suis infection that induces meningitis and brain damage [18–20]. The septicemic phase of S. suis infections is characterized by depression, rough hair coat, swollen eyes, weakness, and death during the first 48 h post-infection. If animals survive this critical step of the disease, they may still develop central nervous system damage and meningitis, with the sudden appearance of nervous signs beginning 3-4 days post-infection, including hyperexcitation, episthotonus, opisthotonus, bending of the head toward one side, and walking in circles [18]. Clinical signs of infection and survival Cytoskeletal Signaling inhibitor were recorded on a daily basis

post-infection for 14 days as previously described [18]. Mice exhibiting extreme lethargy or neurological signs were considered moribund and were humanely euthanized. All experiments involving mice were conducted in accordance with the guidelines and policies of the Canadian Council on Animal Care and the principles set out in the Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Welfare Committee of Université de Montréal. Overall survival rates for the various

groups were calculated using Kaplan-Meier plots. Survival curves were compared using the log-rank test with the Holm-Sidak method used to analyze multiple curves. A p < 0.05 was considered statistically significant. All analyses were performed using the Sigma Plot System (v.11; Systat Software, San Jose, click here CA, USA). Results The S. suis mutant library created selleck chemicals llc by insertion of Tn917 transposon (1,200 mutants) was screened for degradation of the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-pNa. Three mutants (G6G, J9G, and M3G) were found to be devoid of activity (A415 < 0.05) compared to parental strain (A415 = 0.85). With the objective to show that only one transposon insertion was present in mutants, chromosomal DNA was analyzed by Southern blotting using a DIG-labeled probe specific for the erm gene

in the Tn917 transposon. As shown in Figure 1, only one Tn917 insertion occurred in the G6G and M3G mutants. Since the J9G mutant had two insertions, we only used the G6G and M3G mutants for further experiments. The mutations were highly stable, with G6G and M3G still unable to degrade the chromogenic substrate after 35 serial transfers in liquid medium (erythromycin-free). Figure 1 Southern blot of S. suis P1/7 and the Tn 917 mutants. Chromosomal DNA was digested with HindIII restriction Alvespimycin research buy endonuclease and hybridized with a DIG-labeled probe specific for the erm gene. Lane 1, wild-type parent strain P1/7; lane 2, mutant J9G; lane 3, mutant M3G; lane 4, G6G. To identify which gene was inactivated in mutants, the Tn917 insertion sites in G6G and M3G were sequenced. The affected gene corresponded to a gene coding for the SSU0757 protein in the genome of S. suis P1/7 based on a comparison of the sequence with those of the S. suis Sequencing Group at Sanger Institute.

The probe specific membership probabilities of N 1(μ 1 i ,s 1 i 2) represents the null-hypothesis of “”not absent”", which is the hypothesis under test. False discovery rate correction as described by [64] was applied to both the test for quantifying aberrations as well as to the test for quantifying genomic losses. The data was visualized using the Integrated Genome Browser [65]. The final data set including dead probes and conserved, aberrant and absent genes is shown in additional file 3. Acknowledgements We acknowledge Arie Jan van Winkelhoff for help with the study design and useful discussions. We furthermore gratefully acknowledge MLN2238 order the National Institute of Dental and Craniofacial

Research and the Pathogen Functional Genomics Research Centre of the J. Craig Venter Institute (formerly The Institute for Genomic Research) for providing the microarrays. Electronic supplementary material selleck Additional file 1: Conserved core gene set of P. gingivalis. The conserved core genes of P. gingivalis consisting

of 1476 genes and two ambiguous genes, which are called non-aberrant but absent. (DOC 1 MB) Additional file 2: W83-specific genes 65 genes. aberrant in each test strain of which 39 W83-specific genes (marked in red) (DOC 92 KB) Additional file 3: P. gingivalis CGH data set. Table listing each P. gingivalis probe included in the results of this study in the order of geneID, including annotation. Low adjP-values (<0.05) depicted in yellow indicate aberrance in a test strain. High adj Pvals. absent (>0.99) depicted in red indicate absence in the test strain. Black

rows indicate the dead probes as found on the W83 array in this study. Zooming out gives an overview of the whole genomic diversity along the test strains. (XLS 546 KB) References 1. Hugoson A, Sjodin B, Norderyd O: Trends over 30 years, 1973–2003, in the prevalence and severity of periodontal disease. J Clin Periodontol 2008,35(5):405–414.PubMedCrossRef 2. Phipps KR, Chan BK, Jennings-Holt M, Geurs NC, Reddy MS, Lewis CE, Orwoll ES: Periodontal health of older men: the MrOS dental Sitaxentan study. Gerodontology 2009,26(2):122–129.PubMedCrossRef 3. Skudutyte-Rysstad R, Eriksen HM, Hansen BF: Trends in periodontal health among 35-year-olds in Oslo, 1973–2003. J Clin Periodontol 2007,34(10):867–872.PubMedCrossRef 4. Genco R, Offenbacher S, Beck J: Periodontal disease and cardiovascular disease: epidemiology and possible mechanisms. J Am Dent Assoc 2002,133(Suppl):14S-22S.PubMed 5. Grossi SG, Genco RJ: Periodontal disease and diabetes mellitus: a selleck kinase inhibitor two-way relationship. Ann Periodontol 1998,3(1):51–61.PubMedCrossRef 6. Loos BG, Craandijk J, Hoek FJ, Wertheim-van Dillen PM, van der Velden U: Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. J Periodontol 2000,71(10):1528–1534.

The rrsB gene was used as a reference gene for normalization, and the data were analyzed using the 2-ΔΔC T method [37]. The amplicons were obtained using the following primer sets. ada-for (5′-GAAACGCCTGTAACGCTGG-3′) ada-rev (5′-GGCTTTAGGCGTCATTCCG-3′) alkA-for (5′-TGGCGAACGGCTGGATGATT-3′) alkA-rev (5′-TTCAACGGCATACCTAACGCTTT-3′) alkB-for (5′-GCCCATTGATCCGCAAAC-3′) alkB-rev (5′-CTGGAAATCTGGATAGCCCG-3′) aidB-for (5′-GAACGGCTGAATCCCTTGAACTG-3′) aidB-rev (5′-TGAAAACGCACATCG TCCAGAC-3′) Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis

(2-DE) experiments were performed using the IPGphor IEF system (GE Healthcare Life Sciences, Chalfont St. Giles, UK) and Protean II xi Cell (Bio-Rad, Hercules, CA, USA) as described previously [38]. Cell extracts were obtained as reported previously [39]. The protein samples

(200 μg) were applied to the Immobiline PF-4708671 DryStrips (18 cm, pH 3-10 NL; GE Healthcare) using in-gel rehydration in an IPGphor (GE Healthcare) using five phases of stepped voltages from 200 to 8000 V with total focusing of 60 kV·h. The strips were then placed on 12% w/v SDS-PAGE gels prepared by the standard protocol [40]. Protein spots were visualized using a silver staining kit (GE Healthcare) GSK1838705A and the stained gels were scanned by a UMAX PowerLook 2100XL Scanner (UMAX Technologies, Inc., TX, USA). PDQuest 2-D Analysis Software (Bio-Rad) was used to automate the process of finding protein spots within the image and to quantify the density of the spots on a percentage of volume basis. Features resulting from non-protein sources (e.g. dust particles and scratches) were filtered out and protein spots were normalized and pairwise image comparisons were performed. At least triplicate gels of each sample were analyzed. All protein spots exhibiting at least 2-fold differences between the samples were evaluated for statistical significance using the Student’s t-test and all spots with p values of < 0.05 were matched with the corresponding MycoClean Mycoplasma Removal Kit spots on the silver

stained images for identification using LC-MS/MS. LC-MS/MS and data analysis For protein identification by the MS/MS analysis, samples were prepared as described previously [41]. Tryptic peptides (10 μL aliquots) were analyzed by a nano-LC/MS system consisting of an Ultimate HPLC system (LC Packings, Amsterdam, Netherlands) and a quadrupole-time-of-flight (Q-TOF) MS (Micromass, Manchester, UK) equipped with a nano-ESI source as described previously [39]. The MASCOT search server (version 1.8; http://​www.​https://www.selleckchem.com/products/lazertinib-yh25448-gns-1480.html matrixscience.​com/​) was used for the identification of protein spots by querying sequence of the trypsin digested peptide fragment data. Reference databases used for the identification of target proteins were UniProt Knowledgebase (Swiss-Prot and TrEMBL; http://​kr.​expasy.​org/​) and NCBI http://​www.​ncbi.​nlm.​nih.​gov/​.

Adjuncts to this approach including

angiography with selective vessel embolization, computed tomography directed drainage of abscess or biloma, and endoscopic retrograde cholangiopancreatography with biliary stenting have recently been integrated into the nonoperative management strategy of liver trauma with encouraging results [13]. Liver packing, although AZD6094 price a life-saving maneuver is not without complications. Placing sponges between the liver and diaphragm to tamponade bleeding compromises venous return, impairing cardiopulmonary function in patients with already limited reserve. Re-bleeding and intraabdominal abscess formation after pack removal has also been described. In patients who require massive resuscitation, visceral edema and elevated intraabdominal pressures may lead

to subsequent abdominal compartment syndrome with the use of perihepatic packing. Abdominal compartment syndrome may cause compromise of cardiac performance and respiratory function, renal function, splanchnic perfusion, and may impair cerebral perfusion [14–17]. The concepts of damage control laparotomy, multiorgan failure, and abdominal compartment syndrome have lead to the use of temporary selleck kinase inhibitor abdominal closures to allow rapid means of abdominal domain control, in anticipation of delayed, definitive intraabdominal injury repair [13, 18, 19]. Vacuum assisted closure (VAC), also referred to as negative pressure wound therapy, has gained wide acceptance for use in the management of a range of acute and chronic wounds as well as for temporary abdominal closures in cases of abdominal compartment syndrome and damage control laparotomy [20, 21]. VAC therapy combines IMP dehydrogenase several features conducive to wound healing including apposition, drainage and coverage. VAC has been successfully utilized to treat numerous and varied conditions including decubitus ulcers, skin grafts, enterocutaneous fistulae, animal and insect bites, osteomyelitis, urologic and perineal wounds, burns, and post-sternotomy sternal wound infections

[22–30]. Temporary abdominal closure after damage control laparotomy for abdominal compartment syndrome has been successfully managed using VAC and this modality is now used routinely in our Level I trauma center for such cases. The porcine or swine model has been used extensively to simulate, experimentally, human liver injury [31–38]. A reproducible Grade V liver injury has been consistently attained in a number of swine model liver trauma studies by the standardized use of a device well described in the trauma and military literature [31, 33, 34, 36–38]. Given the buy AZD6738 complications associated with traditional hepatic packing, the authors present a novel approach to nonresectional therapy in major hepatic trauma utilizing intraabdominal perihepatic vacuum assisted closure or Liver VAC (L-VAC) therapy in the porcine model.

A549 cells were plated onto 6-well plates one day prior to transfection. Following confirmation of 70%–80% confluence, the cells were transfected with pGL3-Basic without promoter (negative control), pGL3-SVP-229-luc (mutant plasmid), and pGL3-SVP-230-luc (normal plasmid). For cell transfection, A549 cells were transiently transfected with 2 μg plasmids Selumetinib nmr and 0.2. g internal control plasmid pRL-TK by using Lipofectamine 2000™ reagent according to the manufacturer’s instructions. Luciferase reporter gene expression detection Thirty hours after transfection, cells were harvested and lysed with 1 × lysis buffer (Promega),

and then 20 μl of cell extract was assayed for luciferase activity using the Dual-Luciferase assay kit (Promega) according to the manufacture’s instructions. The PD0325901 mw relative level of reporter gene expression was expressed as the ratio of firefly luciferase activity to Renilla luciferase (LU/RL). RNA interference A double strand siRNA oligonucleotide targeting HIF-1α (sense: 5-CUGAUGAC CAGCAACUUGAdTdT-3, antisense: 5-UCAAGUUGCUGGUCAU CAGdTdT-3) was designed based on the reference [21] and synthesized by Shanghai Genepharma Co. Ltd. (China). A pair of negative control siRNA were also designed with sequences

different from siRNA-HIF-1α and not homologous to any sequences found in gene bank (sense: 5-AGUUCAACGACCAGUAGUCdTdT-3, antisense: 5-GACUACUGGUCGUUGA 8-Bromo-cAMP manufacturer dTdT-3). For transfection, cells were plated onto 10 cm2 cell culture dishes and grown to 30–50% confluence

before transfection. through 50 μl of Oligofectamine transfection reagent per dish (Invitrogen) was added, and the cells were incubated at room temperature for 20 min. The cells were then rinsed with Opti-Mem I to remove any residual serum. The siRNA duplexes were diluted to a final concentration of 20 nM in Opti-Mem I (Invitrogen). Cells were incubated with the oligonucleotide duplexes in serum-free conditions for 4 h at 37°C. Serum was then added back to the culture, and cells were incubated in normoxic or hypoxic condition for an additional 48 h. Real Time Reverse Transcription-PCR Total RNAs were isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Twenty-five nanogram total RNA per sample was reverse transcribed by using the Reverse Transcription Reaction Kit (Takara Code: DRR061S) according to the manufacturer’s instructions. Quantitative real-time PCR was performed analyzed on the Applied Biosystems 7300 Real-Time PCR System to determine the relative amounts of survivin, HIF-1α and GAPDH (internal control) mRNAs expressed. The SYBR Green Supermix was used for all real-time PCR reactions.

When appropriate, plates or broths were supplemented with erythromycin (Erm) (5 μg/ml) and/or chloramphenicol (Cm) (5 μg/ml). Growth BI 10773 clinical trial was monitored by measuring optical density of cultures at 600 nm (OD600) at regular time intervals. To investigate the effect of various stress agents on RpoE activity, cells were grown to mid log phase (OD600 = 0.6-0.7) and

treated for different time periods (30 min-1 h) with hydrogen peroxide (5 mM), diamide (100 mM), 0.01% SDS-0.1 mM EDTA or methylene blue (1 μM) in the presence of white light (source of singlet oxygen) [77]. Sequences from the following strains (with Genbank ID) were downloaded for comparative aligments: N.meningitidis_MC58 (AE002098); N.meningitidis_FAM18 (AM421808); N.meningitidis_053442 (CP000381); N.meningitidis_Z2491 (AL157959); N.gonorrhoeae_FA1090 (AE004969); N.gonorrhoeae_NCCP11945 (CP001050); N.cinerea_ATCC_14685 Selleckchem Necrostatin-1 (ACDY00000000); N.flavescens_NRL30031/H210 (ACEN00000000); N.lactamica_ATCC_23970 (ACEQ00000000); N.subflava_NJ9703 (ACEO00000000); N.sicca_ATCC_29256 (ACKO00000000); N.mucosa_ATCC_25996 (ACDX00000000); Streptomyces coelicolor_A3(2)

(AL645882); Rhodobacter sphaeroides_ATCC_17025 (CP000661). Construction of ΔrpoE and ΔNMB2145 mutants of N. meningitidis N. meningitidis H44/76 knock-out mutants of rpoE (NMB2144) and NMB2145 were constructed

using the PCR-ligation-PCR method [79, 80]. All primers used in this study are listed in Oxymatrine Table 1. PCR products were generated with primer pairs CTsE-1/CTsE-2 and CTsE-3/CTsE4 for creating ΔrpoE and primer pairs CT-2145-1/CT2145-2 and CT-2145-3/CT-2145-4 for creating ΔNMB2145, ligated and the ligation products were reamplified with primer pairs CTsE-1/CTsE-4 (for ΔrpoE) and CT-2145-1/CT-2145-4 (for ΔNMB2145). The resulting PCR products were cloned into pCR2.1 (Invitrogen). The EcoRI digested Erm https://www.selleckchem.com/products/azd9291.html resistance cassette from pAErmC’ [81] was introduced into the created unique MfeI restriction site yielding plasmids pCR2.1-NMB2144 and pCR2.1-NM2145. The ΔrpoE and ΔNMB2145 strains were generated by natural transformation of strain H44/76 with pCR2.1-NMB2144 and pCR2.1-NMB2145 respectively, and selection for Erm resistance. Replacement of NMB2144 and NMB2145 by the Erm cassette was confirmed by PCR with primer pair CTsE-5/CTsE-6 (for ΔrpoE) and primer pair 2144-01/CT-2145-6 for ΔNMB2145. The orientation of the Erm cassette was determined by PCR using primer pair JP19/JP20 and mutant strains in which the transcriptional direction of the Erm cassette was in accordance with the transcriptional direction of the deleted genes were selected.

The shift of fluorescence peak obtained for LL-mInlA+ in FACS analysis was significantly higher as compared to NZ9000 JAK inhibitor strain thus confirming successful surface expression of mInlA on L. lactis.

Other invasins, from Gram-positive bacteria, such as MG-132 clinical trial InlA or FnBPA, have already been successfully expressed in L. lactis confirming that the signal peptide for secretion and the anchoring signal are well recognized by the L. lactis machinery. Production of invasins from Gram-negative bacteria, such as Yersinia pseudotuberculosis invasin at the surface of L. lactis has never been successful (Denis Mariat, personal communication). The invasivity was assessed by gentamicin assay in non-differentiated E-cadherin expressing human epithelial cell line Caco-2 cells. This experiment

showed that LL-mInlA+strain is 1000-fold more invasive than NZ9000 strain. Wollert and collaborators (2007) observed a 2-foldincrease in the adhesion and invasion efficiency of L. monocytogenes strain producing mInlA compared to wild-type listeria expressing native InlA by using gentamicin-protection-invasion assays in Caco-2 cells [30]. A confocal image taken after gentamicin assay showed clearly that LL-mInlA+ is capable of adhering to and entering in non-differentiated Caco-2 cells. buy VX-770 The preferential distribution of recombinant bacteria at the periphery of the Caco-2 cell islets can be explained by the fact that E-cadherin is accessible only at the periphery. A similar type of bacterial distribution, around the Caco-2 cell islets, was previously observed when Caco-2 cells were co-incubated with LL-FnBPA+[25]. LL-mInlA+ and LL strains were then transformed with pValac: BLG plasmid, co-incubated with Caco-2 cells and BLG expression was followed 72 h later

by ELISA. BLG was detected in the cytoplasmic fraction of Caco-2 cells which were co-incubated with noninvasive and invasive strains carrying pValac: BLG. This data confirms prior observations that even noninvasive L. lactis can transfer functional plasmids to Caco-2 cells [23]. Cells were also capable of secreting the allergen, which is an interesting characteristic facilitating antigen uptake Y-27632 2HCl and presentation by professional APCs through cross-priming pathways [1]. The use of LL-mInlA+ improved BLG expression around ten times compared to noninvasive strain. Our hypothesis is that invasive lactococci can enter in higher numbers inside epithelial cells and thus deliver more plasmids. Noninvasive and invasive L. lactis, carrying pValac: BLG or not, were orally administered for 3 consecutive days in BALB/c mice. On the fourth day, enterocytes from the small intestine were isolated and BLG production was measured by enzyme immunoassay (EIA). Isolated enterocytes from mice administered with invasive LL-mInlA+BLG produced the same amount of BLG as compared to mice immunized with noninvasive LL-BLG. Thus, we confirmed that noninvasive lactococci are able to transfer a functional plasmid in vivo in mice [27].

(B) Quantification of cell aggregation in S. oneidensis MR-1 wild type and mutants in planktonic culture under minimal medium conditions. The ratio of the optical density measured at 600 nm of wild type and mutant cultures after and before dispersion was used to determine their aggregation phenotypes. These data indicate a possible role for mxdA and mxdB in cell-surface

adhesion when growing in minimal medium. When comparing growth rates in LB to minimal medium, we found no correlation between growth rate and mxd expression, suggesting that a low growth rate, as found under starvation conditions in minimal medium, was most likely not responsible for mxd induction (data not shown). We therefore hypothesized PF-02341066 mouse that limitation for essential nutrients or accumulation of metabolites might be involved in mxd induction, and specifically tested whether carbon or nitrogen limitation induced mxd expression. For this purpose we constructed a MGCD0103 in vitro wild type mxd::lacZ reporter strain (AS832) (see Table 1 and 2). This strain was grown in LB medium to an OD600=0.3. Cells were pelleted, resuspended in minimal medium amended with 50 mM sodium lactate, incubated for 120 minutes at 30°C and subsequently assayed for specific β- galactosidase activity. Similarly, cells were also exposed to minimal

medium without carbon or nitrogen source. As a control, cells were resuspended in the same LB culture medium. As shown in Figure 2 no increase Dimethyl sulfoxide in mxd expression was observed when cells were incubated in the LB culture medium for 120 minutes (Figure 2) and compared to the same sample at t=0 minutes. Similarly, cells exposed to minimal medium void of a nitrogen source also did not show any increase in mxd expression. Cells exposed to minimal medium supplemented with lactate led to minor mxd induction. However, shifting cells to minimal medium void of a carbon source led to significant mxd induction (~400 MU). Thus, starvation for carbon appears to be important for mxd expression

in S. oneidensis MR-1. Table 1 Strains used in this study Strain Relevant genotype or description Source or reference E. coli     S17-lambda pir thi pro recA hsdR [RP4-2Tc::Mu-Km::tn7]lambda pir Tpr Smr [38] AS259 (BW20767) RP4-2-Tc::Mu-1 Kan::Tn7 integrant leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA (deltaMluI)::pir + [12] AS262 S17-lambda pir GSK458 price harbouring pUX-BF13 [39] AS392 S17-lambda pir harbouring pGP704-mini-Tn7(Gm) P A1/04/03-GFPmut3* [39] S. oneidensis     AS93 S. oneidensis MR-1, wild type, tagged with GFPmut3* in a Tn7 construct, Genr [12] AS536 AS93 harbouring pME6031(Tc)::Pmxd -300+1 lacZ (pJM1) This study AS556 AS93 harbouring pME6031(Tc)::lacZ (promoterless) This study AS579 (MR-1) S.

The minimum spanning tree was created based on the categorical coefficient and the priority rule ‘highest number of single-locus variants’. A detailed description of analysis using minimum spanning tree can be found

in the study by Schouls et al. [14]. Results Identification of MIRUs Four [6] and eight MIRU-VNTR [7] have been already described for M. avium and M. paratuberculosis, respectively. Because of the phylogenetic similarity between these species and M. intracellulare, it was predicted that several of these MIRU-VNTR could also be used in typing M. intracellulare isolates [15]. Thus we included these MIRU-VNTR loci, named MIRU 1 through 4 (Bull et al.), MIRU 32, 292, X3, 25, 3, 7, 10, and 47 (Thibault et al.), in S63845 purchase our study. The analysis of the genome of M. avium 104 identified 120 Tandem PCI-34051 manufacturer Repeat (TR) sequences of which 16 were selected based on their degree of homology and their size (MIN 1 through MIN 16). Examination of the 353 contigs of M. intracellulare ATCC 13950 identified 310

TRs of which 17 were used in the study (MIN 17 through MIN 33). Thus a total of 45 TR loci were studied. The polymorphism of the selected 45 TR loci was initially investigated on a set of nine randomly chosen isolates of M. intracellulare (isolates 2 through 10), as well as the reference strain M. intracellulare ATCC 13950 (strain 1). Thirty-four MIRU-VNTR were absent GSK2118436 ic50 during amplification of one or more isolates while four MIRU-VNTR did not demonstrate polymorphism on the isolates tested, they were thus eliminated. One of the 12 MIRU-VNTR already described for M. avium and M. intracellulare, MIRU 3 (Bull at al.), was polymorphic with different allele sizes. None of the new MIRU-VNTR identified from the strain M. avium 104 could be validated on our set of 10 isolates of M. intracellulare.

Consequently, of the 45 candidate MIRU-VNTR only seven, MIRU 3 (Bull et al.), MIN 18, MIN 19, MIN 20, MIN 22, MIN 31, and MIN 33, were present and exhibited polymorphism. The stability of the seven polymorphic MIRU-VNTR markers PRKD3 was studied on four isolates. No difference was seen in the gel profiles before and after 10 passages in MGIT medium. Thus, an MIRU-VNTR scheme was proposed, including seven markers. It allowed unambiguous type assignment using agarose gels, with PCR products ranging in size between 200 and 750 bp. Characteristics of each MIRU-VNTR marker are shown in Table 1. As the genome sequence of M. intracellulare was available only in a contig format and was not annotated, it was impossible to determine where the MIRU-VNTR were located in the genome. The sizes of the unit repeat ranged from 53 to 57 bp. Sequencing of the different size PCR products at each of the seven loci from each of the 10 isolates confirmed the sizes and sequences of individual MIRU-VNTR loci.

tularensis strains were richly streaked on MC plates that were incubated in 37°C and 5% CO2 over night. Bacteria were harvested, serially diluted in PBS and 100 μl of a dilution estimated to give approximately 100 colony forming units per plate were evenly spread on MC plates. The plates were incubated at 37°C in an aerobic or microaerobic milieu and the colony size scored after 2, 3, and 6 days of incubation. OxyBlot assay The OxyBlot Microbiology inhibitor Protein Oxidation Detection Kit (Chemicon International)

is based on VE-822 datasheet a method for detection of carbonyl groups introduced into proteins by oxidative reactions. The carbonyl groups are derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by use of 2,4-dinitrophenylhydrazine (DNPH) and can thereafter be detected by immunostaining. The OxyBlot kit was used to compare the amount of oxidized proteins in LVS and ΔmglA grown in an aerobic or a microaerobic milieu. Samples were collected at an OD600 of 0.6-0.7 and the bacteria were lysed using a buffer containing 2 M thiourea, 7 M urea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), 0.5% ASB-14 (amidosulfobetaine-14), 1.0% DTT, 0.5 × protease inhibitor, and 1% β-mercaptoethanol. The amounts of protein in the samples were determined by use of the Bradford assay (Fermentas, Tideglusib St. Leon-Rot, Germany). The assay was carried out according to the manufacturer’s protocol for Standard Bradford assay in microplates.

Equal amounts of proteins were taken from each sample for derivatization and synthesis of negative controls according to the manufacturer’s protocol. Briefly, samples were incubated with 1 × DNPH solution for 15 min at RT to allow derivatization selleck chemicals of carbonyl-groups to DNP-hydrazone, after which a neutralization solution was added. Negative controls were prepared as the samples with the exception that they were treated with dH2O instead of 1 × DNPH solution, and therefore lack DNP-hydrazone. Negative controls were synthesized in order to ensure the specificity of the antibodies used for detection of DNP-moieties in oxidized proteins. Samples were blotted to PVDF

membranes using a Bio-Dot Microfiltration Apparatus (BioRad), immunostained using a primary Rabbit anti-DNP antibody and a secondary Goat Anti-Rabbit IgG (HRP-conjugated) antibody; and developed with chemiluminescence to visualize the DNP-modifications, as directed by the instructions provided in the OxyBlot Kit. Samples were blotted at a concentration of 2.5 ng of protein in the first well followed by two-fold dilutions thereof. Catalase assay LVS and ΔmglA were cultivated overnight in CDM and thereafter sub-cultured in CDM. When bacteria reached logarithmic growth phase (0.4-0.7 OD600 nm), the OD600 of the cultures were measured and 20-50 μl of culture was withdrawn and transferred to a 96-well UV-clear plate (Greiner Bio-One, Frickenhausen, Germany). To each well, PBS was added to give a final volume of 200 μl.