Responders were

Responders were determined by ELISA with the recombinant proteins and serum samples from patients of both phases of the disease. The reactivity was evaluated as IgG antibodies. Serum was considered MAT positive or MAT negative if agglutination was detected when the sera were tested for their reactivity’s with isolates of the 22 serovars (see Methods). The cutoff values are depicted as horizontal bars and were defined as the mean plus

3 standard deviations obtained for sera from 12 healthy individuals. (A) shows the data for Lsa33, (B) for Lsa25, and (C) depicts the Ilomastat data when both proteins were employed (Lsa33 plus Lsa25). Recombinant proteins adhesion to ECM components The ability of Lsa33 and Lsa25 proteins to mediate host colonization by adhering to extracellular matrix proteins was examined by ELISA. Laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins fetuin and gelatin were BIIB057 mouse immobilized on microdilution wells and recombinant protein attachment was A-1155463 concentration assessed by ELISA using antibodies against the proteins. As shown in Figure 5A, both recombinant proteins exhibited adhesiveness to laminin, while no statistically significant

binding was observed with these proteins when wells were coated with collagen Type I and IV, cellular and plasma fibronectin, gelatin or with the highly glycosylated control

protein fetuin. The interaction of recombinant proteins with laminin was also observed when anti – polyhistidine monoclonal antibodies were employed to probe the ligands (Figure 5B). The binding between Lsa33 and Lsa25 with laminin was also evaluated on a quantitative basis as depicted in Figure 5C. A dose – dependent and saturable binding was observed Sclareol when increasing concentrations of the recombinant proteins (0–6 μM) were allowed to adhere to a fixed laminin concentration (1 μg) (Figure 5C). Binding saturation level was achieved by protein concentration of ~4 and 5 μM for Lsa33 and Lsa25, respectively. Based on ELISA data, the calculated dissociation equilibrium constants (K D) for the recombinant protein Lsa33 and Lsa25 with laminin is 367.5 and 415.4 nM, respectively. The role of laminin sugar moieties in the binding with Lsa33 and Lsa25 was assessed with laminin previously oxidized by increasing concentrations of sodium metaperiodate, ranging from 5 to 100 mM. The effect of metaperiodate concentration on the interaction is displayed in Figure 5D. Laminin oxidation had some effect on the interaction with Lsa25, being the reduction of 40% achieved at the highest metaperiodate concentration employed (100 mM). However, the attachment of Lsa33 to metaperiodate – treated laminin had no interference on the binding.

In this way, we detected a 28S rDNA fragment with a product of ne

In this way, we detected a 28S rDNA fragment with a product of nearly 375 bp in 19 out of the 21 isolates tested. However, applying a semi-nested PCR

system to these DNA see more samples with a new pair of primers specific for Coccidioides spp., we detected bands of sizes compatible with the expected fragment in the DNA of all cultures tested. As a control, the DNA of 41 lineages of other human pathogenic fungi (S. schenckii, P. brasiliensis, H. capsulatum, A. niger, A. fumigatus, A. nidulans, B. dermatitidis, M. canis, T. rubrum, T. mentagrophytes, C. neoformans and C. gattii) were submitted to the same protocol, and all results were negative. The results were also negative when the protocol was applied to DNA from Ganetespib manufacturer bacteria. Our results indicate the high specificity of PCR with these primers and highlight the increased sensitivity, expected in nested PCR reactions

using DNA obtained from soil samples. The next step was to optimize direct PCR with specific primers for detecting Coccidioides spp. in the DNA extracted from our 24 soil samples. The direct PCR method revealed the expected fragment only in 8 (33.3%) soil samples, but when the semi-nested system was used, all the soil samples were positive, thus confirming to be a very sensitive method for detecting Coccidioides spp. 28S rDNA. It is important to note that all of the AZD0156 in vivo positive soil samples were collected in and around armadillo burrows strongly suspected to be heavily contaminated because their disturbance CDK inhibitor caused acute cases of human and canine coccidioidomycosis. It is possible that these restricted sites harbor high concentrations of viable arthroconidia of C. immitis, which are easily detected by animal inoculation, as well as dormant or dead fungal elements with DNA partially preserved, which can only be detected by molecular tools. To evaluate these factors, it should be of interest to analyze soil samples collected in concentric circles from

the center of the focus. As controls for the PCR protocols applied to our soil samples from Piauí, we analyzed DNA extracted from soil samples collected in non-endemic areas of the cities of Goiânia (capital of the state of Goiás) and Brasília (Capital of Brazil), and none presented the 375-bp band, reinforcing our results. Thus, we believe it is important to note that the primer system RFA12 + P2 was able to identify both C. immitis and C. posadasii. The molecular detection of Coccidioides spp. in suspected soil or in clinical specimens has obvious importance for epidemiological studies and laboratory diagnosis of coccidioidomycosis. Furthermore, molecular procedures such as PCR present substantial advantages, as they reduce the biological risk inherent in the classical techniques and reduce the time necessary to identify a suspected environmental focus or diagnose a clinical case to a few hours.

75 DLL “Plan-Apochromat” or a 100×/1 4 DIC objective Metamorph 7

75 DLL “Plan-Apochromat” or a 100×/1.4 DIC objective. Metamorph 7.5 (Molecular Devices) or NIS-Element (Nikon) software was used for digital analysis and treatment of the images to extract the

number, buy BAY 11-7082 specific fluorescence intensity and length of stained bacteria. This system allows enumeration and manual differentiation between individually labeled cells, cells in aggregations and/or auto fluorescent particles which can interfere with automated analysis. For the CV6 procedure, cells were scored as viable if their fluorescence intensity was at least 1.5 times greater than the fluorescence background noise. Under our conditions, detection limits for CV6 measurement were 3 × 104 cells ml-1. Co-culture of L. pneumophila and A. Castellanii Axenic cultures of A. Combretastatin A4 purchase castellanii (ATCC 30234) were check details prepared as previously described [48]. Briefly,

the A. castellanii strain was grown in a 150-cm2 cell culture flask in PYG medium (peptone-yeast extract-glucose) at 30°C for 3 days. Monolayers were developed in 24-well tissue culture plates using Page’s amoeba Saline (PAS) for 24 h at 30°C. Aliquots of 1 × 106 amoebae per well in 24-well tissue culture plates were infected with 10 × 1 ml of L. pneumophila at 1 × 108 cells ml-1 in PAS as described above (MOI 100). The plates were centrifuged at 500 × g for 5 min and incubated for 3 days at 37°C. Then, the monolayer and supernatant were removed and spread on BCYE agar plates. Colonies were counted after 3 days and 10 days of incubation at 37°C. Acknowledgement We thank Gail G. Hardy (Indiana university, Bloomington), Audrey Dumont (CNRS, Marseille), Emilie Fugier (CNRS, Marseille), and Sophie Jarraud (CNRL, France) for discussions and critical reading of the manuscript. This work was supported by a fund from the ANSES (Program ARCL-2005) and a doctoral fellowship (Adrien Ducret) from CIBA

SA. We also thank Yannick Fovet for his helpful comments on the manuscript. We thank Bernard Lascola and Isabelle Pagnier for their generous gifts of LP1 and amoebae. References 1. Fliermans: Ecology of legionella: from data to knowledge Benzatropine with a little wisdom. Microb Ecol 1996, 32:203–228.PubMedCrossRef 2. Muldrow LL, Tyndall RL, Fliermans CB: Application of flow cytometry to studies of pathogenic free-living amoebae. Appl Environ Microbiol 1982, 44:1258–1269.PubMedCentralPubMed 3. Fields BS, Benson RF, Besser RE: Legionella and legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 2002, 15:506–526.PubMedCentralPubMedCrossRef 4. Fields BS: The molecular ecology of legionellae. Trends Microbiol 1996, 4:286–290.PubMedCrossRef 5. Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y: Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 2005, 71:20–28.PubMedCentralPubMedCrossRef 6. Newton HJ, Ang DKY, van Driel IR, Hartland EL: Molecular pathogenesis of infections caused by legionella pneumophila. Clin Microbiol Rev 2010, 23:274–298.

J Allergy Clin Immunol 92(3):387–396CrossRef Bernstein DI, Cartie

J Allergy Clin Immunol 92(3):387–396CrossRef Bernstein DI, Cartier A, Cote J, Malo JL, Boulet LP, Wanner M, Milot J, L’Archeveque J, Trudeau

C, Lummus Z (2002) Diisocyanate antigen-stimulated monocyte chemoattractant protein-1 synthesis has greater test efficiency than specific antibodies for identification of diisocyanate asthma. Am J Respir Crit Care Med 166(4):445–450CrossRef Brandli O, Schindler C, Kunzli N, Keller R, OSI-906 manufacturer Perruchoud AP (1996) Lung function in healthy never smoking adults: Angiogenesis inhibitor reference values and lower limits of normal of a Swiss population. Thorax 51(3):277–283CrossRef Brandli O, Schindler C, Leuenberger PH, Baur X, Degens P, Kunzli N, Keller R, Perruchoud AP (2000) Re-estimated equations for 5th percentiles of lung function variables. Thorax 55(2):173–174CrossRef Budnik LT, Nowak D, Merget R, Lemiere C, Baur X (2011) Elimination kinetics of diisocyanates after specific inhalative challenges in humans: mass spectrometry analysis, as a basis for biomonitoring strategies. J Occup Med 4SC-202 clinical trial Toxicol 6(1):9–18CrossRef Campo P, Wisnewski AV, Lummus Z, Cartier A, Malo JL, Boulet LP, Bernstein DI (2007) Diisocyanate conjugate and immunoassay characteristics influence detection of specific antibodies in HDI-exposed workers. Clin Exp Allergy 37(7):1095–1102CrossRef Curwick CC, Bonauto DK, Adams DA (2006) Use of objective testing in the diagnosis of work-related asthma by physician specialty.

Ann Allergy Asthma Immunol 97(4):546–550 Hendrick DJ (2002) Diagnostic tests for occupational asthma. Am J Respir Crit Care Med 166(4):436–437CrossRef Hur GY, Koh DH, Choi GS, Park HJ, Choi SJ, Ye YM, Kim KS, Park HS (2008) Clinical and immunologic findings of methylene diphenyl diisocyanate-induced occupational asthma in a car upholstery factory. Clin Exp Allergy 38(4):586–593CrossRef Jayet PY, Schindler C, Kunzli N, Zellweger JP, Brandli O, Perruchoud AP, Keller R, Schwartz J, Ackermann-Liebrich U, Leuenberger P (2005) Reference Cyclic nucleotide phosphodiesterase values for methacholine reactivity (SAPALDIA study). Respir Res 6:131CrossRef Jones MG, Floyd A, Nouri-Aria KT, Jacobson MR, Durham SR, Taylor AN, Cullinan P

(2006) Is occupational asthma to diisocyanates a non-IgE-mediated disease? J Allergy Clin Immunol 117(3):663–669CrossRef Kumar A, Dongari N, Sabbioni G (2009) New isocyanate-specific albumin adducts of 4,4′-methylenediphenyl diisocyanate (MDI) in rats. Chem Res Toxicol 22(12):1975–1983CrossRef Lushniak BD, Reh CM, Bernstein DI, Gallagher JS (1998) Indirect assessment of 4,4′-diphenylmethane diisocyanate (MDI) exposure by evaluation of specific humoral immune responses to MDI conjugated to human serum albumin. Am J Ind Med 33(5):471–477CrossRef Maestrelli P, Boschetto P, Fabbri LM, Mapp CE (2009) Mechanisms of occupational asthma. J Allergy Clin Immunol 123(3):531–542CrossRef Malo JL, Chan-Yeung M (2009) Agents causing occupational asthma.

All authors read and approved the final manuscript “

All authors read and approved the final manuscript.”
“Background Currently, reverse genetics-based tools have been largely employed to obtain biological information on genes of unknown function. Nowadays genomic sequence data are easily obtained, but gene function is not always obviously extracted from these data. These tools have been used for many purposes, such as protein subcellular localization [1], this website protein interaction identification [2], protein overexpression [3], gene knockout [4] and gene silencing [5]. These techniques are particularly important in the study of trypanosomatid protozoa. Sexual reproduction, although not

frequent, may play a role in the heterogeneity of several trypanosomatid species. However, these parasites mostly have a clonal population structure [6, 7]. This characteristic precludes the use of forward genetics to study gene function in these parasites. In addition, their protein-coding genes are transcribed in polycistronic mRNAs, not related to bacterial operons, which are further processed to mature monocistronic mRNAs by a trans-splicing mechanism [8]. This process CP673451 results in a short nucleotide sequence (miniexon) being added to the 5′ end of trypanosomatid mRNAs [9]. The same machinery probably scans the intergenic region (IR) to process the upstream transcript and add the poly-A tail [10]. However, no consensus sequence for poly-A tail addition

has been found in trypanosomes. Furthermore, gene expression in these microorganisms is mostly controlled by post-transcriptional events involving RNA processing and stability [8]. Hence, to be expressed in trypanosomatids, transgenes need to be flanked by intergenic regions that contain sequence elements promoting miniexon and poly-A tail addition. Ketotifen Generally, IRs in trypanosomatid plasmid vectors are derived from constitutively expressed genes, such as those encoding glyceraldehyde 3-phosphate dehydrogenase [11, 12], actin, aldolase [5, 13, 14], α-tubulin [15] or ubiquitin [16]. Gene expression in trypanosomatids appears to be ubiquitous and is not dependent on the presence of a typical

RNA polymerase II (pol II) promoter [17]. Although typical pol II selleck promoters have not been found in trypanosomatids, it has been shown that pol II transcription of an entire polycistronic unit initiates upstream of the first gene of the polycistron (in strand-switch regions) [18]. To enhance gene expression, vectors for use in trypanosomatids were constructed to ensure that transcription is directed by strong promoters like RNA polymerase I (pol I) promoters [3, 14, 19–21]. Some vectors were also designed to control gene expression, by combining T7 or pol I promoters with tetracycline-inducible systems [5, 12, 14, 16, 22–26]. These features require the development of reverse genetics strategies to deal with trypanosomatid biology. There are a few examples of vectors designed for use in T.

This procedure leads to dilution of type I persisters whilst stoc

This procedure leads to dilution of type I persisters whilst stochastically build type II persister cell levels should remain constant. As depicted in Figure 2B the percentage of antibiotic tolerant persisters decreased sequentially after 100-fold MIC gentamicin challenge when the bacterial culture was kept in the early growth phase for three cycles. This data indicate that

gentamicin Caspase Inhibitor VI cost tolerant persisters are not or only rarely produced in the early exponential growth phase and that most of the tolerant bacteria represented type I persisters. These were probably ‘left overs’ from the overnight culture and became diluted within repeated cycles of exponential growth. S. suis persister cells also tolerate combinations of different Eltanexor mw antibiotics Antibiotics like penicillin are frequently used to treat S. suis infections, sometimes in combination with other antibiotics like aminoglycosides. However, relapses of S. suis infections in pigs and humans have been reported [36]. Furthermore, penicillin and gentamicin

are widely used in standard antibiotic protection assays to quantify intracellular bacteria in in vitro cell culture experiments. Therefore, we investigated S. suis tolerance against a combination of penicillin (200-fold MIC) and gentamicin (4-fold MIC) that correspond to the concentrations applied in these antibiotic protection experiments. After simultaneous treatment selleck chemical of exponential grown S. suis with penicillin and gentamicin we observed a biphasic killing curve characterized by a rapid decrease of CFU numbers within the first hour and a subsequent plateau of surviving bacteria persisting for more than 8 hours (Figure 3A). The killing kinetics of stationary grown bacteria treated similarly resembled treatment with gentamicin alone, as depicted in Figure 1B. Similar to what we observed after treatment with a single antibiotic, the tolerance to a combination of penicillin

and gentamicin was not inherited, as revealed from heritability tests (Figure 3B). Baf-A1 nmr These data suggest that S. suis persister cells are capable of tolerating not only single antibiotics, but also a combination of penicillin and aminoglycosides. Figure 3 Time-dependent killing after combined antibiotic treatment. (A) Exponential (solid line) and stationary (dotted line) grown S. suis strain 10 was exposed to a combined antibiotic treatment of 200-fold MIC of penicillin and 4-fold MIC of gentamicin over time. (B) This penicillin/gentamicin combination was also used in a heritability test with exponential (solid line) and stationary (dotted line) grown S. suis in three consecutive cycles. The values are means of two biological replicates plated in triplicate. Error bars indicate the standard deviation. Persister cell formation in S.

However, it needs the acquisition of new skills which I did not p

However, it needs the acquisition of new skills which I did not possess. I got the message. The recording of light scattering by intact leaves learnt at Stanford required complex interpretation. I concluded that light scattering revealed alterations of leaf energization (Heber 1969). This was not wrong but decades of further research by others were required to open the view on various complex mechanisms which protect leaves against photo-oxidative damage. The molecular

basis of these mechanisms is still under investigation. Fig. 1 Stacy French around 1970 at the Department of Plant Biology, Carnegie Institution of Washington, Stanford. Courtesy of Jeanette Brown After I had returned to my new position at Düsseldorf, the problem of establishing a balance between research and teaching was not easy to solve. Martha find more Kirk, on sabbatical Selleckchem Quisinostat leave from Berkeley, came to my laboratory with her unique combination of human warmth and A-1155463 nmr Scientific competence. This was of great help. The teaching load of a professor had to be borne, but how to do this without reducing research? Student unrest also interfered. The slogan of the 1968 student generation was ‘Unter den Talaren, der Muff von tausend Jahren’ (Below their gowns, the dust of one thousand years! Did they mean me?). I had little objection against student boycott

of my lectures but warned, successfully, against interference

with my laboratory work. A few postdocs found Düsseldorf attractive. Lina Tyankova from Sofia worked successfully in the frost hardiness field until she decided she had sufficient data and should, before returning to Bulgaria, turn some attention to the elegant shops of Königsallee. Tilberg and Egneus came from Sweden, Umeo Takahama from Kyushu, Japan. He was the first of several Japanese postdocs who were undaunted to do original work in difficult fields (Takahama et al. Vasopressin Receptor 1981). In 1970, I was offered a chair at the Hochschule für Bodenkultur, an Agricultural University in Vienna, Austria. Negotiations proved difficult. A counter-offer kept me in Düsseldorf, now as full professor or ‘Ordinarius’. It also made it possible for me to get, as compensation for too much teaching, half a year’s time for research with Keith Boardman at the Commonwealth Scientific and Industrial Research Organization, in short CSIRO, in Canberra, Australia. There I met Hal Hatch, famous for his work on C4 photosynthesis (Fig. 2). Keith knew all about cytochromes. I hoped for enlightenment and was not disappointed. But of main importance for me was the presence of Robin Hill (Fig. 3) who with his wife Priscilla was guest of Sir Rutherford (Bob) Robertson, President of the Australian Academy of Sciences.

P21 (CIP1/WAF1) is a direct target of p53 [36], thus, p53 mediate

P21 (CIP1/WAF1) is a direct target of p53 [36], thus, p53 mediated induction of p21 (CIP1/WAF1) at least contributed to the inhibitory effect of BBR on cell proliferation and cell cycle arrest. On the other hand, our results suggested that activation of p38 MAPK mediated the BBR-induced FOXO3a protein expression and the selleck kinase inhibitor latter also contributed to the BBR-inhibited cell growth and -induced apoptosis. It is possible that the inhibition of proliferation can be in part a consequence of increased cell apoptosis or vise versa. The FOXO3a is an important tumor suppressor and is

under-expressed in many cancers. There are a number of parallels between FOXO3a and p53, TSA HDAC concentration both play a pivotal role in regulating the cellular response to stress and damage signals, inducing cell cycle GNS-1480 purchase arrest, apoptosis, and DNA repair [37]. Several studies showed that FOXO3a interacts with p53, and that FOXO3a is a p53 target gene [15, 38]. In this study, we demonstrated that the potential interaction and mutually exclusive events of p53 and FOXO3a may contribute to enhance BBR-induced apoptosis and -inhibited cell proliferation. However, the detailed mechanism underlining the regulation of these transcriptional networks in mediating the effect of BBR on the control of lung cancer cell survival

needs to be elucidated. Our results also demonstrated a causative role of FOXO3a in mediated the effect of BBR on p21 (CIP1/WAF1) expression. We showed that the knockdown of FOXO3a blocked, while overexpression of FOXO3a

augmented the increase in p21 (CIP1/WAF1) protein expression in BBR-treated cells. These, together with the observation from silencing of p53 experiments indicated that p21 (CIP1/WAF1) is not only the direct target of p53 but also function as FOXO3a downstream effector, which may be through the p53-independent GBA3 way [17]. p53 and FOXO3a share similar target genes including p21(CIP1/WAF1), FOXO factors bind to the promoter of p21 to induce cell cycle arrest at the G1/S transition [39]. Given the fact that p21 (CIP1/WAF1) is involved in regulation of fundamental cellular processes, such as cell proliferation, differentiation, regulation of gene transcription and apoptosis [40, 41]. BBR-induced FOXO3a expression may contribute to induce cell apoptosis, which could be in part a consequence of inhibition of NSCLC cell growth. Of note, the dual function of p21 (Cip1/Waf1) was observed in cancerogenesis. On the one hand, p21 (Cip1/Waf1) acts as a tumor suppressor; on the other hand, it prevents apoptosis and acts as an oncogene [40, 42]. Therefore, precise understanding the role of p21 (Cip1/Waf1) and relevant signaling pathways involved would help to develop better cancer-treatment strategies. Study showed that activation of p38 MAPK reduced protein expression of cyclin D1, another cell cycle regulator [43].

Forty-six (69 7%)

of 66 male patients were categorized in

Forty-six (69.7%)

of 66 male patients were categorized in the low group, whereas only 15 (44.1%) of 34 female patients were categorized in this group. Table 1 Correlation between serum adiponectin level and clinicopathological characteristics in gastric cancer patients.   Adiponectin high group (n = 39) Adiponectin low group (n = 61) p value Age (y) 63.5 ± 12.1 60.6 ± 13.2 0.275 Gender          Male 20 46 0.013    Female 19 15   BMI 22.1 ± 3.6 23.4 ± 3.9 0.079 Macroscopic type          Elevated 5 6 0.642    Depressed/flat 34 55   Depth MM-102 mw of invasion          T1 15 31 0.227    T2, T3 and T4 24 30   Histological type          differentiated 17 22 0.558    undifferentiated 23 38   Lymphatic invasion          positive 32 42 0.142    negative 7 19   Venous invasion          positive 22 33 0.821    negative 17 28   Lymphatic metastasis          positive 23 34 0.750    negative 16 27   Peritoneal dissemination          positive 9 8 0.196    negative 30 53   Hematogenous metastasis          positive 1 3 0.558    negative 38 58   Stage          I and II 26 41 0.910    III and IV 13 20   AdipoR1/R2 expression in gastric cancer The protein expression of AdipoR1 and AdipoR2 was confirmed by immunostaining of surgically resected gastric cancer tissue specimens (selleck chemicals llc Figure 4). AdipoR1 and AdipoR2 were positively

detected in the cytoplasm as well as the cell membrane of cancer cells. In contrast, normal gastric epithelial cells did not show significant immunoreactivity for either AdipoR1 or AdipoR2. In some parietal cells of normal gastric mucosa, slight reactivity was observed in AdipoR2 expression. This was in accordance with the findings of Ishikawa et al [28]. Figure 4 Representative photomicrographs. Representative photomicrographs of immunohistochemical staining of AdipoR1 (A, normal mucosa; B, cancer tissue)

and AdipoR2 (C, normal mucosa; D, cancer tissue). AdipoR1 and AdipoR2 were expressed in normal gastric mucosa in the cytoplasm as well as in the cell membrane. In gastric cancer tissues, higher intensity of immunostaining compared to normal mucosa was considered positive. Original magnification, ×100. AdipoR1 expression was significantly associated with Endonuclease histopathological type (p = 0.011) (Table 2). In addition, negative AdipoR1 immunostaining was significantly higher in patients with lymphatic metastasis (p = 0.013; Table 2) and peritoneal dissemination (p = 0.042; Table 2). On the other hand, AdipoR2 expression was also associated with the histopathological type (p = 0.001; Table 3). However, no significant differences were observed in other clinicopathological characteristics (Table 3). Table 2 Expression of AdipoR1 and clinicopathological characteristics in gastric cancer patients.

Collectively, these molecules seem to act coordinately to regulat

Collectively, these molecules seem to act coordinately to regulate the development of mature biofilms. Methods Bacterial strains and media The P. gingivalis strains used in this study are shown in Table 4. P. gingivalis cells were inoculated from blood agar plates and grown anaerobically (85% N2, 10% H2, 5% CO2) at 37°C in trypticase soy broth supplemented with 1 mg/ml of yeast extract, 1 μg/ml of menadione and 5 μg/ml of hemin (TSB). At stationary phase, the cells were harvested by centrifugation at 6,000 × g for 7 minutes, resuspended in pre-reduced 10 mM phosphate

buffer containing 0.15 M sodium chloride (PBS; pH 7.4) and then used in the assays. When necessary, the following antibiotics were used at the concentrations shown in parentheses: chloramphenicol (20 μg/ml), erythromycin (10 μg/ml), and tetracycline Combretastatin A4 cost (1 μg/ml). To observe initial attachment and ARN-509 order organization of biofilms, P. gingivalis cells were anaerobically incubated in pre-reduced PBS without a nutrition source [19]. In order to monitor an increase in biovolume due to cell division as well as exopolysaccharide accumulation, bacterial cells were cultured in TSB medium diluted with PBS (dTSB; TSB/PBS ratio, 1:2) [47]. Table 4 P. gingivalis strains used in this study Strain Genotype Relevant properties Reference 33277 Wild type Wild type

ATCC KDP150 fimA::erm Long fimbria (FimA)- deficient [20] MPG67 mfa1::erm Short fimbria (Mfa1)- deficient [18] MPG4167 fimA::erm mfa1::tetQ Long and short

fimbria-deficient [18] KDP129 kgp::cat Kgp-null [20] KDP133 rgpA::tetQ rgpB::erm Rgp-null [20] KDP136 rgpA::erm rgpB::tetQ kgp::cat Rgp/Kgp-null [20] Autoaggregation assay An autoaggregation assay was essentially performed as described previously [48]. Briefly, 1 ml of P. gingivalis suspension (4 × 108 cells) was transferred into a UV-cuvette then incubated at 37°C with stirring. Autoaggregation was monitored by measuring the decrease in optical density at A 550 (OD550) using a UV-visible recording Benzatropine spectrophotometer (UV-265FW; Shimadzu Co. Kyoto, Japan). During the incubation, dA/dt was continuously calculated and recorded by subtraction of At, the absorbance at time t min, from At+, at time (t + 1) min. The maximum value of – dA/dt in this curve was used as the autoaggregation activity [48]. The data LY2874455 concentration represent the mean ± standard error of three separate experiments with each strain in duplicate. Saliva Saliva stimulated by mastication of paraffin balls was collected in a sterile centrifuge tube on ice from healthy donors and pooled, as described previously [49]. Dithiothreitol (Sigma-Aldrich, St. Louis, MO) was added to a 2.5 mM final concentration, then the saliva was gently stirred on ice for 10 minutes and centrifuged at 3,000 × g for 20 minutes at 4°C.