Cotransfected GFP diffusely stained axons (bottom panel). Fig. S3. Cbln1 or Cbln2 directly causes clustering

of NRX1β(S4+). Beads coated with HA-Cbln1, Cbln2, Cbln4, or CS-Cbln1 were incubated with HEK293 cells expressing NRX1β(S4+) for 2 days. Confocal images of HEK293 cells immunostained against NRX1β(S4+) (red or white) and beads (green) are shown. Scale bar, 25 μm. Fig. S4. Cbln1 serves as a direct presynaptic organizer in hippocampal neurons. (A) Accumulation of Navitoclax clinical trial functional presynaptic sites labeled with FM4-64 (red) around HA-Cbln1-coated beads (green). Scale bar, 20 μm. () Presynaptic sites were directly induced by HA-Cbln1-coated beads. Synapsin I-immunopositive terminals (red) were induced around HA-Cbln1-coated beads (arrowheads), which were located at extrasynaptic sites lacking endogenous AMPA receptors (detected by

anti-pan AMPA receptor antibody; green). Scale bar, 20 μm. Fig. S5. Cbln1 and Cbln2 but not Cbln4 induced presynaptic differentiation of cortical neurons. Beads coated with HA-Cbln1, Cbln2, Cbln4, or CS-Cbln1 were cocultured with cortical neurons. Confocal images of neurons immunostained for synapsin I (red or white) and beads (green) are shown. Scale bar, 20 μm. As selleck inhibitor a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset by Wiley-Blackwell. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. “
“Alzheimer’s disease (AD) is the most common dementia-causing disorder in the elderly; it may be related to multiple risk factors, and is characterized pathologically by cerebral hypometabolism, paravascular β-amyloid peptide (Aβ) plaques, neuritic dystrophy, and intra-neuronal aggregation of phosphorylated tau. To explore

potential pathogenic links among some of these lesions, we examined β-secretase-1 (BACE1) alterations relative to Aβ deposition, neuritic pathology Metformin purchase and vascular organization in aged monkey and AD human cerebral cortex. Western blot analyses detected increased levels of BACE1 protein and β-site-cleavage amyloid precursor protein C-terminal fragments in plaque-bearing human and monkey cortex relative to controls. In immunohistochemistry, locally elevated BACE1 immunoreactivity (IR) occurred in AD but not in control human cortex, with a trend for increased overall density among cases with greater plaque pathology. In double-labeling preparations, BACE1 IR colocalized with immunolabeling for Aβ but not for phosphorylated tau. In perfusion-fixed monkey cortex, locally increased BACE1 IR co-existed with intra-axonal and extracellular Aβ IR among virtually all neuritic plaques, ranging from primitive to typical cored forms.

Results: 

Overall, 22.3% of participants indicated that they had pain, aching or stiffness in either of their shoulders. Women, those aged 50 years and over, current smokers and those classified as obese were all significantly more likely to report shoulder pain. Respondents with shoulder pain scored lower on all domains of the SF36. In those with shoulder symptoms, women had more severe pain and worse shoulder function than men, and older people had worse shoulder function than younger people. Conclusion:  Shoulder pain affects almost a quarter of people in the Australian community, see more with a significant detrimental impact on health-related quality of life and physical functioning. “
“The presence of the lupus erythematosus (LE) phenomenon has been generally conceptualized as an in vitro occurrence where numerous damaged cells are present and substantial nucleo-phagocytosis has occurred. In systemic lupus erythematosus (SLE), the positive LE cell phenomenon

selleck screening library has been shown to indicate active disease with major organ involvement which potentially warrants prompt and heavy immunosuppressive therapy. We report a 36-year-old woman with a known history of SLE who presented with fever, left knee effusion, polyserositis, pancytopenia, low complement and high anti-dsDNA antibody levels whose immunosuppressive treatment was escalated in view of the clinically and serologically active SLE, accompanied by the presence of LE cells in her inflammatory yet sterile left knee synovial fluid. Within 3 days of immunosuppressant escalation, her ascites worsened. While microscopic examination of the ascitic fluid also revealed LE cells, culture of the ascitic fluid later grew Candida parapsilosis. The patient subsequently responded to the addition of anti-fungal therapy into her augmented immunosuppressive regime. Coexistence of the LE cell phenomenon and infection tuclazepam in SLE patients has hitherto not been described. This case illustrates that infection remains to be meticulously excluded despite

the presence of the LE phenomenon in the context of clinically and serologically active SLE. “
“We aimed to investigate serum cystatin C (cysC) levels in primary Sjögren’s syndrome (pSS) patients, and evaluate its correlation with renal involment. Eighty-six pSS patients and 65 age- and gender-matched healthy controls were enrolled into the study. Serum cysC, urea, serum creatinine (SCr), creatinine clearance (CrCl), glomerular filtration rates (GFR), Na, K, Mg, Ca, uric acid, P, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), anti-Ro/SS-A, anti-La/SS-B, antinuclear antibodies, 24-h urinary poteinuria and microalbuminuria were evaluated. Mean serum cysC levels did not differ between the patients and healthy controls (P > 0.05). Nine patients with pSS had proteinuria over 150 mg (and microalbuminuria over 30 mg) per 24 h. In patients with proteinuria, serum cysC levels correlated with serum K (r = 0.279, P = 0.024), ESR (r = 0.

Results: 

Overall, 22.3% of participants indicated that they had pain, aching or stiffness in either of their shoulders. Women, those aged 50 years and over, current smokers and those classified as obese were all significantly more likely to report shoulder pain. Respondents with shoulder pain scored lower on all domains of the SF36. In those with shoulder symptoms, women had more severe pain and worse shoulder function than men, and older people had worse shoulder function than younger people. Conclusion:  Shoulder pain affects almost a quarter of people in the Australian community, find more with a significant detrimental impact on health-related quality of life and physical functioning. “
“The presence of the lupus erythematosus (LE) phenomenon has been generally conceptualized as an in vitro occurrence where numerous damaged cells are present and substantial nucleo-phagocytosis has occurred. In systemic lupus erythematosus (SLE), the positive LE cell phenomenon

learn more has been shown to indicate active disease with major organ involvement which potentially warrants prompt and heavy immunosuppressive therapy. We report a 36-year-old woman with a known history of SLE who presented with fever, left knee effusion, polyserositis, pancytopenia, low complement and high anti-dsDNA antibody levels whose immunosuppressive treatment was escalated in view of the clinically and serologically active SLE, accompanied by the presence of LE cells in her inflammatory yet sterile left knee synovial fluid. Within 3 days of immunosuppressant escalation, her ascites worsened. While microscopic examination of the ascitic fluid also revealed LE cells, culture of the ascitic fluid later grew Candida parapsilosis. The patient subsequently responded to the addition of anti-fungal therapy into her augmented immunosuppressive regime. Coexistence of the LE cell phenomenon and infection either in SLE patients has hitherto not been described. This case illustrates that infection remains to be meticulously excluded despite

the presence of the LE phenomenon in the context of clinically and serologically active SLE. “
“We aimed to investigate serum cystatin C (cysC) levels in primary Sjögren’s syndrome (pSS) patients, and evaluate its correlation with renal involment. Eighty-six pSS patients and 65 age- and gender-matched healthy controls were enrolled into the study. Serum cysC, urea, serum creatinine (SCr), creatinine clearance (CrCl), glomerular filtration rates (GFR), Na, K, Mg, Ca, uric acid, P, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), anti-Ro/SS-A, anti-La/SS-B, antinuclear antibodies, 24-h urinary poteinuria and microalbuminuria were evaluated. Mean serum cysC levels did not differ between the patients and healthy controls (P > 0.05). Nine patients with pSS had proteinuria over 150 mg (and microalbuminuria over 30 mg) per 24 h. In patients with proteinuria, serum cysC levels correlated with serum K (r = 0.279, P = 0.024), ESR (r = 0.

Direct and inverted repeat regions were identified with the Repseek software integrated in the MaGe platform (Achaz

et al., 2007). To insertionally inactivate xbpS1, a 736 base pair internal fragment located near the 5′ end of the gene was amplified and subsequently ligated into the EcoRV site of pSTBlue-1 (Novagen). The xbpS1 fragment from the recombinant plasmid was ligated into the PstI-XbaI sites of the conjugal suicide vector pKnock-Cm. The resultant plasmid was transformed into E. coli S17-λpir and subsequently transferred into X. bovienii-SF43. The xbpS1 mutant strain (SF70) was selected on LB supplemented with ampicillin (50 μg mL−1) and chloramphenicol (25 μg mL−1), and gene disruption was confirmed by PCR. The xenorhabdicin activity assay was performed as described previously (Morales-Soto & Forst, 2011). SF31 and TT01 strains (Table 1) were separately subcultured in 5 mL of PF 2341066 LB and grown at 30 °C to an OD600 nm of 0.5–0.6. Cultures were diluted 1200-fold, and 100 μL mixed with 50 μL of each

polyethylene glycol (PEG)-precipitated xenorhabdicin preparations in a 96-well microplate. Experiments were performed in triplicate. Microplate cultures were incubated at 30 °C with shaking. The OD600 nm was measured at 0 and 24 h of incubation. R-type phage tail structures derived from different strains of X. bovienii induced with mitomycin C were analyzed by transmission electron microscopy (Fig. 1). X. bovienii strains, Opaganib in vivo SF43, SF44,

and SF32 isolated from the Steinernema nematodes S. jollieti, S. feltiae, and Dichloromethane dehalogenase S. kraussei, respectively, produced higher levels of phage tail structures (Fig. 1). The xenorhabdicin preparations contained extended tails (Ext), empty sheaths (Emt), and contracted sheaths (CS). Other structures such as uncharacterized filamentous strands were also visualized. SF31 (S. oregonense) and SF35 (S. puntauvense) produced lower levels of phage tail structures, and SF36 (S. intermedium) produced hardly any tail structures. These findings suggest that the contribution of R-type bacteriocin to intraspecies and interspecies competition may vary depending on the level of xenorhabdicin production by the individual strains. As X. bovienii-SF43 produced phage tail structures, its genome was analyzed for P2-like phage clusters. Xenorhabdus bovienii-SF43 contained two P2-type prophage and six other clusters of mostly hybrid lambdoid-like phage genes (Table S1). One P2-type phage locus was a remnant cluster (Fig. 2) consisting of mostly tail synthesis genes (xbp1), while the second cluster (xbp2) also contained capsid, lysis, and replication genes (data not shown). A 400 kb inversion in X. bovienii on the right side of the chromosome (Ogier et al., 2010) places the xbp1 cluster in the opposite orientation in the chromosome relative to X. nematophila.

At least three independent assays were performed, and the results were expressed as the mean ± SD. Statistical analysis was performed using GraphPad Prism

Software version 5.00 C646 concentration for Windows (San Diego, CA). The groups were compared using one-way analysis of variance (anova) followed by the Student–Newman–Keuls multiple comparison post hoc analysis. A P-value of < 0.05 was considered significant. Adhesion of 43 human lactobacilli, isolated from the gastrointestinal tract or from vagina, to mucin was first characterized (Supporting Information, Table S1). Of the 43 strains tested, 27 showed higher adhesion capabilities to mucin than L. rhamnosus GG being statistical significant for 10 of them (P-value < 0.05). In fact, the find more best performing strain, L. plantarum Li70, adhered 51 times more than L. rhamnosus GG. In the rest of the experiments, only the eight most adherent lactobacilli with different RAPD profile were selected (Table 1, Data S1). Strain Lv67 was also selected as a negative control. Adhesion was tested using two epithelial cell lines of intestinal origin (Caco-2 and HT-29) and the vaginal cell line HeLa (Fig. 1). Lactobacillus casei Li71, L. gasseri Lv19, and L. plantarum Li68 were the most

adherent strains to HeLa cells. Lactobacillus vaginalis Lv67, L. plantarum Li68, and L. casei Li71 showed the best adhesion to Caco-2, and finally, L. plantarum Li68, L. plantarum Li69, L. plantarum Li70, L. casei Li71, and, to a lesser extent, L. vaginalis Lv67 were the most adherent to HT-29. All the adhesion values showed statistical differences (P-value < 0. 05) comparing to each control in all the cell lines used. The effect of the lactobacilli and their secreted proteins

Exoribonuclease on the adhesion of the vaginal pathogens C. albicans and A. neuii to HeLa cells was then investigated (Fig. 2). Inhibition values were calculated as adherent bacteria per HeLa cell. Lactobacillus gasseri Lv19 and L. plantarum Li70 increased significantly the adhesion of A. neuii R1 to HeLa cells (P-value < of 0.05 and 0.001, respectively), as well as their extracellular proteins (P-value < 0.001), although the proteins of Lv70 do not show statistical differences (Fig. 2a and b). Conversely, the proteins secreted by L. plantarum Li69 and L. salivarius Lv72 abrogated the adhesion of A. neuii to the same cell line (P-value < 0. 05) (Fig. 2b). Regarding C. albicans, some Lactobacillus strains slightly enhanced the adhesion of the yeast (no significant differences) (Fig. 2c), while their secreted proteins did not have any effect (Fig. 2d). Crude preparations of the proteins secreted by the eight Lactobacillus strains in MRS broth (Fig. 3a) and their surface-associated proteins (Fig. 3b) were resolved by SDS-PAGE.

PCR products were purified using the UltraClean™ PCR Clean-up Kit (MoBio) according to the manufacturer’s instructions. 16S rRNA

gene nucleotide sequences were determined using ABI Prism® BigDye™ dye-terminator chemistry (Applied Biosystems) and an automated ABI Prism® 3700 Genetic Analyzer (Applied Biosystems). Sequences were aligned to known sequences in the GenBank database using blast (Altschul et al., 1990). To identify possible chimeras within the 16S sequences, all sequences were analyzed using the RDP program check_chimera. The sequences obtained in this study were deposited in the GenBank database PI3K Inhibitor Library in vivo under accession numbers GQ332269–GQ332300. The effectiveness, of each biochemical method and for a group of tests, was evaluated based on sensitivity and specificity. One hundred percent sensitivity was sought in order to eliminate

false negatives. Sensitivity and specificity were calculated as follows: sensitivity=[(number of isolates positive as determined by biochemical tests and PCR)/(total number of isolates positive as determined by PCR)] × 100; specificity=[(number of isolates negative as determined by biochemical tests and PCR)/(total number of isolates negative as determined by PCR)] × 100 (Choopun et al., 2002). The environmental conditions in both sampling sites are well described in Celussi & Cataletto (2007): seawater temperature ranged from 6.4 to 25.3 °C following a typical selleck screening library seasonal

progression, while the salinity showed a different trend: in C1, it ranged between 37.0 and 38.2 p.s.u., remaining fairly constant throughout the year, while in D2, we detected strong variations underlined by a wide annual range between 25.5 and 37.7 p.s.u. D2 is, in Interleukin-3 receptor fact, located more close to the Isonzo River mouth and the season-dependent amount of freshwater inputs is reflected in strong variations in salinity. Out of the 269 sucrose-negative isolates subjected to the screening phase, only 171 were confirmed as Vibrio spp. and then analyzed to verify their identity as V. parahaemolyticus. Twenty-three strains died during the analyses; 35 strains showed an arginine dihydrolase-positive reaction that is inconsistent with a V. parahaemolyticus typical response. One hundred and thirteen strains selected as presumptive V. parahaemolyticus were tested using API systems, and even among these, three strains yielded K/K in the KIA test, 32 strains were sensitive to 10 μg Vibriostat O/129 and 40 did not grow in 8% NaCl. API systems characterized only 19 strains as V. parahaemolyticus (Table 1); the urease production was recorded only for one strain (#PVP408). PCR amplification and sequencing of the 16S rRNA gene and the detection of toxR, tlh, tdh and trh genes were carried out on 32 strains (19 presumptively identified as V.

coelicolor FabH with the acetyl-CoA-specific E. coli FabH (YL1/ecFabH mutant) results in a dramatic shift to a fatty acid profile of predominantly straight-chain fatty acids (Li et al., 2005). As predicted, FabH was able to use malonyl-RedQ in place of malonyl-FabC. Under saturating malonyl-RedQ find more conditions, FabH was able to use either acetyl-CoA or isobutyryl-CoA (Table 1). The Km values for each of these were comparable to those observed using

malonyl-FabC, and again there was almost a 40-fold higher catalytic efficiency (kcat/Km) for isobutyryl-CoA compared to acetyl-CoA. However, for both acyl-CoA substrates, the reaction rate kcat was at least 20-fold less using malonyl-RedQ vs. malonyl-FabC (Fig. 2). At fixed isobutyryl-CoA and acetyl-CoA concentrations and variable malonyl-RedQ or malonyl-FabC Hydroxychloroquine ic50 concentrations, similar sets of observations were made. Greater catalytic efficiency was seen with isobutyryl-CoA relative to acetyl-CoA, and for each acyl-CoA substrate, the apparent reaction rate was much faster using malonyl-FabC than with malonyl-RedQ.

This set of analyses also demonstrated that the apparent Km for malonyl-FabC (4.53 μM) and malonyl-RedQ (7.80 μM) was comparable. Thus, the difference in overall catalytic efficiency of FabH using malonyl-ACP substrates arises predominantly from differences in apparent catalytic rates rather than Km values. The ability of FabH to utilize malonyl-RedQ and to have a preference for isobutyryl-CoA click here is consistent with a) genetic data which suggest that FabH can initiate prodiginine biosynthesis in SJM1, the S. coelicolor redP deletion mutant, and b) the observation of a significant

increase in branched-chain alkyl prodiginines in the SJM1 mutant relative to the wild-type S. coelicolor (Mo et al., 2005). A final observation from these analyses is that the maximal kinetic efficiency of FabH (kcat/Km of 9.84 μM−1 min−1 using isobutyryl-CoA and malonyl-FabC) is 66-fold higher than that of RedP (kcat/Km of 0.147 μM−1 min−1 using acetyl-CoA and malonyl-RedQ). This difference might arise from the ability of FabH to utilize isobutyryl-CoA (the enzymes have comparable efficiencies using acetyl-CoA), or because FabH is a primary metabolic enzyme. Initial characterization of many FabH enzymes, including those from streptomycetes, was carried out with a commercially available E. coli ACP (Han et al., 1998; Choi et al., 2000a, b; Khandekar et al., 2001). Subsequent work has revealed that these enzymes have ACP specificity. Improved catalytic activity and in some cases apparent changes in acyl group specificity can be observed when assays are performed using malonyl-ACP generated from the cognate ACP (Florova et al., 2002; Brown et al., 2005).

To test whether an additional nitrogen source could complement the ΔareA mutation, carrot agar was supplemented with nitrate, urea, or ammonium. Ascospores of ΔareA strains did not mature in carrot agar containing nitrate or ammonium, whereas 5 mM urea completely complemented the mutant phenotypes of ΔareA. Both wild-type and ΔareA asci produced eight nuclei through meiosis followed by mitosis (Fig. 4b). The developing asci delimited the nuclei and immature ascospores were formed. However, ΔareA ascospores exhibited defects in maturation and remained in the one-nucleus stage whereas the wild-type nucleus in the developing ascospore divided

into four nuclei. We complemented the ΔareA strain by introducing the GFP-areA-hyg construct where GFP was tagged at the N-terminus EPZ-6438 solubility dmso of AreA.

The ΔareA::GFP-areA strain (KM3) was outcrossed with the mat1r strain to generate ΔareA::GFP-areA;hH1-RFP BGB324 in vivo strains (KM4) in order to visualize both the nuclei and AreA-GFP. Mycelia of KM4 grown in CM for 24 h were transferred to CM, MM supplemented with nitrate, or MM without a nitrogen source. CM is a complete medium that contains rich nitrogen sources from yeast extracts and peptone. The expression levels and localization of GFP-AreA were examined after 12 h of incubation (Fig. 5). Intense GFP fluorescence co-localized with RFP fluorescence, indicating that AreA proteins were localized to nuclei when nitrate was given as a sole nitrogen source. In addition, the expression level of AreA was

higher in nitrogen starvation condition compared with the nitrate. Despite the low intensity of GFP fluorescence, GFP-AreA still localized to nuclei in CM cultures. As a plant pathogenic fungus, the efficient acquisition of nitrogen from host tissues and crop residues is important for the virulence and propagation of G. zeae (Coleman et al., 1997; Snoeijers et al., 2000; López-Berges P-type ATPase et al., 2010). In the present work, we characterized the global nitrogen regulator gene, areA, from G. zeae. Utilization of nitrate was completely repressed but urea was partially utilized (Fig. 1). Ammonium and glutamine were utilized in the ΔareA strains, although they were not utilized efficiently in the wild-type strain. Deletion of areA in G. zeae also triggered various defects in fungal development, including virulence, secondary metabolism, and sexual development. These results suggest that areA is required not only for nitrogen metabolism but also for other fungal development pathways of G. zeae. In A. nidulans, ammonium and glutamine are preferred nitrogen sources over nitrate, nitrite, or proteins (Marzluf, 1997). Loss-of-function mutations in areA trigger an inability to use nitrogen sources other than ammonium and glutamine (Arst & Cove, 1973). In contrast to A. nidulans, ammonium and glutamine are not the preferred nitrogen sources of G. zeae (Fig. 1).

cenocepacia K56-2 after 24 h of exposure. As shown in Fig. 2a, DHA exhibits a concentration-dependent bacteriostatic activity. Upon exposure to DHA, B. cenocepacia K56-2

cells aggregated and formed clusters (Fig. 2b). Moreover, the highest concentrations of DHA screened (50 and 100 mM) caused not only a significant growth inhibition (80–90%) but also death of B. cenocepacia K56-2 cells (8 log10-unit reduction of viable B. cenocepacia cells) (Fig. 2c). Therefore, these results indicate that DHA has a bacteriostatic/killing activity against B. cenocepacia K56-2. To further confirm the in vitro antibacterial effect of DHA (50 mM), we extended our analysis to one representative strain of each of the 17 Bcc species. In addition, click here we also

included two additional clinical isolates (J2315, AU1054) belonging to the B. cenocepacia species. Figure 3 demonstrates that although there is variation in the extent of the antibiotic effect observed, DHA significantly reduces the growth of all Bcc strains studied (40–100% inhibition). Burkholderia cenocepacia J2315, Burkholderia stabilis LMG14294 and Burkholderia anthinia AU1293 were particularly susceptible to DHA, while Burkholderia vietnamiensis PC259, Burkholderia pyrrocinia BC011 and Burkholderia lata 383 possessed the highest levels of resistance (Fig. 3). To determine whether Smoothened antagonist the observed sensitivity/tolerance of the Bcc isolates to DHA was because of hydrophobic interactions with the bacterial cell membrane, the BATH assay was used (Rosenberg et al., 1980). As shown in Fig. 3, a direct relationship was not observed between the degree of cell surface hydrophobicity and DHA sensitivity/tolerance. The in vivo antimicrobial efficacy of DHA against B. cenocepacia was examined in a G. mellonella caterpillar model system.

To mimic a therapy with DHA, larvae were inoculated with a lethal dose of B. cenocepacia K56-2 followed by the administration of a single dose of DHA (50 mM: 190 mg kg−1), given 6 h after infection. The dose of DHA used was within the limits of dosage used in animal studies (Willumsen et al., 1993; Mizota et al., 2001). As shown in Fig. 4a, over a period of 5 days, the treatment with DHA, compared with an infected Vasopressin Receptor control group, prolonged the survival of G. mellonella caterpillars (P < 0.01). Uninfected larvae were also inoculated with 50 mM of DHA, and 100% survival was observed after 5 days (Fig. 4a). We also monitored the growth of B. cenocepacia K56-2 in the hemolymph of infected larvae over a period of 24 h postinfection. We observed a reduced bacterial load (2 log10-unit reduction; P < 0.01) in treated group (administration of DHA) compared with control group (Fig. 4b). Finally, by using quantitative real-time RT–PCR, we determined the expression patterns of four immune-related G. mellonella genes encoding antimicrobial peptides at 10 and 21 h postinfection.

Agglutination was examined by dark-field microscopy and titres were measured as the last dilution where at least 50% of the leptospires were agglutinated (Cole et al., 1973). MAT results were read blind by two expert operators in two different centres; the reactions with LaiWT and LaiMut were tested at the same time. Twenty millilitres of 14-day cultures of LaiWT Selleckchem Belnacasan or LaiMut were centrifuged (3500 g at 4 °C) for

6 min. The pellet was washed twice with sterile distilled water and then resuspended in 700 μL of distilled water, to which 350 μL of 3 × treatment buffer [0.125 M Tris-HCl (pH 6.8); SDS (sodium dodecyl sulphate, 4%); glycerol (20%); 2, β-mercaptoethanol (15%); bromophenol blue (0.1%)] was added. The mixture was heated to 100 °C for 5 min, 1 μg proteinase K was added, and incubated overnight at 60 °C (Hitchcock & Brown, 1983), and the solution was stored frozen until analysis. A discontinuous SDS-PAGE (gradient 6–15%) was used to analyse lipopolysaccharide molecules from LaiWT and LaiMut (Laemmli, 1970). For the

visualization of lipopolysaccharide, polyacrylamide gels were stained using the procedure described by Tsai & Frasch (1982) as modified by Hitchcock & Brown (1983). Following electrophoresis, lipopolysaccharide was transferred to Immobilon-P membranes (Millipore, St. Louis, MO) (Towbin et al., 1979) and probed with mAb F70C7 at a 1 : 100 dilution as the primary antibody and GSK2118436 datasheet alkaline phosphatase-labelled goat anti-mouse immunoglobulin G (Kirkegaard and Perry, MD) at a 1 : 5000 dilution as the secondary antibody. Reactions were visualized colourimetrically with a solution containing 90 μL of NBT (75 mg mL−1 of nitroblue tetrazolium in 70% dimethylformamide), 70 μL of BCIP RG7420 cost (50 mg mL−1 of 5-bromo-4-chloro-3-indolyl phosphate), and 20 mL of alkaline buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5). Each sequence read was trimmed for quality and mapped to a region of the reference genome sequence of serovar Lai (Ren et al.,

2003) representing the lipopolysaccharide biosynthesis locus using sequencher 3.1 (Gene Codes, Ann Arbor, MI). An escape mutant strain of LaiWT was obtained after serial subculture in the presence of mAb F70C7. Strains LaiWT and LaiMut had identical RFLP patterns using either EcoRI or BamHI (data not shown). This near genetic identity was further confirmed by sequencing of the secY gene, which was identical in both strains. The MAT titre of F70C7 against LaiWT was 1280, whereas LaiMut was not agglutinated by F70C7. Additional MAT testing with a set of mAbs and polyclonal sera revealed that the agglutination of LaiMut was decreased by all reactive mAbs and polyclonal sera against serovar Lai, except for mAb F20C4-1, which showed an increased titre.