Our results

revealed a closure of wound within 12h in control siRNA transfected cells, while MDA-MB-231 cells transfected with SPAG9 siRNA failed to close the wound scratch even after 48 h (Figure 4). This data clearly indicated that SPAG9 is involved in cellular motility and early spread of breast cancer cells, suggesting that SPAG9 may be involved in migration and invasion of MDA-MB-231 cells. Figure 4 Down regulation of SPAG9 causes reduction in wound healing capacity of MDA-MB-231 cells. MDA-MB-231 cells transfected with SPAG9 siRNA showed significantly reduced cellular motility even after 48 h. In contrast, MDA-MB-231 cells transfected selleck products with control siRNA revealed closing of wound within 12 h. Results are from three independent experiments. SPAG9 depletion reduced tumor growth in vivo Our in vitro data indicated that ablation of SPAG9 expression by SPAG9 siRNA significantly reduced colony formation which led us to investigate

its effect on human breast xenograft tumor growth in nude mice in vivo. To determine the effect of SPAG9 siRNA or control siRNA on tumor growth, mice were treated with control siRNA or SPAG9 siRNA and were observed for 42 days. A representative photograph shows reduced tumor growth in SPAG9 siRNA treated group compared with control siRNA treated group (Figure 5a). The tumor volume of mice injected Obeticholic Acid concentration with SPAG9 siRNA showed a significant reduction in tumor growth as compared to mice administered with control siRNA (Figure 5b; P < 0.001). Furthermore, in order to investigate whether the reduction of tumor growth is a result of ablation of SPAG9 expression, the xenograft tumors were excised and processed for immunohistochemical staining for SPAG9 protein expression. As depicted in Figure 5c, the SPAG9 protein was ablated in SPAG9 siRNA treated mice compared with mice treated with control siRNA. Furthermore to investigate whether SPAG9 siRNA treated animals which

showed reduced tumor growth was associated with reduced cellular proliferation, serial tumor sections were probed for PCNA expression. Our data revealed that there was significant reduction of PCNA expression (72%; P < 0.0001) in tumors treated with SPAG9 siRNA treated compared with control siRNA as shown in Figure 5c and histograms (Figure 5d). These results Methane monooxygenase suggest that SPAG9 may be a molecular target for novel cancer treatment modalities. Figure 5 Effect of down regulation of SPAG9 expression in breast cancer xenograft model. (a) A representative photomicrograph showing nude mice with tumor (arrows) treated with control siRNA or SPAG9 siRNA plasmid. (b) a graph representing tumor volume calculated on the indicated days revealed significant reduction in the tumor growth in mice treated with SPAG9 siRNA plasmid compared with control siRNA (n = 8; *, P < 0.0001). (c) Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) and SPAG9 protein in control siRNA and SPAG9 siRNA treated tumors.

CrossRef 49. Sanjaq S:Enterobacter sakazakii – Risikoprofil

und Untersuchungen zum Nachweis in Säuglingsnahrungen. Ph. D. thesisGiessen: Justus-Liebig-Universitaet 2007. 50. Ewing WH, Fife MA:Enterobacter agglomerans (Beijerinck) comb. nov. (the herbicola-lathyri bacteria). Int J Syst Bacteriol1972,22(1):4–11.CrossRef 51. Mergaert J, Hauben L, Cnockaert MC, Swings J:Reclassification of non-pigmented Pexidartinib supplier Erwinia herbicola strains from trees as Erwinia billingiae sp. nov. Int J Syst Bacteriol1999,49:377–383.CrossRefPubMed 52. Tamura K, Sakazaki R, Kosako Y, Yoshizaki E:Leclercia adecarboxylata gen. nov., comb. nov., formerly known as Escherichia adecarboxylata.Curr Microbiol1986,13:179–184.CrossRef 53. Beji A, Mergaert J, Gavini F, Izard D, Kersters K, Leclerc H, De Ley J:Subjective synonymy

of Erwinia herbicola,Erwinia milletiae, and Enterobacter agglomerans and redefinition of the taxon by genotypic and GSK-3 beta phosphorylation phenotypic data. Int J Syst Bacteriol1988,38(1):77–88.CrossRef 54. Mergaert J, Verdonck L, Kersters K:Transfer of Erwinia ananas (synonym, Erwinia uredovora ) and Erwinia stewartii to the genus Pantoea emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii (Smith 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov. Int J Syst Bacteriol1993,43(1):162–173.CrossRef 55. Lind E, Ursing J:Clinical strains of Enterobacter agglomerans (synonyms: Erwinia herbicola,Erwinia milletiae ) identified by DNA-DNA-hybridization. Acta path microbiol immunol scand Sect B1986,94:205–213.

56. Grimont PAD, Grimont F, Farmer JJ, Asbury MA:Cedecea davisae gen. nov, sp. nov. and Cedecea lapagei sp. nov, new Enterobacteriaceae from clinical specimens. Int J Syst Bacteriol1981,31:317–326.CrossRef 57. Rezzonico F, Defago G, Moenne-Loccoz Y:Comparison of ATPase-encoding type III secretion system hrcN genes in biocontrol fluorescent Pseudomonads and in phytopathogenic proteobacteria. Applied and environmental microbiology2004,70(9):5119–5131.CrossRefPubMed 58. Jin M, Wright SAI, Beer SV, Clardy J:The biosynthetic gene cluster of pantocin A provides insights into biosynthesis and a tool for screening. Angew Chem Int Ed2003,42:2902–2905.CrossRef 59. Beijerinck MW:Cultur des Bacillus radicicola aus den Knollchen. Bot Zeitung1888,46:740–750. CYTH4 60. Dye DW:A taxonomic study of the genus Erwinia . III. The “”herbicola”" group. N Z J Sci1969,12:223–236. 61. Graham DC, Hodgkiss W:Identity of gram negative, yellow pigmented, fermentative bacteria isolated from plants and animals. J Appl Bacteriol1967,30:175–189.PubMed 62. Leliott RA:Genus XII. Erwinia . Winslow, Broadhurst, Buchanan, Krumwiede, Rogers and Smith 1920. Bergey’s manual of determinative bacteriology 8 Edition (Edited by: RE B, Gibbons NE).Baltimore: The Williams & Wilkins Co 1974, 332–359. 63. Dauga C:Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies.

Still, in some experiments the promoter activity was abolished while others showed only a low activity – a finding that deserves further attention. In this paper we have shown that the part of the hupSL promoter region that gave the highest expression level is limited to a 316 bp DNA fragment stretching from -57 (in relation to tsp) to the translation start site (Fig. 4). Not only does this short promoter confer a high transcription level, it also retains

heterocyst specificity. A loss of heterocyst specificity could have lead to a misleading conclusion of high promoter activity: SCH772984 price the promoter would have shown high total expression, due to expression in all cells, even if the promoter activity was still low. However the fact that this promoter fragment kept heterocyst specificity (Fig. 5) enables us to draw the conclusion that the activity of the shortest promoter is truly higher than for the other promoter fragments. One assumption could be that heterocyst specificity of expression is due to a transcription factor binding to the hupSL promoter

and stimulating transcription in heterocysts. However, another possibility could be that hupSL is constitutively Atezolizumab datasheet transcribed and that vegetative cells contain a repressor lacking in heterocysts which restrain transcription in that cell type. If the heterocyst specificity is mediated by an activator binding the short promoter sequence upstream the tsp (or perhaps the untranslated leader region downstream the tsp) or by a repressor only present in vegetative cells needs to be subjected to further investigations. Further characterization of

this short promoter region will not only give information about what promotes hupSL transcription but can also help answering the question what directs heterocyst specific expression of genes and pattern formation in N. punctiforme, and perhaps other heterocystous, filamentous cyanobacteria. Conclusion The result that the 57 bp promoter is a highly active promoter is most interesting and will be investigated further. This short DNA sequence, and its 258 bp untranslated leader region Arachidonate 15-lipoxygenase downstream the tsp, appears to harbour enough information to make the transcription to occur in heterocysts only. Taken one step further, if this information conferring heterocyst specific transcription can be elucidated it will give clues to what signals are involved in heterocyst specific gene expression and pattern formation in filamentous cyanobacteria. Acknowledgements This work was supported by the Swedish Energy Agency, the Knut and Alice Wallenberg Foundation, the Nordic Energy Research Program (project BioH2), EU/NEST FP6 project BioModularH2 (contract # 043340) and the EU/Energy FP7 project SOLAR-H2 (contract # 212508). References 1. Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wunschiers R, Lindblad P: Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol Rev 2002,66(1):1–20.PubMedCrossRef 2.

Antimicrob Agents Chemother 1977, 11:773–79.PubMed 27. Guerrero C, Stockman L, Marchesi F, Bodmer T, Roberts GD, Telenti A: Evaluation of the rpoB gene in rifampicin-susceptible and -resistant Mycobacterium avium and Mycobacterium intracellulare. J Antimicrob Chemother 1994, 33:661–3.CrossRefPubMed 28.

Bodmer T, Zurcher G, Imboden P, Telenti A: Mutation R788 solubility dmso position and type of substitution in the beta-subunit of the RNA polymerase influence in vitro activity of rifampin-resistant Mycobacterium tuberculosis. J Antimicrob Chemother 1995, 35:345–48.CrossRefPubMed 29. Moghazeh SL, Pan X, Arain T, Stover CK, Musser JM, Kreiswirth BN: Comparative antimycobacterial activities of rifampin, rifapentine, and KRM-1648 against a collection of rifampin-resistant Mycobacterium tuberculosis buy Temsirolimus isolates with known rpoB mutations. Antimicrob Agents Chemother 1996, 40:2655–57.PubMed 30. Miller LP, Crafword JT, Shinnick TM: The rpoB gene of Mycobacterium tuberculosis. Antimicrob Agents Chemother 1994, 38:805–11.PubMed 31. Hillemann D, Kubica T, Rusch-Gerdes S, Niemann S: Disequilibrium in distribution of resistance mutations

among Mycobacterium tuberculosis Beijing and Non-Beijing strains isolated from patients in Germany. Antimicrob Agents Chemother 2005, 49:1229–31.CrossRefPubMed 32. Huang H, Jin Q, Chen X, Zhuang Y: Characterization of rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolated in China. Tubecrulosis 2000, 82:79–83.CrossRef 33. Ozkutuk N, Gazi H, Surucuoglu S, Gunduz A, Ozbakkaloglu B: Characterization of rpoB mutations by Line Probe Assays in rifampicin-resistant Mycobacterium tuberculosis

clinical isolates from the Aegean region in Turkey. Jpn J Infect Dis 2007, 60:211–13.PubMed 34. Bostanabad S, Bahrmand A, Titov LP, Taghikhani M: Identification of mutations in the rpoB encoding the RNA polymerase beta subunit in rifampicine-resistant Mycobacterium tuberculosis strains from Iran. Tuberk Toraks 2007, 55:370–77.PubMed 35. Brossier PIK3C2G F, Veziris N, Truffot-Pernot C, Jarlier V, Sougakoff W: Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance. J Clin Microbiol 2006, 44:3659–3664.CrossRefPubMed 36. Gryadunov D, Mikhailovich V, Lapa S, Roudinskii N, Donnikov M, Pan’kov S, Markova O, Kuz’min A, Chernousova L, Skotnikova O, Moroz A, Zasedatelev A, Mirzabekov A: Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clin Microbiol Infect 2005, 11:531–9.CrossRefPubMed 37.

In fact, MLH1 and ATM genes play a key role in DNA detection and

repair systems and their inactivation may cause genomic DNA to become more unstable and error-prone, increasing the risk of transformation. The MLH1 protein is involved in the DNA mismatch repair system (MMR) and methylation of this gene has been observed in CRC, especially in tumors characterized by MSI, a molecular marker of the presence of defective MMR [25,26]. The ATM protein, a serine/threonine kinase involved in DNA double-strand break repair, is also involved in DNA repair and its inactivation is a highly destabilizing event for the cell, promoting the progression of neoplastic disease [27,28]. It is interesting to note that MLH1 is an independent variable, despite the molecular interaction between MLH1 and ATM in regulating DNA Ulixertinib ic50 repair. This suggests that concurrent inactivation of both genes may also

be important in cancer development. FHIT, a tumor suppressor gene involved in numerous important mechanisms associated with cell cycle response to stress signals and DNA replication control, is another independent variable [29]. Wali reported that the FHIT gene loses its ability to produce its specific protein in the early stages of lung, head and neck, esophageal, colorectal, breast, and cervical cancer [30]. The diminution or loss of FHIT protein expression appears to be influenced by the extensive promoter methylation program manifested in CIMP-high CRC cases [31]. TP73 and BRCA1 genes, both Sirolimus manufacturer related to a higher risk of recurrence, are also involved in cell cycle control and DNA repair. In particular, TP73 is a homolog of TP53 tumor suppressor gene, known to be involved in the regulation of cell proliferation and

apoptosis [32-34], while BRCA1 represents a key regulator in the repair of double-stranded DNA breaks [26,35]. In Huang et al.’s 2010 study on 110 stage I to IV CRC patients, TP73 and BRCA1 were identified from a panel of 15 radiation-related genes as prognosis-related markers on the basis of their significant correlation with clinical prognostic variables [36]. In our study, methylation status analysis of a combination of the three most significant genes (MLH1, ATM, PRKACG FHIT) confirmed that they could be used to accurately identify patients at a higher risk of recurrence. Moreover, it is worthy of note that these genes (MLH1, ATM, FHIT, TP73 and BRCA1) were not among those most frequently methylated in our case series, suggesting that the risk of recurrence is related to specific molecular characteristics. In fact, higher aberrant methylation (more than 70% of cases with methylation levels higher than 20%) was noted for ESR1 and CDH13, which are not associated with a risk of recurrence.

J Pathol RXDX-106 research buy 2008, 214:283–293.PubMedCrossRef 27. Deryugina EI, Quigley JP: Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 2006, 25:9–34.PubMedCrossRef 28. Folkman J: Tumor angiogenesis and tissue factor. Nat Med 1996, 2:167–168.PubMedCrossRef 29. Hembrough TA, Swartz GM, Papathanassiu A, Vlasuk GP, Rote WE, Green SJ, Pribluda VS: Tissue factor/factor VIIa inhibitors block angiogenesis

and tumor growth through a nonhemostatic mechanism. Cancer Res 2003, 63:2997–3000.PubMed 30. Koomagi R, Volm M: Tissue-factor expression in human non-small-cell lung carcinoma measured by immunohistochemistry: correlation between tissue factor and angiogenesis. Int J Cancer 1998, 79:19–22.PubMedCrossRef 31. Yu JL, May L, Lhotak V, Shahrzad S, Shirasawa S, Weitz JI, Coomber BL, Mackman N, Rak JW: Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis. Blood 2005, 105:1734–1741.PubMedCrossRef 32. Versteeg HH, Spek CA, Peppelenbosch MP, Richel DJ: Tissue factor and cancer metastasis: the role of intracellular and extracellular signaling pathways. Mol Med 2004, 10:6–11.PubMedCrossRef 33. D’Andrea MR, Derian CK, Santulli RJ, Andrade-Gordon P: Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts SB525334 price of normal, benign, and malignant human tissues. Am J Pathol 2001, 158:2031–2041.PubMedCrossRef 34. Dorsam RT, Gutkind JS:

G-protein-coupled receptors and cancer. Nat Rev Cancer 2007, 7:79–94.PubMedCrossRef 35. Widmann C, Gibson S, Jarpe MB, Johnson GL: Mitogen-activated

protein kinase: conservation of a three-kinase module from yeast to human. Physiol selleck compound Rev 1999, 79:143–180.PubMed 36. Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, Greenberg ME: Regulation of neuronal survival by the serine-threonine protein kinase AKT. Science 1997, 275:661–665.PubMedCrossRef 37. Shoji M, Sun A, Kisiel W, Lu YJ, Shim H, McCarey BE, Nichols C, Parker ET, Pohl J, Mosley CA, Alizadeh AR, Liotta DC, Snyder JP: Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa. J Drug Target 2008, 16:185–197.PubMedCrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions XC and GQ have contributed to the research design, the data collection and manuscript writing. CW, WL, SW, ZN, XQ and WJ have contributed to manuscript writing. FN has contributed to the research design and manuscript writing. All authors read and approved the final manuscript.”
“Background Tumor-associated macrophages (TAMs) are the most abundant cancer stromal cells involved in the host immune system [1, 2]. In recent years, increasing attention has focused on TAMs, unique macrophage populations that play pivotal roles in tumor immunosuppression, and provide a suitable microenvironment for cancer development and progression[3].

Case report. J Gastrointestin Liver Dis 2006, 15:297–299.PubMed 2. Oida Y, Motojuku M, Morikawa G, Mukai M, Shimizu K, Imaizumi T, Makuuchi H: Laparoscopic-assisted resection of gastrointestinal stromal tumor in small MG 132 intestine. Hepatogastroenterology 2008, 55:146–149.PubMed 3. Miettinen M, Sobin LH, Lasota J: Gastrointestinal stromal tumors presenting

as omental masses–a clinicopathologic analysis of 95 cases. Am J Surg Pathol 2009, 33:1267–1275.PubMedCrossRef 4. Sornmayura P: Gastrointestinal stromal tumors (GISTs): a pathology view point. J Med Assoc Thai 2009, 92:124–135.PubMed 5. Steigen SE, Bjerkehagen B, Haugland HK, Nordrum IS, Løberg EM, Isaksen V, Eide TJ, Nielsen TO: Diagnostic and prognostic markers see more for gastrointestinal stromal tumors in Norway. Mod Pathol 2008, 21:46–53.PubMedCrossRef 6. Wilson SL, Wheeler WE: Giant leiomyoma of the small intestine with free perforation into the peritoneal cavity. South Med J 1992, 85:667–668.PubMedCrossRef 7. Shah SN: Malignant gastrointestinal stromal tumor of intestine: a case report. Indian J Pathol Microbiol 2007, 50:357–359.PubMed 8. Huang CC, Yang CY, Lai IR, Chen CN, Lee PH, Lin MT: Gastrointestinal stromal tumor of the small intestine: a clinicopathologic study of 70 cases in the postimatinib era. World J Surg 2009, 33:828–834.PubMedCrossRef 9. Kingham TP, DeMatteo RP: Multidisciplinary treatment of gastrointestinal stromal tumors. Surg Clin North Am 2009, 89:217–233.PubMedCrossRef

10. Annaberdyev S, Gibbons J, Hardacre JM: Dramatic response of a gastrointestinal stromal tumor to neoadjuvant imatinib therapy. World J Surg Oncol 2009, 7:30.PubMedCrossRef Competing interests The authors

declare that they have no competing interests. Authors’ contributions UD participated in the conception, design of the study, sequence alignment and drafted the manuscript. SD carried out the immunohistochemical studies. DK participated in the clinical and surgical management. KKD helped to draft the manuscript. All authors read and approved the final manuscript.”
“Introduction Intra-abdominal infections (IAIs) include a wide spectrum of pathological conditions, ranging from uncomplicated appendicitis to fecal Dichloromethane dehalogenase peritonitis. In the event of complicated IAI [1], the infection proceeds beyond a singularly affected organ and causes either localized peritonitis (intra-abdominal abscesses) or diffuse peritonitis. Effectively treating patients with complicated intra-abdominal infections involves both source control and antimicrobial therapy [2, 3]. Study design The aim of the CIAO Study was to describe the epidemiological, clinical, microbiological, and surgical treatment profiles of community-acquired and healthcare-associated complicated intra-abdominal infections (IAIs) based on data collected over a 6-month period (January-June 2012) from 68 medical institutions throughout Europe (see Figure 1). Figure 1 Geographic distribution of the CIAO Study.

The MTT method is a quantitative colorimetric toxicity

test, based buy Osimertinib on the transformation of yellow, soluble tetrazolium salts (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to purple-blue insoluble formazane. This process occurs naturally in mitochondria of living cells. After 48 h incubation with compounds, cell cultures were supplemented with 10 μl of 5 mg ml−1 MTT solution per well, and further incubated for 4 h at 37 °C. Afterwards, 100 μl of water solution, including 50 % dimethylformamide and 20 % SDS, per well was added and after the all-night incubation the absorbance was measured by the 96-well plastic plate reader (Organon Teknika) at wavelengths of λ = 540 and 620 nm. The medium with

DMSO at tested concentration range without the tested compound served as control––it was not toxic to vero cells line. The experiments were carried out in duplicates. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Agrawal Small molecule high throughput screening A, Murphy TF (2011) Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era. J Clin Microbiol 49:3728–3732PubMedCentralPubMedCrossRef Amer FAA, El-Behedy EM, Mohtady HA (2008) New targets for antibacterial agents. Biol Rev Camb Philos Soc 3:46–57 Armbruster CE, Hong W, Pang B, Dew KE, Juneau RA, Byrd MS, Love CF, Kock ND, Swords EW (2009) LuxS promotes biofilm maturation and persistence of nontypeable Haemophilus influenzae in vivo via modulation of

lipooligosaccharides on the bacterial surface. Infect Immun 77:4081–4091PubMedCentralPubMedCrossRef Bassler BL (1999) How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr Opin Microbiol 2:582–587PubMedCrossRef Bekhit AA, Abdel-Aziem T (2004) Design, synthesis and biological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial Clostridium perfringens alpha toxin agents. Bioorg Med Chem 12:1935–1945PubMedCrossRef Bjarnsholt T (2013) The role of bacterial biofilms in chronic infections. APMIS doi: 10.​1111/​apm.​12099 Bjarnsholt T, Givskov M (2007) Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens. Philos Trans Royal Soc Lond B 362:1213–1222CrossRef Black CT, Kupferschmid JP, West KW, Grosfeld JJ (1988) Haemophilus parainfluenzae infection in children with the report of a unique case. Rev Infect Dis 10:342–346PubMedCrossRef Bottone EJ, Zhang DY (1995) Haemophilus parainfluenzae biliary tract infection: rationale for an ascending route of infection from the gastrointestinal tract.

The complete ORF of MaAC encoded a predicted protein LBH589 research buy of 2,169 amino acids (aa) with a molecular mass of 542.0 kDa. An analysis using SignalP

suggested that the N-terminal sequence of MaAC had no signal peptide. The predicted protein had a high similarity to the adenylate cyclase gene (ACY) of Metarhizium anisopliae (98% identity, EFY97222.1), the adenylate cyclase gene of Cordyceps militaris (98% identity, EGX90508.1), MAC1 of M. oryzae (96% identity, AAC34139.1) and SAC1 of S. sclerotiorum (95% identity, ABF71879.1). A fungal phylogenetic tree was established using MEGA 4.0 (Figure 1). MaAC was most similar to the sequence of the entomopathogenic fungus M. anisopliae, belonging to the Sordariomycetes. All species were members of the subdivision Pezizomycotina

in the division Ascomycota. Figure selleck chemicals llc 1 Neighbor-joining tree inferred from  MaAC  protein sequence alignment. Numbers on the nodes represent the results of bootstrap analyses (1,000 replicates), using the neighbor-joining method. Fungal species: M. acridum (JQ358775), Metarhizium anisopliae (EFY97222.1), Cordyceps militaris (EGX90508.1), Gibberella zeae (XP_381410.1), Gibberella intermedia (AAY79378.1), Colletotrichum lagenarium (BAD04045.1), Magnaporthe oryzae (AAC34139.1), Grosmannia clavigera (EFW99531.1), Chaetomium globosum (XP_001221049.1), Neurospora crassa (BAA00755.1), Neurospora tetrasperma (EGZ77248.1), Blumeria graminis (CAC19663.1), Sclerotinia sclerotiorum (ABF71879.1), Botryotinia fuckeliana (CAB77164.1), Paracoccidioides

brasiliensis (AAS01025.1), Ajellomyces dermatitidis (XP_002624019.1), Coccidioides posadasii (EFW21958.1), Penicillium marneffei (XP_002146654.1), Aspergillus niger (XP_001394156.2), Spathaspora passalidarum (EGW29847.1), Aspergillus fumigates (CAC81748.1), Aspergillus clavatus (XP_001268121.1), Spathaspora passalidarum (EGW29847.1). Knocked-down MaAC transcription by RNAi We conducted an RNA interference (RNAi) strategy to study the function of MaAC. Phosphinothricin-resistant transformants of M. acridum were generated by transformation with the vector pK2-Pb-MaAC-RNAi HAS1 (Figure 2A). To investigate the efficiency of RNAi, the wild type and RNAi mutants of MaAC were analyzed by quantitative RT-PCR. Compared to the wild type, MaAC transcription in the RNAi mutants was downregulated by 66.0%, 43.5%, 23.1%, 36.2% and 38.8%, respectively (Figure 2B). These results demonstrated that the transcription of MaAC was efficiently knocked down. Figure 2 Construction and quantitative RT-PCR analysis of the AC-RNAi mutant. A. Maps of pPK2-Pb-MaAC-RNAi, the silencing vector for MaAC. PgpdA: promoter of gpd from A. nidulans; bar: herbicide resistance gene; TtrpC: terminator of trpC from A. nidulans; AC: partial sequence of the adenylate cyclase element gene in M. acridum. B. Relative expression of MaAC in the wild type (calibrated as 100%) and three RNAi strains. Error bars denote standard deviations of three trials.

For confocal analysis five animals of each developmental stage were investigated. Confocal laser scanning microscopy (CLSM) Midguts were dissected from individuals and gut content washed out in sterile PBS. Subsequently the midgut samples were fixed on microscopic slides and permeabilized as described previously [13]. Hybridization was carried out by default with FITC-labeled oligonucleotide Bfl172 specific for B. floridanus 16S rRNA which had been used successfully in a previous study for fluorescent in situ hybridization studies [13]. The probe was labeled with the dye

at the 5′end as described by the manufacturer (Metabion International AG, Planegg-Martinsried, Germany). Alternatively, red fluorescent Cy3-labeled Bfl172 was used. For CLSM with a Leica DMR laser scanning microscope (Leica Microsystems SCH727965 ic50 Obeticholic Acid clinical trial GmbH, Wetzlar, Germany) these labeled oligonucleotides were applied in combination with SYTO Orange 83 (Molecular Probes Inc.) with a concentration of 2.5 – 5 μM in TE buffer, pH 7.4, resulting in unspecific nucleic acid counterstaining of cytoplasm as well as mitochondria and nuclei after 30 minutes post-FISH incubation and 5 minute washing in TE buffer

at room temperature. For actin-staining 0.5 ng/μl FITC-Phalloidin (Invitrogen Inc.) was used (the B. floridanus specific probe was coupled to Cy3 instead of FITC in the respective experiments). The dyes were used according to the manufacturers’ protocols. Confocal images were analyzed

with the Leica Application Suite Advanced Fluorescence Software (Leica Microsystems GmbH, Wetzlar, Germany). Each of the images shown is representative Methane monooxygenase for a series of preparations from the respective host stage with very similar appearances. For the quantification of Blochmannia population densities of ant guts in different larval developmental stages, exemplarily shown in Figure 1 to 10, were calculated as follows: optical sections of gut preparations were recorded by CLSM (see above). Images in the Leica-specific lif file format were opened as ImageJ hyperstacks [31] making use of the LOCI bio-formats plugin (http://​loci.​wisc.​edu/​software/​bio-formats). The stack corresponding to the FITC channel was thresholded and binarized. The area fraction of labeled Blochmannia symbionts was thus measured within each confocal slice. Area fractions were collected for each slice of a stack, summed up, and normalized for the number of slices. The resulting value was termed volume fraction of symbionts (Figure 12). Differences in volume fractions among developmental stages were compared using a one-factorial ANOVA, after homogeneity of variances had been confirmed by Levene’s test implemented in SPSS 15.0 (SPSS Inc. Chicago, Illinois, USA). Acknowledgements We thank Dagmar Beier and Achim Paululat for critical reading of the manuscript and Adrian Mehlitz for help with confocal microscopy.