used combinations of the procedures shown in 2O + 0.1% formic acid; eluent B, CH3CN + 0.1% formic acid; gradient, 2% to 60% B in 40 min, then to 98% B in 6 min for 4 min, and re-equilibration to 2% B for 24 min. MS conditions were as follows: source DESI Omni Spray  used in nanospray mode with positive ions; ion spray voltage, 2400 V; interface capillary temperature and voltage, 220°C and 42 V. The “lock mass” option was enabled for accurate mass measurements in MS mode. For CID fragmentation in multi-stage MS (MSn) mode, normalized collision energy was set at 35%. Full MS “survey” scans (m/z 400-2000) were run using the Orbitrap at resolution 60,000 at m/z 400. Each survey scan was followed by ion trap (IT) MSn analysis in “data-dependent” or “targeted” mode, as follows. For data-dependent analysis, low-resolution MS2 scans were acquired by the LTQ for the four most abundant precursor ions with isolation width 3 m/z, AGC target value of 4 x 104, exclusion of singly-charged ions, and 30 s dynamic exclusion. For targeted MSn analysis, MS survey scans were followed by “targeted” MS2 scans of a pre-selected glycopeptide precursor ion. Each MS2 scan was followed by a MS3 scan of the third isotopomer of the “Peptide+HexNAc” fragment ion generated during the MS2 step.The various digests were directly analyzed with an LTQ Orbitrap XL™  interfaced with a 1200 series capillary pump . Peptides/glycopeptides were separated on a C18 reverse-phase column ; flow rate, 2 µl/min; eluent A, HThe Mascot search engine  was used to identify non-glycosylated and deglycosylated peptides in the various digests. MS2 ion search was done against the NCBInr database 20120531. All other details are shown in Averaged high-accuracy, high-resolution MS spectra over given LC retention time-ranges were deconvoluted to obtain the singly-charged monoisotopic molecular mass of the multiply-charged glycopeptides, using Xtract for Qual Browser 2.0 (Thermo Scientific).+ ions).The Qual Browser 2.0 Isotope Envelope Simulation tool was used to examine the correspondence between the experimental and theoretical isotope envelopes of each putative peptide glycoform (multi-charged MH+) of peptide glycoforms were entered into GlycoMod together with the sequence of the γ-conglutin precursor (Q9FSH9), the digesting enzyme (trypsin), the protein chemical modification , 3 ppm tolerance for the theoretical vs. experimental mass match, and no restriction on monosaccharide composition.GlycoMod (http://web.expasy.org/glycomod/)  was usedAfter reduction , γ-conglutin was directly infused  into the LTQ Orbitrap at 2 µl/min flow rate. MS conditions were: source DESI Omni Spray in nanospray mode with positive ions; ion spray voltage, 2400 V; interface capillary temperature and voltage, 225°C and 32 V. MS spectra were acquired in the Orbitrap at mass range 200-2000 m/z, resolution 100,000. Mass spectra charge-deconvolution was done with Xtract for Qual Browser 2.0.–MS spectrum of the whole in-gel trypsin digest of the large subunit was first charge-deconvoluted over the entire m/z range (LC time range: 10-40 min) to obtain a global view of the monoisotopic singly-charged pseudo-molecular ions. Considering trypsin cleavage also before proline, the molecular mass of the shortest non-glycosylated peptide encompassing the two N-glycosylation sequons (N131NT and N132TC) is 4085 Da. Above 4000 m/z the deconvoluted mass spectrum showed two main series of five peaks  spaced by mass differences possibly due to single monosaccharide residues, i.e. 132.04 Da , 162.05 Da , 203.08 Da , and again 203.08 Da. We thus hypothesized that the five peaks in series I and II represented the same five glycoforms (hereafter named A to E) of two partially overlapping tryptic peptides that encompass both potential glycosylation sites (N131 and N132), i.e. Pept127-165 and Pept111-165 . The A glycoforms would thus represent Pept 127-165 or Pept111-165 carrying a short glycan residue , while B to E would have the same saccharide composition as A, with the following increasingly complex composition: B=A+Pent, C=B+Hex, D=C+HexNAc, and E=D+HexNAc. We also noted two minor glycoform series of Pept127-165 and Pept111-165 (named here B′ to E′) which are discussed below.The averaged LC127-165 eluted late (around 35 min), and slightly earlier than the non-glycosylated peptide   covering the elution of both glycoform series (as well as non-glycosylated Pept127-165) . The mas127-165) , and is 127-165 and Pept111-165 obtained by LC-MS were used to hypothesize the structure of the attached N-glycans by interrogating GlycoMod. A unique matching glycopeptide was obtained for each experimental mass. The predicted oligosaccharide compositions  matched a single specific isomeric N-glycan structure (Man2(Fuc) GlcNAc2) [In plant organisms, the composition of our A glycoform (Hex GlcNAc2) ,38. The specific structures that we propose are based on the unique-matching GlycoSuiteDB entries, and should thus be taken as the most probable within the context of plant N-glycosylation knowledge. The A to E glycoforms of γ-conglutin correspond to well-known N-glycan structures often seen in plants [We confirmed the oligosaccharide composition and some sequence features of the A to EN-glycans by MSn (see below), but the n plants ,37,39. Dn plants  and horsn plants , respect127-165 and Pept111-165, we verified the correspondence of the experimental and theoretical isotopic envelopes. To further substantiate the identity of the A to E glycoforms of Pept131N132TCGLISSNPVTQE (Pept122-145) and PGCHN 131N132TCGLISSNPVTQE (Pept127-145).To support the identity of the major A to E glycoforms, we analyzed shorter V8/trypsin glycopeptides by combined data-dependent LC–MS2 and targeted LC–MSn analysis. The shortest predicted peptides encompassing the two potential N-glycosylation sequons were STTSRPGCHN The correct isotopic envelope of the MH3+ ions of the A to E glycoforms of both Pept122-145 and Pept127-145 appeared in the full MS spectra (not shown) within 1 ppm of the following theoretical monoisotopic mass of the MH3+ ions: 1165.170, 1209.184, 1263.202, 1330.895 and 1398.588 m/z for Pept122-145, and 987.750, 1031.764, 1085.782, 1153.475 and 1221.168 m/z for Pept127-145. The glycoform pattern of Pept122-145 and Pept127-145 was identical to that of Pept127-165 and Pept111-165,  survey scan by Orbitrap (400-2000 m/z); 2) MS2 scan targeting for CID fragmentation the triply-charged ion of one selected glycoform; 3) MS3 scan targeting the MS2-product ion at m/z 1411 (z=2), a fragment corresponding to the intact peptide carrying a single HexNAc residue [122-145 glycoforms (122-145 (1 fragment corresponding to Pept+HexNAc (m/z 1411 (z=2) and m/z 941 (z=3)); 2) the loss of a terminal non-reducing HexNAc residue from the intact glycopeptide ion in the glycoforms D and E; 3) the loss of core dHex (fucose) from the intact glycopeptide; 4) the sequential cleavage of all glycosidic bonds within the glycan, leading to ladders of abundant Y ions  [The V8/trypsin digest was repeatedly analyzed by LC–MSn, each time targeting a different glycoform of Pept residue  (hereaftycoforms . The MS3(122-145  have in t Y ions . In the –HexNAc) . For all–HexNAc) . Other i122-145 by fragmenting their common MS2 product ion Pept+HexNAc . The MS3 fragmentation pattern clearly indicated that the peptide moiety is indeed STTSRPGCHNNTCGLISSNPVTQE. 131 and N132 (positions 10 and 11 in Pep122-145) carries the N-glycosylation. However, we indicate N131 as the glycosylation site in Identical MS3 spectra were obtained from the different glycoforms of Pep127-165 and Pept111-165 , which supports the core position of fucose, was abundant in all the B to E glycoform MS2 spectra (122-145 that carry one carbamidomethyl group in excess (+57.02 Da) [A first compositional evidence of the minor variants of the B to E glycoforms lacking fucose (named here B′ to E′) was obtained for both tryptic peptides Peptt111-165 . The dat spectra , but abs127-165 and Pept111-165 remained substantially unchanged after PNGase F, but were both completely deglycosylated by PNGase A . The MS spectra of the putative deglycosylated Pept111-165 showed multicharged MH+ ions (z=4-7) that corresponded, after deconvolution and deisotoping, to a monocharged monoisotopic ion at 5954.675m/z. This indicates a +0.984 Da mass shift from the theoretical value of the non-glycosylated peptide (5953.698 m/z), supporting the actual N-deamidation, and thus N-deglycosylation, of Pept111-165 after PNGase A treatment. The same +0.984 Da mass shift was observed for PNGase A-treated Pept127-165.To obtain supporting evidence of the core α1-3 fucose in the A to E glycoforms, we used PNGase F (which cannot remove N-glycans that carry α1-3-linked core fucose) in parallel with PNGase A . The A to E glycoforms of PeptPNGase A . The B′ 131-Asn-Thr and Asn132-Thr-Cys [131 of Pept127-145 by MS2 analysis . The laanalysis . Our resLupinusalbus γ-conglutin subunits has been documented earlier by Duranti’s group [111-126 and Pept127-165) derived from the non-canonical “trypsin/P” cleavage  at R126–P127 (126–P127G…).An additional finding of this study is the extensive sequence coverage of γ-conglutin subunits provided by the global LC–MS2 analysis of the in-gel tryptic peptides. While the N-terminal sequence of the two ’s group , availab’s group ,47. Our 126–P127 . The lat452) identified here, we found – in addition to the most abundant N-terminal peptide S301YHESSEIGGAMITTTNPYTVLR that confirms the N-terminal sequence described earlier [299SSYHESSEIGGAMITTTNPYTVLR and S297SSSSYHESSEIGGAMITTTNPYTVLR. The existence of three major N-terminus variants in the native protein small subunit  with one, three, or five N-terminal serines implies that the small subunit variants had theoretical monoisotopic molecular masses of 16407.21, 16581.28 and 16755.34 Da, respectively. We confirmed this by measuring the accurate molecular mass of the intact small subunit variants with direct-infusion Orbitrap MS  or from successive steps of N-terminal proteolytical trimming of serines from the largest subunit variant having S297 as N-terminus.The in-depth γ-conglutin sequence coverage helped explain the heterogeneity of the small subunit. While the C-terminus described by Duranti’s group  was conf earlier  – two aditrap MS . The seqWe characterized the N-glycosylation profile of γ-conglutin, and also defined the major N-terminal sequence variants of the heterogeneous small subunit.2(Xyl) (Fuc) GlcNAc2 and Man3(Xyl) (Fuc) GlcNAc2) and two less abundant “complex-type” N-glycans (D and E: GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2) [a) epitope [By providing a general view of the structure and relative abundance of γ-conglutin glycoforms, we observed the prevalence of two “paucimannosidic-type” glycoforms (B and C: ManGlcNAc2) . We did  epitope .a-containing “complex-type” N-glycans) [N-glycans of the “paucimannosidic-type”, typical of vacuolar and seed storage glycoproteins, are truncated forms of “complex-type” N-glycans that lack terminal non-reducing GlcNAc ,28. The glycans) .in vitro allergy tests with non-homologous (but CCD-carrying) glycoproteins [The four most abundant N-glycans (B to E) alternatively attached to γ-conglutin carry two independent glyco-epitopes  that are widespread in plants but absent in humans ,51. Glycproteins ,51,52. Tproteins ,51,52,53Protocol S1Details are given for the following procedures: 1) Purification of γ-conglutin; 2) Proteolytic digestion; 3) N-deglycosylation; 4) Data analysis.(DOCX)Click here for additional data file.Table S1Mass list of the deconvoluted spectrum shown in Figure 1.(XLSX)Click here for additional data file.Table S2Relative abundance of the main series of the γ-conglutin glycoforms (A to E) based on the intensity of the deisotoped molecular ion in the deconvoluted Orbitrap MS spectra.(DOCX)Click here for additional data file.Table S3Linkage details of the plant N-glycan GlycoSuiteDB entries that univocally match the predicted composition of γ-conglutin A to E glycoforms.(DOC)Click here for additional data file.Table S4MASCOT peptide identification details.(XLS)Click here for additional data file.Figure S1SDS-PAGE analysis of purified γ-conglutin under non-reducing and reducing conditions.(TIF)Click here for additional data file.Figure S2127-165 by LC–MS2 and Mascot.Identification of non-glycosylated PeptThe identification was obtained analyzing the tryptic peptide/N-glycopeptide mixture obtained from in-gel digested γ-conglutin.(TIF)Click here for additional data file.Figure S3Comparison of experimental and theoretical isotope envelopes.Representative Orbitrap MS spectra (top panels) and simulated (bottom panels) isotope envelopes are shown for MH5+ of Pept127-165 glycoform C  and MH7+ of Pept111-165 glycoform B .(TIF)Click here for additional data file.Figure S4122-145.Reverse-phase LC–MS analysis of A to E glycoforms of PeptExtracted ion chromatograms  are shown for the third isotopomer of the MH3+ ions. The signal intensity was normalized to the most abundant glycoform, to highlight the approximate relative abundance of the five glycoforms (B=C>D>E>A). The structures shown in the insets represent the N-glycans first hypothesized by MS and GlycoMod, then confirmed by targeted MSn experiments .(TIF)Click here for additional data file.Figure S5111-165 shown as an example.Effect of PNGase A on the A to E glycoforms, with PeptLC–MS extracted ion chromatograms are shown for the A-E glycoforms and the N-deglycosylated (N-deamidated) Pept111-165. The MH6+ ions were extracted using the theoretical m/z values of the fifth isotopomer (± 2.5 ppm). The A-E glycoforms of Pept111-165, clearly visible in the control sample (left panel), completely disappeared after PNGase A-treatment (right panel), while the expected large amount of the previously absent N-deamidated (i.e. N-deglycosylated) Pept111-165 appeared at the expected retention time, i.e. right after the smallest (A) glycoform. All glycoform traces are normalized to the highest intensity (3.5E6) for easier comparison. The intensity of the N-deamidated peptide peak is 2.2E7.(TIF)Click here for additional data file.Figure S6127-145, which encompasses the two potential N-glycosylation sequons (N131NT and N132TC).Mascot identification of the N-deamidation site within PeptThe MS2 fragments show that N-deamidation occurred at N131 and not N132, since there is a +0.98 Da mass shift in all the y and b fragments that include N131 , but not in the fragments including N132 but not N131 (y3 to y14).(TIF)Click here for additional data file.Figure S7In-depth sequence coverage of γ-conglutin.The deduced sequence of γ-conglutin chain  is shown in black, while the sequence covered by peptide identification by Mascot with “semitrypsin” as enzyme is in blue or red. The N-terminal region of γ-conglutin corresponds to the large subunit (blue), and the C-terminal region to the small subunit (red). Dots indicate the short unidentified amino acid sequences. Detailed Mascot results are given for all the identified peptides in Table S2. The three underlined red serine (S) positions indicate the three alternative N-termini of the small subunit that were first suggested by peptide identification, then confirmed by the accurate mass of the intact small subunit variants analyzed by direct-infusion Orbitrap MS .(TIF)Click here for additional data file.Figure S8Enlarged view of the MS spectrum of γ-conglutin small subunit variants.The averaged spectrum showed – after charge-deconvolution and deisotoping – three abundant ions with monoisotopic neutral mass values  that are within 3 ppm from the theoretical values of the three sequence variants of the small subunit.(TIF)Click here for additional data file.


To understand the role of c-Myc in the mycoplasma induced apoptosis prevention and malignant cell transformation, 32D cells, an IL-3 dependent cell line, were infected and transformed by different species of mycoplasmas. The expression of Myc and ras gene families, apoptosis and the cell cycle during the infection and transformation were examined. Results showed that c-Myc expression was significantly increased in mycoplasma transformed 32D cells. Withdrawal of IL-3 substantially decreased c-Myc expression and led to cell cycle arrest at the G1 phase followed by rapid apoptosis. Infection by M. fermentans or M. penetrans not only alleviated the sharp decrease of c-Myc expression, rescued 32D cells from cell-cycle arrest and prevented apoptosis in IL-3-free culture, but also induced autonomous growth of 32D cells. Although M. hominis and M. salivarium had the ability neither to prevent apoptosis nor to induce malignant transformation, they were still able to rescue the cells from cell cycle arrest. The expression of ras family did not change significantly during the infection and transformation. These results suggest that constitutive expression of c-Myc appears to be associated with the continuous growth and malignant transformation of 32D cells induced by M. fermentans and M. penetrans, but not with rescuing the cell cycle arrest by the mycoplasmas.Our previous studies showed that mycoplasmas prevented apoptosis and induced the malignant transformation of mammalian cells. Other studies indicate that  Withdrahe cells . We haveude mice . We beliude mice . In the c-Myc proto-oncogene was first described in 1982 as the cellular homologue to the transforming sequences of the avian myelocytomatosis retrovirus  and PG18 strains, respectively . 32D/GTU/c, 32D/MI/c and 32D/PG/c are IL-3 independent 32D cell lines established by transforming 32D cells with ectively .M. fermentans (PG 18), M. salivarium and M. hominis PG21T were obtained from National Institute of Health, Bethesda, Maryland; M. penetrans (GTU-54) and M. fermentans MI were isolated from a patient with AIDS in this laboratory  according to the manufacturer’s instruction . RNAs wein vitro transcription from multiple DNA template sets , which include two housekeeping genes, L32 and GAPDH  as loading controls. An equal amount of total RNA (20 μl for each sample) was hybridized overnight to the 32P-labeled RNA probes. Unhybridized RNAs and RNA probes were digested with RNases A and T1. The protected mRNAs and probes were precipitated with ethanol and resolved on a 6% denaturing urea polyacrylamide gel. Undigested 32P-labeled probes serve as molecular size markers. The gel was dried and exposed to X-ray film as well as to a phosphoimage screen. The expression of mRNA transcript of a specific gene was quantified on a Storm 860 phosphoimage scanner followed by the analysis with software ImageQuant .mRNAs of myc and ras gene families were analyzed by the RNase protection assay (RPA) using RiboQuant multi-probe kit  following instructions of the manufacturer. In brief, 32P-labeled specific anti-sense RNA probes were synthesized by 6 cells/ml and frozen at -20°C for at least 24 h before the assay. To measure nucleosome, cell lysates were added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate that has been coated with anti-nucleosome antibodies. The absorbed nucleosomes on the plate were detected by a biotinylated detector antibody and streptavidin-peroxidase conjugate, and quantified on an ELISA reader.Cell apoptosis was assessed by measuring nucleosome levels in cell lysates using a nucleosome ELISA kit . Briefly, Cells from 5 to 10 ml of culture were counted, harvested by centrifugation and lysed in 1 ml of lysis buffer. The lysates were diluted with the lysis buffer to equivalent 106 cells were resuspended in 1 ml of 0.1M Tris-HCl, pH7.2 containing 0.07M NaCl, to which equal volume of 10μM 4',6-diamidino-2-phenylindole (DAPI) in 800mM Na2HPO4 was added. The cell suspension was incubated at 4°C overnight. Fluorescence of DAPI stained cells was measured on a flow cytometer. The percentages of cells in G0/G1, S and G2/M phases were determined.Cells were collected and washed once with phosphate buffer saline (PBS) by centrifugation, and then fixed in 50% ethanol. The fixed cells were stored at 4°C until analyzed by flow cytometry. To determine cell cycle distributions, the fixed cells in 50% ethanol were washed with PBS once. To stain cells, about 10sin 3, c-Myc, L-Myc, L-Myc, B-myc, Max, mad, mxi, mad3 mad4 and mnt) in mycoplasma-transformed 32D cell lines 32D/GTU/c, 32D/MI/c and 32D/PG/c was examined by using the multiple probe RPA. Parental 32D cells were used as control. In the presence of IL-3, control 32D cells expressed a relatively high level of c-Myc and moderate level of sin3 and Max as well as a low level of mxi, mad4 and mnt. The expression of B-myc, L-Myc, N-Myc, mad, mad3 and mad4 was not detected in these cells  of myc oncogene family . Results indicated that 32D cells expressed substantial amount of ras mRNAs. However, the expression level was not significantly increased in mycoplasma-transformed 32D cells , activate transcription and regulate cell biological functions. Although early studies indicated that c-Myc and N-myc were equally potent in transforming mammalian cells and L-myc also had weak transformation ability  RNA or c-Myc repressors, such as FUSE-binding protein-interacting repressor (FIR)  , 32.c-Myc promote proliferation and transformation of mammalian cells, but it is also involved in apoptosis. Rat-1 fibroblasts with the high level of c-Myc proteins are more prone to programmed cell death upon serum deprivation   rather than the c-Myc pathway. Since M. hominis and M. salivarium failed to rescue the IL-3-free 32D cells from dying of apoptosis, c-Myc could be critical in releasing cell arrests at other check points of the cell cycle. (F-MEL) . Withdrae arrest , 39. Thegression . The res1 phase arrest caused by negative growth factors is easily overcome by supplying corresponding growth factors or by adding a growth factor inducer. When the cell cycle is stopped by DNA damage, DNA repair may restore the cell cycle or the cells undergo apoptosis. Our previous studies have revealed that mycoplasmas and mycoplasmal associated membrane proteins (LAMPs) induce the expression of many cytokines (Generally speaking, withdrawal of growth factors can cause cell cycle arrest at the G1 phase by affecting the G1 phase checkpoint. DNA damage may provent cells from entering the S phase and the G2 phase by affecting DNA replication checkpoint . The G1 ytokines . Some ofytokines . Therefo


Methadone also stimulated P-gp ATPase and inhibited verapamil-stimulated P-gp ATPase activity under therapeutic concentrations. These results may provide a possible explanation for higher methadone dosage requirements in patients carrying variant-type of P-gp and revealed the possible drug-drug interactions in patients who receive concomitant drugs which are also P-gp substrates.Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp’s interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein expression levels of human P-gp were confirmed by real-time quantitative RT-PCR and western blot, respectively. Utilizing rhodamine 123 efflux assay and calcein-AM uptake study, methadone was demonstrated to be an inhibitor of wild-type human P-gp via non-competitive kinetic (IC Opioid addiction is a severe mental disorder, of which chronic and relapsing course is also a heavy burden of patients and their families and even societies Methadone has been demonstrated to be a substrate of P-gp ABCB1 gene, which is a highly polymorphic gene with more than 50 single nucleotide polymorphisms (SNPs) identified in the NCBI database. The most frequently studied SNPs in ABCB1 gene were c.1236C>T (rs1128503), c.2677G>T/A (rs2032582) and c.3435C>T (rs1045642). It has been demonstrated that these SNPs may be associated with P-gp expression, drug responses and disease susceptibility ABCB1 genetic polymorphisms on methadone maintenance therapy has been studied and inconsistent results were reported ABCB1 gene influence this interaction requires elucidation.P-gp is encoded by the human ABCB1 haplotype combinations on methadone-P-gp interactions, we established cell lines carrying human P-gp with haplotype combinations of ABCB1 c.1236C>T, c.2677G>T/A, and c.3435C>T. By this reproducible and validated model, the underlying mechanism of methadone-P-gp interaction and the functional effects of ABCB1’s genetic polymorphisms were studied.In order to investigate the functional effect of ABCB1 cDNA in pMDRA1 was provided by the Riken BRC DNA bank (RDB No. 1372) . All restriction enzymes were purchased from New England Biolabs .Rhodamine 123, calcein-AM, R-(+)-Verapamil and racemic methadone were purchased from Sigma Chemical Co . The Flp-In ™ system, Flp-In™-293 cells (human embryonic kidney cells), zeocin, hygromycin B and all cell culture medium and reagents were obtained from Invitrogen . Human ABCB1 cDNA was digested with Sac I and Xho I, and the ABCB1 cDNA fragment was ligated into pET21a(+) which was pre-cut with Sac I and Xho I. The recombinant plasmid was then digested with Bam HI and Xho I, and the insert was purified by 0.8% agarose gel and further ligated into the mammalian expression vector pcDNA5/FRT pre-cut with Bam HI and Xho I (pcDNA5/ABCB1). The QuickChange™ Site-Directed Mutagenesis Kit  was used to introduce point mutations  into the pcDNA5/ABCB1 according to the instructions. Triple mutations (1236T-2677T-3435T and 1236T-2677A-3435T) were created to evaluate the effect of these genotypes on human P-gp function. The sequences were confirmed by direct sequencing.Plasmid pMDRA1 containing a full-length human 2. The pcDNA5/ABCB1 containing different genotypes of ABCB1 cDNA and pOG44 (the Flp recombinase expression plasmid) were co-transfected into the Flp-In™-293 cells. Stable cell lines were selected on the basis of hygromycin B resistance. The expressions of Pgp were confirmed by Western blot analysis and the genotypes of the transfected cells were confirmed by sequencing.Flp-In™-293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and selected with 100 µg/mL zeocin at 37°C, 95% humidity and 5% COMembrane proteins (2 µg) were separated by sodium dodecyl sulphate-polyacrylamide (10%) gel electrophoresis and transferred to PVDF membrane  using a Transblotter™ . After blocking by 5% (w/v) nonfat powdered milk, the membrane was incubated with the anti-P-gp monoclonal antibody C219  at 1∶500 dilution in Tris-buffered saline supplemented with 0.1% Tween 20 (T-TBS) at 4°C over night, washed four times in T-TBS and incubated with goat anti-mouse IgG at room temperature for one hour. The C219 is a monoclonal antibody to P-gp and recognizes an internal, highly conserved amino acid sequence found in both protein isoforms, P-gp and MDR3 P-gp ABCB1 mRNA levels were evaluated by real-time quantitative RT-PCR utilizing an Applied Biosystems Taqman assay using 1 µg of total RNA. Total RNA was extracted from the transfected cells by the Qiagen RNeasy kit . Taqman primers and probes for human ABCB1 and GAPDH were Assay on Demand reagents  and were analyzed using an ABI Prism 7900 Sequence Detection System. The relative expression of ABCB1 mRNA was normalized to the amount of GAPDH in the same cDNA by using the standard curve method described by the manufacturer.Cytotoxicities of methadone at concentrations used in the present study were determined by MTT assay. Briefly, the cells were seeded into 24-well plates and cultured overnight. Various concentrations of methadone were then added and incubated for 48 hours. After incubation, the medium was removed and added fresh culture medium 200 µl containing 0.5 mg/ml MTT and then incubated at 37°C for 4 hours. After incubation, the medium was removed and 200 µl DMSO was added, and incubation for 30 minutes at room temperature. After incubation, 100 µl of solution was transferred to a 96-well plate and absorbance was measured using an ELISA plate reader  at 570 nm with a reference wavelength of 650 nm.The effect of methadone on P-gp ATPase activity was examined by Pgp-GIO assay system  according to manufacturer’s protocol. To be brief, in a 96-well untreated white plate, 25 µg recombinant human Pgp was incubated with P-gp-GIO assay buffer (untreated control) or 200 µM verapamil (positive control of drug induced P-gp ATPase activity) or 100 µM sodium orthovanadate (selective inhibitor of P-gp ATPase activity) or 1–40 µM methadone. The reaction was initiated by adding 5 mM MgATP and incubated for 40 min at 37°C. After removing the plate from 37°C heat source, the luminescence was initiated by adding 50 µl of ATP detection reagent. The luminescence was read after 20 min of incubation at room temperature on the SpectraMax Gemini XS microplate spectrofluorometer . Each experiment was performed at least three times, each in triplicate on different days.5 cells were harvested after trypsinization by centrifugation and resuspended in fresh culture medium. Rhodamine123  was added and the cells were incubated in a water bath at 37°C in the dark. Cells were pelleted at 1, 5, 10, 15, 30, 45, 60, and 90 min. After washed with ice-cold PBS, cells were resuspended in 1-ml, ice-cold PBS prior to FACS analysis using CellQuest software . Each cell type was performed at least three experiments, each in triplicate on different days.For FACS analysis, 5×105 cells were seeded on 96-well plates and cultured for 24 hours. After incubation, cells were washed with warm Hanks’ balanced salt solution (HBSS) and pre-incubated with HBSS for 30 min, and subsequently incubated in 1 µM rhodamine123 for 30 min with or without methadone in triplicate. After washed with warm PBS, cells were allowed to efflux rhodamine123 for 10 min at 37°C in the incubator. Samples (150 µl) were collected and transferred to 96-well black plates. Rhodamine123 concentrations were measured using a set of standards (from 62.5 nM to 8 µM) in HBSS analyzed by the SpectraMax Gemini XS microplate spectrofluorometer  with the excitation set at 485 nm and the emission set at 535 nm. The measured rhodamine123 concentrations were normalized to the total protein content in the cells. Total protein concentrations were measured by a microplate assay protocol , and bovine serum albumin was used as the standard. Each experiment was repeated at least three times across cell passages to ensure stability of transfection and consistency and reproducibility of the experiments.For efflux assays, 1×105 cells were plated on 96-well black plates and cultured for 24 hours. Before the uptake assay, cells were washed with warm Hanks’ balanced salt solution (HBSS) and pre-incubated with HBSS for 30 min, and subsequently with methadone for 30 min in triplicate. After pre-incubation, calcein AM  was added and incubated at 37°C in the incubator for 30 min. After washed by ice-cold HBSS, cells were lysed with 1% Triton X-100 and calcein fluorescence generated within the cells was analyzed by the SpectraMax Gemini XS microplate spectrofluorometer  with the excitation set at 485 nm and the emission set at 535 nm. Each experiment was performed at least three times, each in triplicate on different days.For calcein-AM uptake study, 1×10For ATPase Assay data analysis, basal P-gp activity, verapamil-stimulated P-gp ATPase activity, methadone effect on P-gp ATPase activity and fold stimulation by methadone were calculated as following equations:Basal P-gp activity:Verapamil-stimulated P-gp ATPase activity:Methadone effect on P-gp ATPase activity:Fold stimulation by methadone:50 values were calculated by the following equation:0, the efflux in the absence of inhibitor; I, the inhibitor concentration; IC50, the concentration that caused 50% inhibition of the maximal drug effect and s is the slope factor.For evaluation of inhibitor potency, ICmax, the maximal efflux rate; Km, the Michaelis-Menten constant and C is the substrate concentration.For kinetic studies, kinetic parameters were estimated by nonlinear regression using Scientist v2.01  according to the following equation:Statistical comparisons among different cells were performed by using SYSTAT v10 . Statistical differences were evaluated by ANOVA and post hoc analysis (Tukey’s test) and the statistical significance was set at p value <0.05.ABCB1 and the two variant types, and little expression of ABCB1 mRNA and protein was found in the Flp-In™-293 cells  values for methadone in wild-type P-gp, P-gp with 1236T-2677T-3435T genotype, and P-gp with 1236T-2677A-3435T genotype were 2.17±0.10 µM, 2.97±0.09 µM and 4.43±1.10 µM, respectively  were incubated with recombinant human P-gp. Methadone was demonstrated to stimulate P-gp ATPase activity by 1.5 fold higher than basal ATPase activity and inhibited 200 µM verapamil-stimulated P-gp ATPase activity .The inhibition kinetics of methadone was further investigated in the wild type and the two variant types of P-gp by Lineweaver-Burk plot . In the ABCB1 gene, c.1236C>T, c.2677G>T/A and c.3435C>T, were demonstrated to influence not only the kinetics of interaction between methadone and P-gp but also the P-gp inhibition potency of methadone.P-gp, is one of the major transporters expressed in human physiological barriers, such as intestine, kidney and BBB. It has been demonstrated that inhibition of efflux transporters may cause clinically relevant drug-drug interactions and lead to insufficient treatment or adverse drug reactions −/− mice were 15- to 23-fold higher than in Mdr1a+/+ mice, indicating that methadone is a good substrate of murine P-gp Several studies have evaluated the interaction between methadone and P-gp ABCB1 genetic polymorphisms on the interaction between methadone and P-gp, c.1236C>T, c.2677G>T/A and c.3435C>T were demonstrated to be associated with methadone plasma concentrations and maximum maintenance doses ABCB1 c.3435C>T 50 values for wild-type P-gp, P-gp carrying 1236T-2677T-3435T and P-gp carrying 1236T-2677A-3435T were 2.17 µM, 2.97 µM and 4.43 µM, respectively. Our results provide a possible explanation for the higher methadone maintenance dose in patients with variant genotypes of P-gp. It is possible that less P-gp may be inhibited in patients with variant genotypes of P-gp; therefore, more methadone would be pumped out and, consequently, higher methadone doses may be required to maintain the therapeutic level of methadone.In terms of the effect of Another concern for the variant genotypes of P-gp in patients under methadone maintenance therapy is the treatment of HIV infection, which is a common complication in these patients. Protease inhibitors are the key component in the Highly Active AntiRetroviral Treatment (HAART) for HIV treatment. It has been demonstrated that the widely used protease inhibitors, such as indinavir, lopinavir, nelfinavir, ritonavir and atazanavir, are substrates of P-gp ABCB1 gene may affect both the potency and kinetics of this interaction. Furthermore, methadone was demonstrated to be a P-gp ATPase stimulator under clinical plasma concentration. Although the present study revealed the important methadone-P-gp interaction, lack of clinical data to support the hypothesis of drug-drug interaction is the major limitation. Therefore, further clinical study is needed.The strength of the present study may include 1) unveiling the kinetic mechanism of methadone-P-gp interaction, and 2) providing evidence that variants of the In conclusion, the present study demonstrated that methadone is a human P-gp inhibitor and showed distinctive mechanism in variant-type human P-gp. These results provide basic evidence for drug-drug interactions, which physicians should take into account when combination therapy is applied. Nonetheless, further clinical studies are warranted to confirm the impact of the P-gp mediated drug-drug interactions as well as the unique effect of the variant-type P-gp.


Vibrio cholerae, ubiquitous in aquatic environments, is responsible for cholera; humans can become infected after consuming food and/or water contaminated with the bacterium. The underlying basis of persistence of V. cholerae in the aquatic environment remains poorly understood despite decades of research. We recently described a “persister” phenotype of V. cholerae that survived in nutrient-poor “filter sterilized” lake water (FSLW) in excess of 700-days. Previous reports suggest that microorganisms can assume a growth advantage in stationary phase (GASP) phenotype in response to long-term survival during stationary phase of growth. Here we report a V. cholerae GASP phenotype (GASP-700D) that appeared to result from 700 day-old persister cells stored in glycerol broth at −80°C. The GASP-700D, compared to its wild-type N16961, was defective in motility, produced increased biofilm that was independent of vps (p<0.005) and resistant to oxidative stress when grown specifically in FSLW (p<0.005). We propose that V. cholerae GASP-700D represents cell populations that may better fit and adapt to stressful survival conditions while serving as a critical link in the cycle of cholera transmission.Toxigenic  V. cholerae strains, generally in serogroups O1 and O139, are the cause of epidemic cholera. V. cholerae has two life styles, including transient passage through the human intestine where it causes profuse diarrhea (i.e. cholera), and a second existence in aquatic environments, including fresh, estuarine and marine environments V. cholerae in the environments, particularly during inter-epidemic period, is poorly understood.Cholera is a major public health threat worldwide, particularly in countries where safe drinking water, adequate sanitation and hygiene are suboptimal V. cholerae can enter into a viable but non-culturable state (VBNC) in response to nutrient starvation and/or cold temperature V. cholerae can also switch from a smooth colony type to a “rugose” (wrinkled) variant characterized by copious production of exopolysaccharide conferring resistance to chlorine, osmotic and oxidative stresses V. cholerae in epidemic cholera is limited because not all V. cholerae strains are capable of switching to rugose variant even in a medium promoting high-frequency rugose production In this context, it has been suggested that V. cholerae assume what we have termed a “persister” phenotype in a “filter sterilized” lake water (FSLW) microcosm model V. cholerae N16961S strain grown overnight statically in FSLW at room temperature will be henceforth termed as N16961S-24 (Table1).Amid this conundrum, we recently reported that a subset of culturable V. cholerae the positive association of polar flagellum to biofilm formation has been demonstrated V. cholerae and to compare the differences, if any, between N16961S-24 and GASP-700D, we investigated the role(s) of novel flagella elicited by N16961S-24 and GASP-700D, respectively Persister cells in other human pathogens exhibited biofilm formation conferring resistance to environmental stresses V. cholerae wild-type strain N16961S and its isogenic mutants  used in this study are listed in V. cholerae N16961 persister cells (in excess of 700 days) in “filter sterilized” lake water microcosm model. Briefly, aliquots (500 ml) of lake water were sterilized using Nalgene 0.22 μm membrane filter units , and the microcosms were prepared as follows: 50 ml of “filter sterilized” lake water (FSLW) was transferred into a sterile 250 ml Erlenmeyer flask; for inoculum preparation a single colony of V. cholerae N16961strain, obtained from L-agar grown overnight at 37°C, was inoculated into 3 ml of L-broth. The culture was incubated overnight at 37°C with a shaking speed of 250 rpm, spun down and the resulting pellet was washed 2X in saline (0.85% NaCl), reconstituted in 3 ml saline, appropriately diluted, and 50 μl of diluted culture was inoculated into the microcosm flasks containing 50 ml FSLW. As confirmed by plate counts, initial V. cholerae concentrations in the microcosms ranged from 104 to 106 cfu/ml. The culturable cells from microcosm were determined at intervals using standard plate counts. The 700 day-old cells were subcultured on L-agar and stored in glycerol broth at −80°C. While we cannot be certain that this is true for all GASP-700D cells, we observed that GASP-700D exhibited small colony phenotypes on L-agar for at least four consecutive days of subculture both at room temperature and at 37°C. However, when the cells were inoculated into L-broth and incubated overnight at 37°C with a shaking speed of 250 rpm, a mixture of small and large colonies were observed on L-agar upon plating. All the strains used in this study were subcultured from glycerol stock at −80°C onto L-agar and incubated overnight at 37°C before being used for specific experiments. As needed, antibiotic was added to the bacterial cultures as follows: ampicillin (100 μg/ml) and polymyxin B (50 U/ml).Bacterial strains, including flaA mutant  vpsR were engineered. Each PCR product carries a restriction endonuclease site at its 5′ end; however, 3′-ends of the forward and reverse PCR products carried a common restriction site to facilitate deletion mutation. For vpsR, SacII and XbaI sites were introduced at 5′ and 3′ ends, respectively, of the forward PCR amplicon (560-bp) while 5′ and 3′ ends of reverse PCR product (520-bp) were introduced with XbaI and BamHI sites, respectively. Primers aa212 and aa213 , resulting in a plasmid pAA73 containing a 420-bp internal deletion of vpsR. The plasmid was transformed into DH5α. Subsequently, plasmid pAA73 was digested with SacI and SalI to retrieve a 1080-bp fragment and the fragment was gel purified. The purified fragment was ligated into a similarly digested suicide vector, pCVD442, E. coli S17 λ pir resulting in a plasmid pAA78  were verified by PCR and DNA sequencing as described previously vpsA gene in the background of N16961S, N16961R and GASP-700D, resulting in the mutants AA218, AA219 and AA220, respectively. Primers  used to create null mutation in vpsA are listed in A Δt AA212;  was creat AA212;  using a nd aa213  were useid pAA78 . E. colid AA215, , RΔvpsR,V. cholerae strains was determined using motility agar plates as described previously flaA mutant were grown in L-broth and incubated overnight statically at room temperature. As for FSLW, the strains were first subcultured onto L-agar; a single colony from L-agar was grown in 3 ml L-broth and incubated overnight statically at room temperature. Subsequently, the cultures were spun down at 7,000 rpm for 5 min in a table top centrifuge; the pellet was washed 2X with FSLW and resuspended into 3 ml FSLW and the culture was incubated overnight statically at room temperature. An inoculating wire was dipped into each culture and then stabbed into the motility agar plate. The plates were incubated for 8 h and overnight at 37°C. Zones of migration of bacterial strains around the inoculating sites were measured at 8 h and after overnight incubation of the plates. If no zone was detected, a block of agar was cut around the inoculating site, homogenized in saline (0.85% NaCl), appropriately diluted in saline, and then plated on L-agar to determine if any culturable cells were survived in the inoculation site.Motility of V. cholerae strains, including N16961S-24, N16961R-24 and GASP-700D  and presented relative to V. cholerae N16961S-24 strain that has arbitrarily been taken as 1  in a well of plastic plate; the culture was incubated overnight statically at room temperature. Following overnight incubation the cultures were discarded, and the wells were rinsed vigorously with distilled water to remove non-adherent cells, filled with 600 μl of a 0.1% crystal violet solution , allowed to incubate for 30 min at room temperature, and the wells were again rinsed vigorously with water. Quantitative biofilm formation was determined by measuring the optical density at 570 nm (OD 570) of a solution produced by extracting cell-associated dye with 600 μl of dimethyl sulfoxide (DMSO) .Quantitative assessment of biofilm produced by 00DΔvpsA  were exa00DΔvpsA  were invVibrio cholerae strains, including N16961S-24, SΔvpsR, SΔvpsA, N16961R-24, RΔvpsR, RΔvpsA, GASP-700D, GASP-700DΔvpsR and GASP-700DΔvpsA (8 cfu/ml) in 4-well cell culture plates  containing 500 μl FSLW. To provide bacterial attachment platform, a 12 mm round glass cover slip  was dipped into each culture well, and the cultures were incubated overnight statically at room temperature. Next day, the cover slips were washed three times with Dulbecco’s PBS (DPBS) , and fixed in 10% neutral buffered formalin solution . They were washed again with DPBS and stained using 300 μl/well of 1∶1000 SYTO 9 dye . Following three consecutive DPBS washings, glass cover slips were mounted onto 75×25 mm microscopic slides . The cover slips were analyzed on a confocal microscope  with an excitation and emission wavelengths of 484 and 500 nm, respectively. The biofilm thickness was measured as an average of 5 non-overlapping fields per slide with a 20X HCX PL APO lambda blue magnifying objective. Images were digitally reconstructed with z-projections of x–y sections using Leica Application Suite Advanced Fluorescence  and DAIME softwares To perform confocal microscopic analysis on possible biofilm formation by 00DΔvpsA  were groV. cholerae strains N16961S-24, N16961R-24 and GASP-700D (For transmission electron microscopic (TEM) analysis, ASP-700D  were gro8 cfu/ml) was inoculated in 3 ml FSLW and incubated as described above. For oxidative stress, 20 mM H2O2   was added to each culture and the resistance of each culture to H2O2 was recorded for every 5 min for 15 min. The culturable bacteria survived the stress were determined using standard plate count at each experimental time point. Similarly, for osmotic and chlorine stresses, V. cholerae’s culture in FSLW was exposed to 2.5 M NaCl   and 3 mg free chlorine per liter [3 ppm] , respectively. Resistance of each V. cholerae strain to osmotic and chlorine stresses was determined by measuring the culturable bacteria present (i) for every 15 min for one hour Stress resistance of GASP-700D, including both oxidative and osmotic stresses, and stress to chlorine was assessed as described earlier One-way ANOVA was performed in STATA v 12  to determine the significant differences in diverse traits assessed in the study. Equal variance within groups was assessed using Barlett’s test, and a Bonferroni correction was implemented to control type I error for multiple comparisons between the wild-type and its isogenic mutants or variants. A p-value of <0.005 was considered as statistically significant.Vibrio cholerae carries a single polar flagellum required for its motility. Since we are the first to describe that V. cholerae can switch, in response to nutrient-starvation in FSLW, from a canonical single polar flagellum to bipolar and peritrichous flagella in N16961S-24 and GASP-700D, respectively ΔflaA mutant strain that is non-motile because it lacks the major flagellin subunit V. cholerae (N16961R). When the bacterial strains were grown in L-broth before inoculating into motility agar, both N16961S (smooth variant) and N16961R (rugose variant) were motile , flrC (encodes regulator of Class III flagellar genes), motY and motB (encode flagellar motor) and flrA  were repressed in GASP-700D relative to N16961S-24. A previous study reported that phoB and Pst system genes of V. cholerae were expressed in nutrient-poor FSLW compared to nutrient-rich L-broth, whereas ctxA and tcp genes were repressed under the same conditions As GASP-700D exhibited no motility in soft agar, we further investigated using qRT-PCR to determine whether flagellar genes, including phoB, Pst-system genes, ctxA and tcp genes by N16961S-24 and GASP-700D grown in nutrient-poor FSLW to that of wild-type V. cholerae N16961S grown in nutrient-rich L-broth in otherwise identical growth conditions  in GASP-700D compared to N16961S-24, suggesting that flagellar synthesis is down-regulated in GASP-700D , N16961R (RΔvpsR and RΔvpsA) and GASP-700D (GASP-700DΔvpsR and GASP-700DΔvpsA) in nutrient-rich L-broth incubated overnight statically at room temperature. In L-broth, the rugose N16961R variant produced about 40- and 120-fold more biofilm (p<0.005) than its smooth counterparts N16961S and GASP-700D respectively; as expected, the N16961R mutants, RΔvpsR and RΔvpsA were defective for biofilm formation  under nutrient-poor conditions (FSLW) compared to that of under nutrient-rich conditions (L-broth)  and 1.5-fold (p = 0.24) higher biofilm compared to N16961S-24 and N16961R-24 in FSLW, respectively. In contrast to biofilm production in nutrient-rich L-broth, the ΔvpsR mutants (except for SΔvpsR) and ΔvpsA mutants produced increased biofilm formation (p<0.005) in FSLW compared to N16961S-24. Our observations suggest that GASP-700D and vpsR and vpsA mutants produced biofilm in response to nutrient stress (in FSLW), and that this biofilm is somewhat independent of VPS-mediated biofilm formation as described previously We next examined comparative biofilm forming abilities among the L-broth) , demonstV. cholerae strains in FSLW described above using scanning confocal laser microscopy (SCLM). As shown in vpsR, all ΔvpsR and ΔvpsA mutants examined exhibited patchy biofilms in FSLW with GASP-700DΔvpsR and GASP-700DΔvpsA displayed much higher patchy biofilms  compared to N16961S-24.To gain further insights into the three dimensional and architectural appearances of biofilms, we examined biofilm produced by biofilms . Specifiectively . Figure We stained biofilms formed by N16961S-24, N16961R-24 and GASP-700D in FSLW with ruthenium red, and examined the biofilm matrix using transmission electron microscopy (TEM) . CopiousV. cholerae rugose variants, that produce copious amounts of exopolysaccharide and biofilm, can resist chlorine, oxidative, and osmotic stresses 2O2, chlorine, and NaCl stresses. We note that there were no obvious growth differences among V. cholerae strains grown in L-broth and examined in this study (data not shown). Interestingly, we observed that, like N16961R-24, GASP-700D was more resistant to H2O2 in FSLW (p<0.005) compared to N16961S-24  have transitioned to what appeared to be a growth advantage in stationary phase (GASP) phenotype. Compared to its wild-type strains (N16961S-24 and N16961S), GASP-700D phenotype of V. cholerae exhibited: (i) non-motile phenotype, (ii) enhanced exopolysaccharide production and biofilm formation that are specific to FSLW, and independent of vps, (iii) resistance to oxidative stress, and (iv) small colony phenotype. The storage and subculture of persister cells in glycerol broth at −80°C may have influenced the observed phenotype seen with GASP-700 as described above.Recently, we reported a V. cholerae may have adopted two responses, including: (i) assume “persister” phenotype We hypothesize that, during long-term survival (700 days) in stressful stationary culture, V. cholerae even before its transition to GASP-700D in a phase-dependent manner. The smooth variant exhibited reduced motility in soft agar after 24 h growth in FSLW. In contrast, the rugose variant, which normally shows reduced motility in comparison with the smooth variant, was unaltered for motility after 24 h growth in FSLW. Once the bacteria have transitioned into GASP-700D, however, they appear non-motile in this assay  to select GASP mutants as seen with other microorganisms GASP-700D produces a well-developed biofilm in FSLW that appears predominantly coalesced rather than scattered. In contrast, the rugose variant forms well-developed but scattered biofilms . However mutants . This su in FSLW , in contFigure S1Comparative analysis of the differential gene expression among V. cholerae8 cfu/ml), and the cultures were incubated overnight statically at room temperature. strains N16961S and GASP-700D using qRT-PCR. N16961S was grown both in nutrient-rich L-broth and in nutrient-poor FSLW (N16961S-24) (ca. 10 GASP-700D was grown (ca. 108 cfu/ml) in FSLW only. Expression of each gene was normalized to that of toxR, and subsequently compared to that of the wild-type N16961S grown in L-broth. Data represent the average results of three independent experiments and error bars indicate as means ± standard deviation (SD).(TIF)Click here for additional data file.Table S1Oligonucleotide primers used in this study.(DOCX)Click here for additional data file.


The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the C-terminal domains of HIV-1 RT.We previously demonstrated Full-length RT sequences in plasma obtained pre- and post-therapy were compared in 23 participants who received AZT monotherapy from the AIDS Clinical Trials Group study 175. Five of the 23 participants reached a primary study endpoint. Mutations significantly associated with AZT monotherapy included K70R (p = 0.003) and T215Y (p = 0.013) in the polymerase domain of HIV-1 RT, and A360V (p = 0.041) in the connection domain of HIV-1 RT. HIV-1 drug susceptibility assays demonstrated that A360V, either alone or in combination with thymidine analog mutations, decreased AZT susceptibility in recombinant viruses containing participant-derived full-length RT sequences or site-directed mutant RT. Biochemical studies revealed that A360V enhances the AZT-monophosphate excision activity of purified RT by significantly decreasing the frequency of secondary RNase H cleavage events that reduce the RNA/DNA duplex length and promote template/primer dissociation.The A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance. Zidovudine  was the first antiviral drug approved by the U.S. Food and Drug Administration for the treatment of HIV infection. AZT is a nucleoside reverse transcriptase (RT) inhibitor (NRTI) that, after metabolism by cellular kinases to its triphosphate form (TP) in cells, competes with the natural substrate TTP for binding and incorporation by HIV-1 RT into the nascent viral DNA. Because AZT lacks a 3′-OH on the ribose sugar, its incorporation into viral DNA results in chain-termination.HIV-1 resistance to AZT was first reported in 1989 et al reported that the N348I mutation is highly associated with TAMs, the lamivudine mutation M184V/I, and the non-nucleoside inhibitor resistance mutations K103N and Y181C/I Recent studies have demonstrated that AZT resistance can be increased by mutations in the connection domain of HIV-1 RT. For example, the G333D/E, G335C, N348I, A360V/I, T369V and A371V mutations have all be shown to augment AZT resistance alone or in combination with TAMs in vitro selection experiments by serial passage of HIV-1 in increasing concentrations of AZT to determine whether AZT selects mutations in the connection and/or ribonuclease H (RNase H) domains of RT We previously carried out Pre-therapy and last-on-therapy serum samples were available from 5 participants who reached a primary study endpoint see  on AZT mTo determine whether the A360V mutation conferred AZT resistance, we first cloned full-length RTs from participant samples 4, 11 and 29 into pxxLAI-3D, and selected a single clone for phenotypic analyses. Recombinant virus derived from participant sample 29 did not replicate in cell culture, and was excluded from subsequent analyses. The genotypes of the individual clones and population sequences obtained from the participant serum are shown in TAMs in HIV-1 RT confer AZT resistance by enabling the enzyme to excise the chain-terminating AZT-MP moiety from the 3′-end of the DNA primer using ATP as a phosphate donor et al reported that failure of AZT and nevirapine therapy was associated with the emergence of N348I, TAMs, and the non-nucleoside RT inhibitor resistance mutations K103N, V108I, Y181C/I and G190A/S et al reported that N348I predominantly emerged in participants receiving lamivudine and AZT et al reported that N348I emerged on AZT- and/or ddI-containing therapies Recent studies show that RT inhibitors select for drug resistance mutations in the connection domain of RT including N348I and the A360V mutation reported here. The drug or drugs responsible for the selection of these connection domain mutations, however, are not well defined. For example, Yap in vivo and in the xxLAI molecular clone. This is the first study to take a systematic experimental approach to determine whether AZT alone selects for mutations in the C-terminal domains of HIV-1 RT. As a result, both the selection of A360V during AZT monotherapy and resistance to AZT from A360V are unequivocally shown.In the current study, we compared paired pre- and on-therapy full-length RT sequences from 23 participants who had received only AZT monotherapy in ACTG study 175. Our analyses reveal that the A360V mutation in the connection domain emerged with similar or higher frequency as TAMs M41L, D67N, L210W and K219E/Q. Longitudinal analyses also showed that A360V was detected at the same time or after the appearance of TAMs but not before. Importantly, we also show that the A360V mutation confers ∼2.0-fold AZT resistance both in the full-length RT sequence context it was selected in vitro selection experiments HIV-1 selected for two novel mutations – A371V in the connection domain and Q509L in the RNase H domain – in combination with D67N, K70R and T215F that together conferred greater than 100-fold AZT resistance in vitro experiments cannot always be extrapolated in vivo.We previously reported that in Of note, the N348I mutation was not identified in any of the sequences in this study. Although our sample size is small (N = 23), this is surprising given that N348I and A360V mutations decrease AZT susceptibility to the same extent  supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES buffer, 50 IU/mL of penicillin and 50 µg/mL of streptomycin. The P4/R5 HeLa reporter cell line was maintained in Dulbecco's Modified Eagle Medium, Phenol Red Free  supplemented with 10% FBS, 50 IU/mL of penicillin, 50 µg/mL of streptomycin and 0.5 µg/mL of puromycin . P4/R5 cells were provided by Dr. Nathaniel Landau . MT-2 cells and AZT were obtained from the NIH AIDS Research and Reference Reagent Program. AZT-TP was purchased from TriLink Biotechnologies . RNA and DNA oligonucleotides were synthesized by Integrated DNA Technologies .All samples were obtained from participants enrolled in the AIDS Clinical Trials Group (ACTG) study 175  For the current study, samples were selected from participants who were randomized to receive AZT monotherapy, had reached a study endpoint and for whom stored serum samples were available at pre-therapy (week 0) and from at least 1 of 3 longitudinal time points . Samples were also obtained from participants who were randomized to receive AZT monotherapy but did not reach a study endpoint. HIV-1 RNA in serum was quantified using the Roche Amplicor Monitor HIV-1 Ultrasensitive Assay Kit 1.5 .Taq High Fidelity  using first-round RT-PCR primers: 5′-AGGAAGAT GGAAACCAAAAATGATAG-3′ and 5′-CCTTGACTTTGGG GATTGTAGGGAA-3′ and second round PCR primers: 5′-AGGAAGATGGAAACCAA A AATGATAG-3′ and 5′-CACAGCTGGCTACTATTTCTTTGC- 3′. PCR products were purified with ExoSAP-IT® , population sequenced using the Big Dye terminator kit (v.3.1) on an ABI 3100 automated DNA sequencer  and compared to consensus subtype B RT . Bidirectional sequences were assembled and analyzed using Sequencher 4.9 software . Sequencing peaks that were greater than 25% of the total peak heights were counted as mutations. All sequences were HIV-1 subtype B . RNA was converted to cDNA and full-length HIV-1 RT was amplified using SuperScript™ III One-Step RT-PCR System with Platinum® BI, MluI, HpaI, NgoMIV and SgrAI, respectively (pxxLAI-3D). Next, an RT defective cloning vector pxxLAI-3DΔnp was developed by deletion of RT polymerase domain codons 113 to 315, using restriction sites NsiI to PflmI, as described previously I and SgrAI were first added to the 5′ and 3′ ends of genotyping amplicons using the primers: Bcl-forward (5′-GTTTTATCAAAGTAAGACAGTATGATCAGATAC-3′) and Sgr-reverse (5′- CTACCACCGGTGGCAGGTTA -3′). PCR products and pxxLAI-3DΔnp were digested with BclI and SgrAI to form 1.89 kb full-length RT inserts and a 9.87 kb pxxLAI-3D cloning vector. Vector and full-length RT inserts were ligated with T4 DNA ligase  and transformed into C2925 cells . Individual recombinant clones or bulk plasmid populations were isolated and DNA sequenced to confirm similarity with the RT sequences in the serum sample from which they were derived. To clone the DNA polymerase domain of participant derived RTs into pxxLAI-3DΔnp, the restriction enzyme sites BclI and BstBI were first added to the 5′ and 3′ ends of genotyping amplicons using the primers: Bcl-forward (5′-GTTTTATCAAAGTAAGACAGTATGATCAGATAC-3′) and Bst-reverse (5′- CTATTAAGTCTTTCGAAGGGTCATAATAC -3′). PCR products and pxxLAI-3DΔnp were digested with BclI and BstBI to form 1.08 kb full-length RT inserts and a 10.68 kb pxxLAI-3D cloning vector. Vector and full-length RT inserts were ligated and transformed, as described above. The bulk plasmid population of participant-derived, RT polymerase domain recombinant clones was isolated and DNA sequenced to confirm similarity with the RT sequences in the serum sample from which they were derived.The full-length infectious HIV-1 clone pxxLAI Site-directed mutagenesis  was used to revert the 360 V mutation (codon GTC) to the wildtype amino acid  in participant-derived full-length recombinant clones. Individual recombinant clones were then isolated and sequenced to confirm reversion. Infectious virus for AZT susceptibility assays was generated and titered as described previously D67N/K70R or M41L/L210W/T215Y (TAM-1) mutations in the p6HRT plasmid. The mutated RT sequences were then cloned into pxxLAI and infectious virus was generated and titered as described previously The A360V mutation was also introduced by site-directed mutagenesis  into the backbone of wildtype HIV-1 RT and into RTs that contained the 50) was determined from 3–11 independent experiments.The AZT susceptibility of participant-derived recombinant virus and site-directed mutants were determined in P4/R5 cells as described previously 5′-CCTGTTCGGGCGCCACT GCTAGAGAT-3′) was 5′-radiolabeled with [γ-32P]ATP and chain-terminated with AZT-MP to generate PAZT. PAZT was then annealed to either a 35-nucleotide DNA  or RNA (TRNA: 5′-AGAAUGGAAAAUCUCUAGCAGUGGCGCCCGAACAG-3′) template. Excision assays were carried out by first incubating 20 nM TRNA/PAZT or TDNA/PAZT with 3 mM ATP, 10 mM MgCl2, 1 µM dTTP and 10 µM ddCTP in a buffer containing 50 mM Tris-HCl (pH 7.5) and 50 mM KCl. Reactions were initiated by the addition of 200 nM wildtype or mutant RT. Aliquots were removed at defined times and analyzed as described previously 32P-end-labelled. Assays were carried out using 20 nM TRNA/PAZT, 3 mM ATP and 10 mM MgCl2 in a buffer containing 50 mM Tris-HCl (pH 7.5) and 50 mM KCl. Reactions were initiated by the addition of 200 nM wildtype or mutant HIV-1 RT.HIV-1 RTs containing the A360V, TAM-1 or TAM-1/A360V mutations were purified to homogeneity as described previously 50) were log10 transformed and compared for significant differences  using the two-sample Student's t test.The statistical significance of HIV-1 RNA levels in pre-therapy and last-on-therapy serum samples was calculated using the Mann-Whitney rank sum test. To identify mutations selected by AZT monotherapy, paired pre-therapy and last-on-therapy sequences from each participant were examined for known NRTI resistance mutations in the polymerase domain using the International AIDS Society – USA (IAS-USA) 2010 resistance table Text S1AIDS Clinical Trials Group Study 175 Protocol Team.(DOCX)Click here for additional data file.


Vibrio cholerae, the causative agent of cholera in humans. To form biofilms, V. cholerae produces an extracellular matrix composed of proteins, nucleic acids and a glycoconjugate, termed Vibrio exopolysaccharide (VPS). Here, we present the data on isolation and characterization of the polysaccharide part of the VPS (VPS-PS), which has the following structure: The ability to form biofilms is important for environmental survival, transmission, and infectivity of  Vibrio cholerae is responsible for cholera, a significant disease in developing countries and areas impacted by natural and man-made disasters V. cholerae is able to form biofilms - i.e., matrix-enclosed surface-associated communities, which is critical for it environmental survival, transmission and infectivity V. cholerae forms biofilms on surfaces of phytoplankton, zooplankton, aquatic plants, crustaceans, and insects V. cholerae as clumps or biofilm-like aggregates, and removal of particles >20 µm in diameter from water can decrease cholera incidence by half V. cholerae, by providing nutrients and protection from protozoan grazing and phage predation The facultative human pathogen V. cholerae also forms biofilms while inside infected individuals. Stool samples from cholera patients contain V. cholerae both in biofilm-like clumps and in a planktonic form V. cholerae biofilms are important in the disease process i.e., exopolysaccharides, proteins, and nucleic acids Pseudomonas aeruginosa has the capacity to produce several polysaccharides that contribute to biofilm formation. One of them is alginate, produced by mucoid P. aeruginosa strains isolated from patients with cystic fibrosis. Alginate is a partially O-acetylated unbranched heteropolymer of β- 1–4 linked D-mannuronic acid and L-guluronic acid residues. P. aeruginosa strains with enhanced alginate production form biofilms that have highly structured architecture,and are resistant to the antibiotic tobramycin P. aeruginosa strains produce Psl and/or Pel. Psl polysaccaride is consist of a branched pentasaccharide repeating unit composed of D-mannose, D-glucose and L-rhamnose residues Biofilm formation depends on production of a biofilm matrix Staphylococcus aureus and Staphylococcus epidermidis biofilm development E. coliPolysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG), consisting of partially N-deacylated poly-1,6-β-GlcNAc, plays critical roles in Vibrio fischeri uses symbiosis polysaccharide biofilms,Syp, to colonize its host, the squid Euprymna scolopesV. parahaemolyticus polysaccharide CPSA contains approximately equal amounts of fucose, galactose, glucose and N-acetylglucosamine and is critical for biofilm formation The importance of exopolysaccharides in biofilm formation has also been recognized in Vibrio spp. V. cholerae biofilm matrix is Vibrio exopolysaccharide (VPS) V. cholerae O1 El Tor  vps genes and phenotypic analysis of vps mutants has revealed that most of the genes of the vps clusters are required for VPS biosynthesis and biofilm formation in vivo biofilm formation V. cholerae’s life cycle.One of the major components of V. cholerae O1 El Tor V. cholerae O1 El Tor rugose variant, A1552R, secreting high levels of VPS V. cholerae biofilm matrix sets the stage for further structure-function analysis of the biofilm matrix and will facilitate identification of targets to combat this deadly pathogen.We previoulsy reported composition and linkage analysis of the crude VPS preparations from rugose variant of Vibrio cholerae rugose variant, A1552R (FY_VC_2), which produces high level of VPS Cells of the To isolate biofilm matrix, overnight-grown cultures were spread on cellulose dialysis membranes placed on the surface of M9 agar plates (150×15 mm) supplemented with glucose (10 mM), casamino acids (0.5%) and MEM vitamins and incubated for 48 h at 30°C. Bacteria from these plates are harvested and suspended in phosphate-buffered saline (PBS). The samples are shaken on a rotary shaker at 4°C for 24 h. During this time, the biofilm matrix separates from the cells and crude biofilm matrix was isolated and processed as described below.Biofilm cells were collected by centrifugation . The supernatant was clarified with additional centrifugation  and dialyzed for 2 days against distilled water containing 0.02% sodium azide. We used cellulose tubing MWCO 12–14 kDa, with repeated change of the water The dialyzed sample was lyophilized to give crude VPS extract . This material was dissolved in water at about 1 mg/mL  and treated with DNAse, RNAse , and then Proteinase K  lipopolysaccharide (LPS) was then removed by ultracentrifugation at 150 000 g for 3 h, solution dialyzed against water for 3 days, to give clear and not very viscous solution, which was lyophilized to give VPS. The yield from the material after enzymatic treatment was about 50%.VPS (50 mg) was dissolved in 37% HCl (2 mL) to give the clear solution a violet color. It was kept for 15 min at room temperature, poured into 100 mL of water and lyophilized. Dried material was washed with water; the precipitate was removed by centrifugation; and the solution passed through a Biogel P6 column to produce polymeric material. This contained VPS-PS and a small amount of the O-polysaccharide, which was removed by anion-exchange chromatography on Hitrap Q column in a gradient of NaCl from 0 to 1 M in 1 h. The VPS-PS was eluted as a broad peak over 10 min and collected in 3 parts, each of which had the same purity and structure. The yield of the VPS-PS from VPS was about 50–70%. A small amount of water-insoluble material was obtained, which we were able to partially dissolve in chloroform, with the rest soluble in DMSO.1H) spectrometer with 3 mm gradient probe at 25–50°C with acetone internal reference (2.225 ppm for 1H and 31.45 ppm for 13C), using standard pulse sequences gCOSY (gradient COrrelation SpectroscopY), TOCSY  (mixing time 120 ms), NOESY (Nuclear Overhauser Effect Spectroscopy) (mixing time 300 ms), gHSQC (gradient Heteronuclear Single Quantum Coherence), and gHMBC (gradient Heteronuclear Multiple Bond Coherence) (100 ms long range transfer delay). AQ time was kept at 0.8–1 sec for H-H correlations and 0.25 sec for HSQC. 256 increments were acquired for t1 in all 2D spectra, except 512 for gCOSY.NMR experiments were carried out on a Varian INOVA 500 MHz  or a Biogel P6 column (2.5×60 cm) in pyridine-acetic acid buffer (4 mL:10 mL:1 L water), and monitored by refractive index detector (Gilson). Anion exchange chromatography was done on an Hitrap Q column , with UV monitoring at 220 nm in a linear gradient of NaCl  at the 3 mL/min. Fractions of 1 min were collected and additionally tested for carbohydrates, by spotting on an SiO−1.Samples with added inositol standard were hydrolyzed with 3 M TFA at 120°C. Monosaccharides were converted to alditol acetates by conventional methods and identified by GC-MS on a Trace GC Ultra apparatus, coupled with a DSQ II Mass spectrometer (Thermo Scientific), and equipped with a TR-5MS capillary column (30 m×0.25 mm ID×0.25 µm film) with helium carrier gas, using a temperature gradient 170°C (3 min), 250°C at 5°C·min2O - 0.3 mL pyridine , dried again using a stream of air, and analyzed by GC-MS on a Varian Saturn 2000 instrument (EI-ion trap) on a DB-17 capillary column, at a temperature gradient of 170–270°C at 4°C/min.A sample (<0.5 mg) in methanol (0.5 mL) was cooled in dry ice and 0.05 mL of acetyl chloride was added, the vial closed and heated at 90°C for 3 h, dried and acetylated with 0.3 mL AcR)-2-BuOH (0.2 mL) and acetyl chloride (0.02 mL) were added at room temperature, heated at 90°C for 2 h, dried by air stream, acetylated, analyzed by GC-MS as described above. Standards were prepared from monosaccharides of known configuration with (R)- and (S)-2-BuOH.To the polysaccharide sample (0.2 mg) (4 (20 mg) was added. This solution was kept for 24 h. Ethylene glycol (0.2 mL) and the excess NaBD4 were added, with the solution kept for 1 h, treated with 0.2 mL of AcOH, and desalted on a Sephadex G-15 column. The product was hydrolyzed with 2% AcOH 2 h at 100°C, and desalted on a Sephadex G-15 column to give OS1.VPS-PS (10 mg) was dissolved in water (2 mL) and NaIOV. cholerae A1552R VPS was first identified and characterized in a rugose variant of Vibrio cholerae rugose variant and an isogenic strain lacking vps gene clusters vps-I and vps-II (Δ-VPS) were grown in a chemically defined medium which did not contain HMW carbohydrates. The crude extracellular (EC) material was recovered from the biofilm matrix. Aqueous solutions of the crude EC material of the rugose strain were very viscous, 100 mg of the material dissolved in 50 mL of water produced gel-like solution. The viscosity of a corresponding sample from the Δ-VPS strain was much less pronounced. Crude r-VPS had a higher carbohydrate/protein ratio than the corresponding Δ-VPS sample , respectively). UV measurements (260 and 280 nm) of the non-dialyzable extracellular material indicated that the protein-nucleic acid content was about 30%. NMR spectra of the whole high-molecular mass extracellular material showed signals characteristic for proteins, lipids, and nucleic acids. No significant signals of the anomeric protons of sugars (4.5–6 ppm) were observed in 1H NMR spectra at neutral, acidic or basic pH  were tried in order to isolate the polysaccharide part of the VPS so that it could be used for further analysis, all with no success. Alkaline treatment produced a small amount of partially depolymerized VPS-PS (polysaccharide part of VPS), which was initally used for the structural analysis. Occasionally, it was found that treatment of the VPS with 37% HCl at room temperature for a short time releases a polysaccharide VPS-PS, suitable for NMR analysis.V. cholerae O1 LPS O-chain. VPS-PS was eluted along a NaCl gradient, resulting in several broad peaks. Three fractions were collected. Each of the fractions had identical NMR spectra, and probably differed in molecular mass. Monosaccharide analysis of the VPS-PS showed the presence of glucose, galactose, and a smaller amount of glucosamine. All these monosaccharides had a D-configuration, determined by GC of 2-butyl glycoside acetates.VPS-PS was additionally purified by anion-exchange chromatography to remove minor impurities of the typical P. aeruginosa PA14 O-specific polysaccharide VPS-PS structure was analyzed using 2D NMR spectroscopy. Four major monosaccharide spin-systems were identified in the spectra. Three of them belonged to α-Gal, α-Glc and β-Glc , Table 1The H-3 signal of the aminoguluronic acid residue B was shifted to the low-field (5.21 ppm), due to the O-acetylation at O-3. A minor variant of the non-O-acetylated residue B was also observed in the spectra. The O-acetyl signal was observed at 2.19/22.0 ppm (H/C).The HSQC spectrum of the VPS-PS contained a set of non-sugar signals at 3.9–4 ppm (H)/43.4 ppm (C), correlating to a carbonyl signal at 175.5 ppm. Position of these signals indicate a glycine spin system. Glycine was linked to GulNAcA by the amide linkage between GulNAcA C-6 and Gly N-2, which was confirmed by the observation of the HMBC correlation between Gly H-2 and GulNAcA C-6 at 172.2 ppm. The presence of an amide was also confirmed by the recording of the HSQC spectra at different pH , which showed that GulNAcA signals were not sensitive to acidity and, consequently, its carboxyl group cannot ionize due to amidation.The sequence of the monosaccharides was determined using NOESY data, where the following correlations were observed: A1:B4; B1:E3,4,5; D1:A4; E1:D4, indicating the sequence as shown on 1H NMR spectra of VPS-PS contained several small signals in the anomeric region  led to the disappearance of nearly all minor signals . The nature of this lipid remains unknown.Cleavage of the VPS using concentrated HCl produced a minor amount of the precipitate, which was partially dissolved in chloroform and the rest in DMSO. However, NMR spectra of these compounds were not informative. They contained large signals of fatty acids and messy small signals elsewhere. GC analysis of methanolysis products of the precipitate led to the identification of C16, Δ-C16, C18, and Δ-C18 acids. Acetylated methanolysate contained additionally derivatives of the monosaccharides found in VPS-PS, but no acetylated hydroxy-fatty acids. MALDI mass spectrum of the precipitate showed two major peaks at m/z 730 and 758. The mass difference of 28 indicated that signals belong to a lipid  encodes a protein predicted to function as UDP-glucose/GDP-mannose dehydrogenase, which catalyzes formation of UDP-glucuronic acid or GDP-mannuronic acid. This is in good agreement with the presence of the acidic sugar GulNAcA in the VPS structure. Two genes involved in vps biosynthesis operon - vpsC (VC0919) and vpsG (VC0923) - are predicted to encode serine acetyltransferase-related proteins that may be involved in VPS O-acetylation. The presence of GlcNAc is consistent with our previous report showing that VPS could be stained with a Cy3-labeled wheat germ agglutinin, which recognizes GlcNAc VPS-PS contains a novel monosaccharide, 2-acetamido-2-deoxy-L-guluronic acid, which is present in the polymer as its amide with glycine - another unusual component of natural polysaccharides. 2-Acetamido-2-deoxy-L-guluronic acid could be biosynthesized from 2-acetamido-2-deoxy-D-mannuronic acid by the enzymatic inversion of C-5 configuration, like it happens in the conversion of D-mannuronic acid into L-guluronic acid in alginate biosynthesis V. cholerae: O139 (Different structures of capsular polysaccharides have been reported for two strains of V. cholerae biofilms as part of the pathogen’s normal transmission route. The presence of the VPS in these biofilms may influence progression of the disease, and/or development of an immune response against V. cholerae. Thus, characterization of the complete chemical structure of VPS and better understanding of VPS biosynthesis could lead to development of inhibitors of biofilm formation and, thus, reduce transmission of the pathogen.Humans ingest


SH2D1A/SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis protein (XIAP) genes, respectively. The diagnosis of XLP in individuals with EBV-associated HLH (EBV-HLH) is generally difficult because they show basically similar symptoms to sporadic EBV-HLH. Although EBV-infected cells in sporadic EBV-HLH are known to be mainly in CD8+ T cells, the cell-type of EBV-infected cells in EBV-HLH seen in XLP patients remains undetermined.X-linked lymphoproliferative syndrome (XLP) is a rare inherited immunodeficiency by an extreme vulnerability to Epstein-Barr virus (EBV) infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP are now divided into type 1 (XLP-1) and type 2 (XLP-2), which are caused by mutations of in situ hybridization or quantitative PCR methods.EBV-infected cells in two patients (XLP-1 and XLP-2) presenting EBV-HLH were evaluated by in EBER-1 + B cells, whereas EBV-infected CD8+ T cells were very few.Both XLP patients showed that the dominant population of EBV-infected cells was CD19In XLP-related EBV-HLH, EBV-infected cells appear to be predominantly B cells. B cell directed therapy such as rituximab may be a valuable option in the treatment of EBV-HLH in XLP patients. SH2D1A/SLAM-associated protein (SAP) gene in 1998 [X-linked inhibitor of apoptosis protein (XIAP) gene can also lead to the clinical phenotype of XLP in 2006 [Hemophagocytic lymphohistiocytosis (HLH) is clinically characterized by prolonged fever, hepatosplenomegaly, hypertriglyceridemia, systemic hypercytokinemia and cytopenia . HLH con in 1998 -6, and m in 2006 . XLP is + T cells, are the primary cellular target of EBV infection in sporadic EBV-HLH [+ T cells in sporadic EBV-HLH. Patients with sporadic EBV-HLH are usually treated with immunochemotherapy, including dexamethasone, cyclosporine A and etoposide, and this therapy can be curable [+ B cells, which appears to be distinct from sporadic EBV-HLH cases.In addition to B cells, EBV can infect other cell types, including epithelial cells, T cells and natural killer (NK) cells . Studies EBV-HLH ,10, whic curable . In cont curable . It is p2 copies/ml to 1.4 × 105, 5.7 × 103 and 1.4 ×106 copies/ml in patients 1, 2 and 3, respectively. Blood samples from the patients were obtained using standard ethical procedures with the approval of the Ethics Committee of the University of Toyama, and an analysis of the SH2D1A and XIAP genes was performed. Patient 1 showed a one-nucleotide insertion (239_240insA) in the SH2D1A gene that resulted in a frameshift and a premature stop codon (80KfsX22). Patient 2 carried a two-nucleotides deletion (1021_1022delAA) in the XIAP gene that resulted in a frameshift and a premature stop codon (N341YfsX7). Patient 3 had no mutations in the SH2D1A or XIAP gene.Three patients presented with clinical features of HLH, including persistent fever, hepatosplenomegaly, cytopenia, abnormal liver function, hyperferritinemia and elevated levels of soluble interleukin-2-receptor Table . The cli+ B cells, CD56+ NK cells, CD4+ T cells and CD8+ T cells were purified by positive selection from the lymphocytes using the respective mAb-coated magnetic beads. The purity of each isolated cell population was assessed by flow cytometriy analysis, and each sorted population was found to be higher than 85% pure.Peripheral blood mononuclear cells (PBMCs) were isolated from the the patients using Ficoll-Hypaque gradient centrifugation. Lymphocytes were prepared from the PBMCs by depleting the monocytes using anti-CD14 monoclonal antibody (mAb)-coated magnetic beads  . The CD1in situ hybridization (ISH) method as described previously [The presence of EBV was estimated by measuring the EBV-encoded small RNA 1 (EBER-1) mRNA using the eviously . The sorBALF5 gene, which encodes the viral DNA polymerase. The primers for amplifying the BALF5 gene sequences were as follows: forward, CGGAAGCCCTCTGGACTTC, and reverse, CCCTGTT TATCCGATGGAATG. The TaqMan probe was FAM-TATACACGCACGAGAAATGCGCC-BFQ. The PCR conditions were as follows: denaturation at 95°C for 2 minutes, annealing at 58°C for 15 seconds, and extension at 72°C for 15 seconds, and the products were subjected to 45 cycles of PCR amplification. The EBV DNA copy number was considered to be significant when more than 500 copies/μg of DNA were observed.Quantitative PCR was performed using AmpliTaq Gold and a real-time PCR 7300 system  as described previously . The PCR+ or CD8+ cells for each receptor family.Flow cytometry analysis of the T cell receptor (TCR) Vβ repertoire was performed as described previously . In brie+ T cells, CD8+ T cells, CD19+ B cells and CD56+ NK cells were sorted using the immunomagnetic bead method and the presence of EBV was evaluated in each lymphocyte subpopulation by EBER-1 ISH   Figure .+ T cells, CD8+ T cells, CD19+ B cells and CD56+ NK cells in patient 2 were determined by quantitative PCR. The number of EBV DNA genome copies in the CD19+ B cells was 1.8 × 104 copies/μg, and the copy number in the CD8+ T cells was 1.0 × 103 copies/μg. The EBV DNA genome could not be detected in either the CD4+ T cells or the CD56+ NK cells that were isolated from patient 2.The viral loads in the CD4+ T cells of patients with sporadic EBV-HLH [+ T cells was demonstrated by mAb in patient 3, but TCR Vβ13.3 was predominantly found in the CD8+ T cells by complentarity-determining region 3 spectratyping [Flow cytometry analysis of the TCR Vβ repertoire revealed a polyclonal pattern in patients 1 and 2  was performed in patients with XLP-1 . Two XLPIn situ Hybridization; mAb: Monoclonal antibody; NK: Natural killer; PBMC: Peripheral blood mononuclear cells; SAP: SLAM-associated protein; TCR: T cell receptor; XIAP: X-linked inhibitor of apoptosis; XLP: X-linked lymphoproliferative syndrome.EBER: EBV-encoded small RNA; EBV: Epstein-Barr virus; HLH: Hemophagocytic lymphohistiocytosis; HSCT: Hematopoietic stem cell transplantation; ISH: The authors declare that they have no competing interests.XY and HK wrote the manuscript. XY, TW, KI and NN performed the experimental studies. TM, MF, HK and FK managed the patients' care. SF, AY, XDZ and TM revised the manuscript. XY, TW and KI contributed equally to this study. All the authors read and approved the final manuscript.


After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G Single-chain Fv antibody (scFv) is composed of immunoglobulin heavy- and light-chain variable regions connected by a short peptide linker -3. ScFv 4S)n are the most widely used  primer mix, and RS primer mix were purchased from Pharmacia Biotech.IC-2 and CCL-187 cells were cultured using standard conditions. IC-2 is a murine hybridism cell line that secretes the monoclonal antibody ND-1, specific for human colorectal carcinoma. CCL-187 is a human colorectal carcinoma cell line. The pET28a (+) expression vector and 6 IC-2 hybridism cells and cDNA synthesized by reverse transcription using random primers. VH and VL genes were separately amplified from cDNA by PCR using a heavy and light chain primer mix. The VH and VL gene fragments were recovered and mixed in equimolar ratios for two PCR reactions, with the first one using a linker primer mix for 7 cycles, followed by a second one using a RS primer mix for 30 cycles. As a result, VH and VL gene fragments were linked to form a scFv construct by extension, with overlapping splicing PCR. The resulting ND-1 scFv-n construct was cloned into pMD18-T and transformed into E. coli JM109, and positive clones identified by colony PCR and DNA sequencing.ND-1 scFv-n was constructed as previously described. Briefly, mRNA was extracted from 5 × 10EcoRI sites at the 5'-end of ND-1scFv-n, and a HindIII site, or SalI site at the 3'-end. S1: 5'-CTGAATTCATGGCCCAGGTGCAGCTGCAGC-3'; S2: 5'-CGCAAGCTTCTAGTCGACTTTCCAGCTTGGTC-3'. pMD18-T-ND-1scFv-n was used as a template, and the product cloned into the vector pET28a(+) after digestion with EcoRIand HindIII, and transformed into competent E.coli BL21 cells for protein expression.Oligonucleotide primers S1 and S2 were designed to add 4S)n-VL is:The amino acid sequence of the wild-type VH and wild-type VL are listed below , and illMAQVQLQQSGPGLVAPSQSLSITCTVSGFSLTTYDVHWVRQPPRKGLEWLGLVWANGRTNCTSALMSRISITRDTSKNQVFLTMNSLQTDDTAMYYCARGSYGAVDFWGQGTTVTVSS(GGGGS)nDIELTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK.The amino acid sequence of a protein determines its high-level structure. Determining high-level protein structure relies on the identification of one or more known protein "templates" that resemble the structure of the query sequence, and alignment of the query sequence residues to the template residues. Swiss-Models can be used for homology modeling to search protein sequence and structure databases, such as the Protein Data Bank (PDB) -21. A thPDB files were obtained from Swiss-Model with the videotext coordinate system (in which the atomic coordinates are located), in order to facilitate protein structure comparison. The coordinate systems were constructed with Matlab7.0.The molecular weight of the atoms in the protein was used to calculate molecular weight, and the centric was obtained using the atomic location of each atom. The centric is the origin of the new coordinate system .N: the number of all the atoms; M = 12.01 + 14.01 + 16.00 + 32.07 + 1.00;Mi: molecular weight of atoms;k, Yk, Zk]: the original three-dimensional coordinates of atoms;: the origin of the new coordinate system.: 3D coordinates of each atom.[XS were calculated, and the eigenvector calculated corresponding to the maximum eigenvalue as the first axis , with the eigenvector corresponding to the second largest eigenvalue as the second axis , and similarly for the Z axis.The eigenvalues and eigenvectors of The End-to-end distance is the distance between the first and the last α-carbon atom in a protein. We obtained this information and the X/Y/Z coordinates of the atoms from the PDB database. The algorithm used is as follows:4S)n.A. Locate the first and last α-carbon atoms in the wild-type VH and VL, and the same in the protein after introduction of (G4S)n.B. Calculate End-to-end distance of wild-type VH (VL) and mutant VH (VL) after introduction of -ND-1scFv-n plasmids were constructed as expression vectors and transformed into n proteins were detected by western blot analysis. BL21 transformed with pET-28a(+)ND-1scFv-n was incubated separately in loading buffer  for 5 min at 100°C, separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and electro blotted onto PVDF membrane . Non-specific binding sites were blocked for 1 h with 5% nonfat milk in TPBS (PBS contained 0.05% Twin 20), and the membrane incubated overnight at 4°C with primary antibody. After washing 3X in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG for 2 h at room temperature, and washed 2X with TPBS. Immunoblot signal was detected by autoradiography using an enhanced chemiluminescence detection kit.ND-1scFv-4) were grown in 96-well micro titer plates at 37°C for 24 h, fixed with 2.5% glutaraldehyde and blocked with 1% BSA, followed by incubation with ND-1IgG or ND-1scFv at 37°C for 2 h. After washing 3X with PBS, anti-His6 antibody was added to wells with ND-1scFv-n and incubated. The plate was washed and HRP-labeled goat anti-mouse IgG was added into both ND-IgG and ND-1scFv wells. After incubating at 37°C for 2 h, TMB substrate was added, and samples incubated in darkness for 30 min. The reaction was terminated with 1 M H2SO4. PBS was used as a negative control.CCL-187 cells  on VH and VL can be represented in the following equation: r were obtained from the corresponding linker length. The r-values were 36.8161, 8.0150, 0.8415, 22.1579, 24.4747, 582.2451, 46.8344, 88.6852, and 112.3846, with a median value of 24.4747.The End-to-end distance of VH (AB), VL (CD) and linker (BC), at different n = 3, the r-value was the smallest, and the structure of fusion proteins was closest to the ideal state. The r-values increased when n increased and hence the linker length increased, in which VH and VL structure would be impacted to a greater extent. When n was 6, the r value was the most unsatisfactory.The results suggest that when E. coli BL21, and protein expression induced with IPTG. Western blot analysis indicated that BL21 lysates expressed scFv-n proteins with bands of 30 kDa  were transformed into a Figure . The seqThe immunoreactivity of purified ND-1scFv-n was determined by ELISA. scFv-n exhibits an immunoreactivity similar to the parental ND-1 antibody, and demonstrated good binding to CCL-187 cells expressing colorectal carcinoma associated antigen LEA. This suggests that scFv-n retains good specificity and activity.450 value) and r-values. The immunoreactivity declined with increasing r-values. It changed significantly when the r-value was less than 42.3716. When the r-value exceeded this value, immunoreactivity became relatively stable  ,28. HoweIt is recognized that fusion proteins have varied affinity and anti-tumor activity compared to the original molecules, due in large part to the structural alterations of the fusion proteins ,28-31. T4S)n as tethers. Linkers comprising repeats of G4S have been used to construct bivalent single-chain antibodies targeting colorectal cancer with linkers of 5-15 amino acids [In previous studies -37, the no acids ,36. With4S)n on the structure and function of VH and VL, and to find the relationship between VH/VL’s End-to-end distance and n (or BC) on bivalent single-chain antibodies targeting colorectal cancer. A multi-factor relationship model was established to evaluate VH and VL structural factors using the following formula: r-values. The immunoreactivity declines as the r-value increases. Fusion protein structure is ideal when the r = 3. When the n value is 6, protein structure is least satisfactory. However, further exploration of this relationship is needed. Indeed, the expression level and activity of scFv depends largely on the length and sequence of linker. Thus, successful construction of a scFv depends on the selection of a linker that neither interferes with the folding and association of VH and VL domains nor reduces the stability and recognition abilities of the Fv molecule.It should be pointed out that the impact of linker length on the activity and affinity of engineered antibodies depends strongly on the distance between the N- and C-terminal of the VH domain . A certaIn summary, based on the databases of natural protein structures and their associated functions, we predicted the structure and function of fusion proteins by homology modeling and further conducted biological experiments to validate our calculations. Thus, a dual approach that incorporates molecular modeling and linker design of engineered antibodies with quantitative determination of antibody affinity is useful to optimize construction. Our approach provides not only a rationale for designing novel engineered antibodies using molecular modeling, but also provides new insight into quantifying antibody binding affinity, especially at low protein concentration. A combination of bioinformatics and genetic research may therefore be beneficial in exploring new agents for genetic engineering of antibodies.scFv: Single-chain Fv antibody; MCS: Multi-cloning site; PCA: Principal component analysis.The author(s) declare that they have no competing interests.JH Zhang drafted the manuscript. ZG Shang participated in the design of the study and performed the statistical analysis. L Shi conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.