Sakamoto CHIR98014 price K, Iwashita K, Yamada O, Kobayashi K, Mizuno A, Akita O, Mikami S, Shimoi H, Gomi K: Aspergillus oryzae atfA controls conidial germination and stress tolerance. Fungal Genet Biol 2009,46(12):887–897.PubMedCrossRef 45. Novodvorska M, Hayer K, Pullan ST, Wilson R, Blythe MJ, Stam H, Stratford M, Archer DB: Trancriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing. BMC Genomics 2013, 14:246.PubMedCentralPubMedCrossRef Competing

interests The authors declare the absence of competing interests. Authors’ contributions ÅS performed the majority of the laboratorial work. ÅS and PM performed all experiments with exception of the RNA extraction from dormant conidia and conidia in early stages of germination, performed by MRL, and the SEM studies, performed by JD. ÅS and PM conceived and designed the study and wrote the manuscript. All authors read and approved the final manuscript.”
“Background The increase in carbapenemase-producing Enterobacteriaceae and Pseudomonas

aeruginosa is a significant threat to modern medicine [1]. As treatment options are very limited, infection control measures are important to contain carbapenemase-producing isolates in health care settings. Rapid detection of carbapenemase-producers is a decisive for adequate infection control measures to be undertaken. The methods used so far for the detection of carbapenemases have been phenotypic methods or PCR [2, 3] Recently, Matrix Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) has been AZD2171 molecular weight introduced in clinical microbiology for species identification and during the last two years a few studies have shown the proof of concept regarding the detection of β-lactamases using this DOCK10 technology [4–6]. These studies have either analyzed a small set of strains [4] or focused on the detection of hydrolysis rather than the verification of specific enzymes [5–8]. All studies have used different protocols and different sets of species/enzyme combinations. In the present study we present a method for the simultaneous detection and discrimination of KPC from the metallo-β-lactamases

(MBL) NDM and VIM in Klebsiella https://www.selleckchem.com/products/elafibranor.html pneumoniae and the possibility of verification of VIM in Pseudomonas aeruginosa through a time dependent hydrolysis assay and the addition of specific inhibitors, APBA (3-aminophenylboronic acid) and DPA (2.6-Pyridinecarboxylic acid). Results Stability of ertapenem Ertapenem was stable after one week and six months when stored at −20°C, but degraded after one week when stored at +4°C. The frozen aliquots were used for further analysis. Klebsiella pneumoniae (n = 40) All the KPC producing K. pneumoniae (n = 10) displayed the specific ertapenem hydrolysis peak pattern after 15 min incubation (Figure 1, middle). As no potassium was included in this assay only the sodium ions of hydrolysed ertapenem with the m/z ratios of 450.5, 472.5, 494.5, 516.5 and 538.5 were detected.

In the first three

cases ATP production is low and for the latter the possibility of spending a lot of energy to transport the heme compound into the cell, which also results in a low ATP balance. So, in all cases, the cell would probably optimize energy expenditure for survival. Whereas the process of pathogenesis demands the production of various enzymes, proteins and other compounds, these observations suggest that these processes will not be realized because of their high energy consumption. Consequently, although the cell survives reasonably well, both in vitro and in planta, it will not develop the disease and thus no external symptoms will be observed. Finally, the fact that the mutant hemB had a growth curve in planta very similar to wild type may be an indication that it is performing aerobic metabolism due to internalization of heme compounds click here from the host and not causing the disease because the energy balance is not favourable, since transport through the membrane consumes so much energy. Histidine kinases are proteins that can play a major process in bacterial metabolism. These proteins, together with their cognate response regulators (RR), can be part of two component systems (TCS), which constitute a signal transduction process in which bacteria sense, respond, and adapt

TPX-0005 cell line to changes in their environment or intracellular State. Signal transduction starts when a histidine kinase senses a signal, e.g., by

binding or reacting with a signaling molecule or due to a physical stimulus, and phosphorylates downstream proteins in the phosphorylation cascade that modulate the activity of a final set of protein targets, which then modulate protein activity or differential gene expression. Based on their components, two TCS exist: prototypical and phosphorelay systems [35]. In the phosphorelay TCS pathway, a stimulus activates autophosphorylation of a hybrid histidine kinase, Epigenetics inhibitor namely, a histidine kinase containing Gefitinib cost a phospho-accepting receiver domain, typically at the C-terminal end of the protein. The catalytic and ATPase (HATPase – PF02518 – Pfam A accession – http://​pfam.​sanger.​ac.​uk/​help) domain of the histidine kinase is responsible for binding ATP and catalyzing autophosphorylation of a conserved histidine found within the dimerization and histidine phosphotransferase (HisKA – PF005121) domain. The HisKA domain mediates homodimerization and serves as the phosphodonor for a C-terminal receiver domain (response regulator – PF00072), similar to that found in response regulators. A histidine phosphotransferase (HPT – PF01627) then shuttles the phosphoryl group from the hybrid kinase to a soluble response regulator containing an output domain through protein-protein interaction or protein-DNA interactions leading to differential gene expression [36–38].

More recent perspectives of the OA movement were discussed during the seminar held in Granada in May 2010, Open Access to science information: policies for the development of OA in Thiazovivin mouse Southern Europe [6], attended Cell Cycle inhibitor by the delegates (researchers and information specialists) of six Mediterranean countries of South Europe (France, Italy, Turkey, Greece, Portugal). This

seminar stressed the importance of the following actions: link the open digital archives to the National Research Anagrafe; guarantee high quality standards of the OA journals; reduce the cost of publications by moving from the paper to the digital publishing; define common standard to facilitate the gathering and aggregation of metadata. Moreover, a new service announced at the Berlin 8 Conference on Open Access held in Beijing

in October 2010 and intended to implement OA strategies is about to be launched by OASIS (Open Access Scholarly Information Sourcebook) in 2011: The open access map [7] a world map and chronology which shows all OA projects, services, initiatives and their development over the last ten years. Open access in Italy As far as Italy is concerned, an important breakthrough for the academic world was marked by the Messina Declaration, in 2004, the first institutional action on the part of the chancellors of the Italian universities in favour of OA. This event represented the starting point of an action towards the statement of policies requiring Everolimus researchers to deposit their papers in institutional repositories and to publish research articles in OA journals. Among the most recent Italian initiatives aimed at promoting the OA philosophy, it is worth mentioning the launch in 2008 of the Italian wiki on open access [8], conceived as Palbociclib cost a reference point on Italian projects and best practices. Another reference point

is also the DRIVER wiki containing a section devoted to Open access in Italy [9] while the state of the art of the OA initiatives is described in Open Access in Italy: report 2009 offering a wide overview on the ongoing projects and experiences [10]. Open access in science and medicine A decisive impulse to the unrestricted availability of research results (scientific publications and data sets) is represented by the OpenAIRE Project (Open Access Infrastructure for Research in Europe) [11]. This Pilot Project, financed by the European Commission and covering the 27 member states of the European Union, has been conceived to deliver both a technical and a networking infrastructure to the benefit of the research community. The former infrastructure is aimed at collecting and providing access to the research articles reporting on outcomes of FP7 and European Research Council (ERC) projects, while the second one, based on the creation of a European Helpdesk System, has been designed to best support the practice of archiving in each EU member state.

O115 Heparanase Role in Oral Cancer Prognosis

and Cellular Differentiation Yoav Leiser 1,4 , Imad Abu-El-Naaj1, Edmond Sabo3, Dan Deutsch5, Philip Lazarovici6, Micha Peptide 17 in vitro Peled1,2, Israel Vlodavsky4 1 The Department of Oral and Maxillofacial Surgery, Rambam Medical Center, Haifa, Israel, 2 The Faculty of Medicine, Technion, check details Haifa, Israel, 3 Department of Pathology, Rambam Medical Center, Haifa, Israel, 4 The Bruce Rappaport Faculty of Medicine, Cancer and Vascular Biology Research Center, Haifa, Israel, 5 Dental Research Laboratory, Institute of Dental Sciences, The Hebrew University Faculty of Dental Medicine, Hadassah Medical Center, Jerusalem, Israel, 6 Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel Background: Numerous studies have shown that metastases formation depends on the ability of tumor cells to invade basement membranes and tissue barriers in a process involving enzymes capable of degrading extracellular matrix (ECM)

components. One of these enzymes is heparanase, an endoglycosidae which degrades heparan sulfate. Purpose: Examine the expression of heparanase in oral carcinomas and establish selleck products whether its extent, intensity and cellular localization can be of prognostic value in predicting the outcome of oral cancer patients

and explore its role during cellular differentiation. Methods: Biopsy specimens from 50 oral carcinoma patients were immunohistochemically analyzed for the expression and cellular localization of heparanase, PC12 (pheochrocmocytoma) cultures were used as an in-vitro model of cellular differentiation induced by NGF. Results: Nuclear localization of heparanase was observed in all oral verrucous carcinomas, a very well differentiated tumor that rarely metastasize, as opposed to only 28% of nuclear localization detected in oral squamous cell carcinomas. Heparanase expression level also significantly correlated with the degree of tumor differentiation. Moreover, while cytoplasmic localization Protein tyrosine phosphatase of heparanase was associated with high grade carcinomas, nuclear localization of the enzyme was found primarily in low grade, well differentiated tumors. Heparanase was suggested to be involved in the differentiation of PC12 cell and was up regulated 6.5 fold during NGF induced cellular differentiation. Furthermore, NGF receptor TrkA seems to be involved in heparanase up regulation in PC12. Conclusion: In rarely metastasizing verrucous carcinomas, heparanase was expressed in the cell nucleus, as opposed to metastasizing oral squamous cell carcinomas which exhibited mostly cytoplasmic localization of the enzyme.

Emerging evidence suggests that radiation-induced modifications of the tumor microenvironment may contribute to the therapeutic effects of radiotherapy. Recurrence after radiotherapy, however, is associated with increased local invasion, metastatic spreading and poor prognosis. We are investigating whether radiation-modified mTOR inhibitor tumor microenvironment may possibly contribute to the increased aggressiveness of relapsing tumors. Irradiation of the prospective tumor bed

results in a sustained impairment of growth factor-driven and tumor angiogenesis without disrupting the preexistent vasculature, through sustained inhibition of proliferation, induction of senescence and inhibition

of migration and sprouting of endothelial cells. Using xenografts tumor models and an orthotopic model of murine breast cancer, we observed AZD6094 purchase that tumors growing within a preirradiated stroma have reduced growth while they display increased hypoxia, necrosis, local invasion and lung metastasis. Mechanisms of progression involve adaptation of tumor cells to local hypoxic conditions as well as the selection of escape variantsretaining an invasive and metastatic phenotype upon returning to normoxia. Though gene expression analysis experiments, Levetiracetam we have identified the matricellular protein CYR61 and αVβ5 integrin as molecules that cooperate to mediate lung metastasis, as well as a gene expression signature associated with tumor hypoxia and predictive for a shorter relapse-free survival after adjuvant radiochemotherapy in human breast cancer. The αV integrin small molecular inhibitor Cilengitide GS-9973 prevented lung

metastasis formation without impinging on primary tumor growth. Radiotherapy also modify the recruitment of bone marrow derived / immune cells known to contribute to tumor angiogenesis and metastasis. Taken together these results demonstrate the impact of radiotherapy-induced modifications of the tumor microenvironment in determining tumor evolution and identify candidate therapeutic targets. We are currently investigating additional cellular and molecular determinants of tumor escape and progression after radiotherapy, and at this conference we will present the latest results.

[http://​www.​hotthyroidology.​com] Hot Thyroidology 2005. 32. Tognella C, Marti U, Peter HJ, Wagner HE, Glaser C, Kampf J, Simon F, Hauselmann HJ, Paulsson M, Ruchti C, et al.: Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis. J Endocrinol 1999, 163:505–514.PubMedCrossRef 33. Beech SG, Walker SW, Dorrance AM, Arthur JR, Nicol F, Lee D, Beckett GJ: The role of thyroidal type-I iodothyronine

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K: Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5. Cytokine 1991, 3:125–130.PubMedCrossRef 37. Gossrau R, Graf R: Protease cytochemistry in the murine rodent, guinea-pig and marmoset placenta. Histochemistry 1986, 84:530–537.PubMedCrossRef VX-689 38. Kotani T, Aratake Y, Ogata Y, Umeki K, Araki Y, Hirai K, Kuma K, Ohtaki S: Expression of dipeptidyl aminopeptidase IV activity in thyroid carcinoma. Cancer Lett 1991, 57:203–208.PubMedCrossRef 39. Hadler-Olsen E, Fadnes B, Sylte I, Uhlin-Hansen L, Winberg JO: Regulation of matrix https://www.selleckchem.com/products/NVP-AUY922.html metalloproteinase activity in health and disease. FEBS J 2011, 278:28–45.PubMedCrossRef 40. Turk B, Turk D, Salvesen GS: Regulating cysteine protease

activity: essential role of protease inhibitors as guardians and regulators. Curr Pharm Des 2002, 8:1623–1637.PubMedCrossRef 41. van der Hoorn RA, Leeuwenburgh MA, Bogyo M, Joosten MH, Peck SC: Activity profiling of papain-like cysteine proteases in plants. Plant Physiol 2004, 135:1170–1178.PubMedCrossRef 42. Boonacker E, Van Noorden CJ: The multifunctional or moonlighting protein CD26/DPPIV. European journal of cell biology 2003, 82:53–73.PubMedCrossRef PAK6 43. St Leger RJ, Cooper RM, Charnley AK: Analysis of aminopeptidase and dipeptidylpeptidase IV from the entomopathogenic fungus Metarhizium anisopliae. J Gen Microbiol 1993, 139:237–243.PubMedCrossRef 44. Gossrau R, Lojda Z: Study on dipeptidylpeptidase II. Histochemistry 1980, 70:53–76.PubMedCrossRef 45. Bernier-Valentin F, Trouttet-Masson S, Rabilloud R, Selmi-Ruby S, Rousset B: Three-dimensional organization of thyroid cells into follicle structures is a pivotal factor in the control of sodium/iodide symporter expression. Endocrinology 2006, 147:2035–2042.PubMedCrossRef 46.

Wu ZJ, Song CF, Guo J, Yu BJ, Qian LM: A multi-probe micro-fabrication apparatus based on the friction-induced BTSA1 in vitro fabrication method. Front Mech Eng 2013,8(4):333–339.Rapamycin manufacturer CrossRef 16. Hendrickson J, Helfrich M, Gehl M, Hu D, Schaadt D, Linden S, Wegener M, Richards B, Gibbs H, Khitrova G: InAs quantum dot site-selective growth on GaAs substrates. Phys Status Solidi C 2011, 8:1242–1245.CrossRef 17. Song CF, Li XY, Yu BJ, Dong HS, Qian LM, Zhou ZR:

Friction-induced nanofabrication method to produce protrusive nanostructures on quartz. Nanoscale Res Lett 2011, 6:310.CrossRef 18. Fang TH, Chang WJ, Lin CM: Nanoindentation and nanoscratch characteristics of Si and GaAs. Microelectron Eng 2005, 77:389–398.CrossRef 19. Taylor CR, Malshe AP, Salamo G, Prince RN, Riester L, Cho SO: Characterization of ultra-low-load (μN) nanoindents in GaAs (100) using a cube corner tip. Smart Mater Struct 2005, 14:963–970.CrossRef 20. Sung IH, Yang JC, Kim DE, Shin BS: Micro/nano-tribological characteristics Selleck Ulixertinib of self-assembled monolayer and its application in nano-structure fabrication. Wear 2003, 255:808–818.CrossRef 21. Song CF, Li XY, Yu BJ, Dong HS, Qian LM, Zhou ZR: Maskless and low-destructive nanofabrication on quartz by friction-induced selective etching. Nanoscale Res Lett 2013,

8:140.CrossRef 22. Guo J, Song CF, Li XY, Yu BJ, Dong HS, Qian LM, Zhou ZR: Fabrication mechanism of friction-induced selective etching on Si (100) surface. Nanoscale Res Lett 2012, 7:152.CrossRef 23. Suedu-Bob CC, Saied SO, Sullivan JL: A X-ray photoelectron spectroscopy study of the oxides of GaAs. Appl Surf Sci 2006, 183:126–136.CrossRef 24. Ghidaoui D, Lyon SB, Thompson GE, Walton J: Oxide formation during etching of gallium arsenide. Corrosion Sci 2002, 44:501–509.CrossRef 25. Zardo I, Yazji S, Marini C, Uccelli E, Morral AF, Abstreiter G, Postorino P: Pressure tuning of the optical properties of GaAs nanowires. ACS Nano 2012,6(4):3284–3291.CrossRef 26. Gotoshia

SV, Gotoshia LV: Laser Raman spectroscopy of phase transformation in GaAs induced by radiation defects. Phys Status Solidi C 2013, 4:646–649.CrossRef 27. Pizani PS, Lanciotti F, Jasinevicius RG, Duduch JG, Porto AJV: Raman characterization of structural disorder and residual strains in micromachined GaAs. J Appl Phys 2000, 87:1280.CrossRef 28. Attolini G, Francesio L, Franzosi P, Pelosi C, Gennari S, triclocarban Lottici PP: Raman scattering study of residual strain in GaAs/InP heterostructures. J Appl Phys 1994, 75:4156.CrossRef 29. Champagnon B, Martinet C, Boudeulle M, Vouagner D, Coussa C, Deschamps T, Grosvalet L: High pressure elastic and plastic deformations of silica: In situ diamond anvil cell Raman experiments. J Non-Cryst Solids 2008, 254:569–573.CrossRef 30. Kiravittaya S, Heidemeyer H, Schmidt OG: Growth of three-dimensional quantum dot crystals on patterned GaAs (001) substrates. Phys E 2004, 23:253–259.CrossRef Competing interests The authors declare that they have no competing interests.

Attenuation of MKP-1 levels by shRNA did not affect proliferation, whereas it significantly increased T-ALL cell death following drug treatment or serum starvation. Importantly, tumorigenesis of MKP-1 deficient T-ALL cells was markedly impaired compared to controls. Our results elucidate a novel mechanism downstream of Notch3 P505-15 by

which the direct interplay between endothelial and tumor cells promotes survival of T-ALL cells. O24 Role of Foxm1 Transcription Factor in Tumor Microenvironment Tanya Kalin 1 , David Balli1, Fu-Sheng Chow1, Vladimir Kalinichenko1 1 Pulmonary Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USA The Forkhead Box m1 (Foxm1) protein is induced in a majority of human cancers, including non-small cell lung cancers. Increased Foxm1 expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion during lung tumor formation

MG-132 mouse remain unknown. In this study, we examined the role of Foxm1 in tumor microenvironment using conditional knockout mouse models with Foxm1 deficiency in macrophages (LysM-Cre Elafibranor solubility dmso Foxm1fl/fl mice; macFoxm1 −/−) or endothelial cells (Tie2-Cre Foxm1fl/fl mice; enFoxm1 −/−). Lung tumors in mice were induced using two experimental protocols: 3-methylcholanthrene (MCA) / butylated hydroxytoluene (BHT) or urethane. Chlormezanone Conditional deletion of Foxm1 from macrophages caused a significant decrease in lung inflammation during induction of lung tumors, leading to reduction in the number and size of lung adenomas. Decreased lung tumorigenesis in macFoxm1 −/− mice was associated with diminished proliferation of tumor cells, decreased numbers of tumor-associated macrophages and reduced expression of pro-inflammatory cytokines in the lung and bronchoalveolar lavage fluid. Furthermore,

we demonstrated that Foxm1 −/− mice displayed a dramatic decrease in proliferation and migration of macrophages in vivo and in vitro. In our studies, we also demonstrated that deletion of Foxm1 from endothelial cells resulted in accelerated lung tumorigenesis. The increased numbers and sizes of lung tumors in enFoxm1 −/− mice resulted from increased endothelial leakage and infiltration of inflammatory cells into lung tissue. The enFoxm1 −/− mice displayed increased tumor cell proliferation and increased mRNA levels of cell cycle regulator cMyc and cyclin D1. Deletion of Foxm1 from endothelial cells caused reduced expression of Foxf1 and Foxf2 transcription factors, both of which are critical regulators of endothelial cell functions and VEGF signaling. Altogether, our studies demonstrated that Foxm1 plays a dual role in tumor microenvironment: it controls cellular permeability in endothelial cells and induces inflammation and migration of macrophages into lung tumors.

These results MM-102 solubility dmso suggest that ceramide might specifically modify the levels of interaction or the cell surface distribution of TEM. In this regard, it has been shown that gangliosides play an important role in the organization of CD82-enriched microdomains [57]. Ceramide enrichment may also induce clustering of CD81 leading to an increased binding of MT81w mAb. In accordance with this hypothesis, it has been shown that high levels of ceramide induce large-scale clustering/capping of death receptors (e.g. Fas/CD95)

required to initiate efficient formation of death-induced signalling complex [58, 59]. Alternatively, MT81w may recognize an epitope of CD81 that is more exposed following ceramide enrichment. Further analyses are necessary to evaluate these hypotheses. HCV and Plasmodium are two major pathogens targeting the liver. Both use the glycosaminoglycans for their initial attachment on the surface of Pictilisib nmr hepatocytes [11, 60–64], and lipidic transfer properties of scavenger receptor class B type I regulate infection

of both pathogens [9, 65, 66]. CD81 is required for HCV and Plasmodium life cycle. Antibodies to CD81 or CD81 silencing strongly reduce the infection of hepatic cells and CD81-deficient mouse hepatocytes are resistant to infection by Plasmodium [26]. Using CD81/CD9 chimeras, it has been recently shown that CD81 LEL plays a critical role in sporozoite infection and a stretch of 21 amino VEGFR inhibitor acids is sufficient to confer susceptibility to infection [66]. In contrast to HCV infection, it seems that CD81 does not act directly as a receptor but is rather involved indirectly, likely by modulating the activity of an associated protein. This hypothesis is supported by the fact that CD81 associated to multiple proteins in the tetraspanin web plays a major role in sporozoite infection, since modulation of cellular cholesterol levels, which changes tetraspanin

microdomain organization, has been shown to also modify the extent of CD81-dependent sporozoite infection [23]. In contrast, in our study, we demonstrated that TEM-associated CD81 is not used by HCV, indicating Tideglusib that these two pathogens, while using the same molecules, invade their host by different mechanisms. Methods Antibodies 5A6 (anti-CD81 kindly provided by S. Levy); ACAP27 (anti-HCV core, kindly provided by JF Delagneau); MT81 (anti-CD81), MT81w (anti-TEM associated CD81), 8A12 (anti-EWI-2) and TS151 (anti-CD151) mAbs were used in this study. The anti-Claudin-1 (JAY.8) was from Zymed, the anti-SR-BI (NB400-104H3) was from Novus, the anti-LDL receptor was from Progen, the anti-transferrin receptor antibody was from Biolegend (Ozyme) and the anti-hCD81 (1.3.3.22) was from Santa Cruz Biotechnology. Alexa488-conjugated goat anti-mouse was from Jackson Immunoresearch.

The inhibition of the fluid-phase uptake was analysed in the presence of several inhibitors, including (a) 3 μM amiloride (AMIL), which is an ion exchange inhibitor that is used as an inhibitor of macropinocytosis [21, 22], (b) 0.1 μM wortmannin (WORT), a PI3K inhibitor [23] and (c) 3 μM cytochalasin D (CD), a known inhibitor of actin A-1210477 molecular weight polymerisation [24]. All of the inhibitors were purchased from Sigma. Each inhibitor was added to the respective

cellular suspensions 30 min prior to treatment and was not removed during the experiment. The cells were processed as previously mentioned, and the resultant RFUs were recorded. The B-cell Captisol research buy line viability in the presence of these inhibitors was monitored during the experiment. The cell viability

was assessed by staining an aliquot with 0.2% trypan blue and calculating the percentage selleck compound of cells that were not dyed. The viability in the control (no inhibitor) and treated cells reached 95%. The fluid-phase uptake data were analysed for statistical significance using one-way analysis of variance (ANOVA) using the SigmaStat software. P values ≤ 0.01 were considered statistically significant. The inhibition of the bacterial uptake was also analysed in the presence of amiloride using a protocol similar to that used in the previous experiments. Concentrations of 1, 3 and 5 mM of amiloride were added to the cells 30 min prior to the addition of the bacteria; the inhibitor was maintained in the samples throughout the 90 min during which the bacterial uptake occurred. A set of untreated cells were infected with the same bacterial suspension for control. At the Liothyronine Sodium end of the incubation, the extracellular bacteria were removed by centrifugation, and the CFUs were determined as described previously. The cell viability was also assessed at the end of the experiment and was found to reach >90% regardless of the concentration of inhibitor that was used. Transmission electron microscopy (TEM) Some

of the features of the infection of B cells with M. tuberculosis, M. smegmatis, and S. typhimurium were analysed by TEM. Because PMA is known to act as a macropinocytosis inducer [25], the features of B cells under PMA treatment were also analysed. B-cell suspensions were treated with 1.0 μg/mL of PMA for 1 h or infected for 1 and 24 h with the following bacterial suspensions: M. tuberculosis at an MOI of 10:1; M. smegmatis at an MOI of 10:1, and S. typhimurium at an MOI of 20:1. After treatment and infection, the suspension cells were washed four times by centrifugation at 1,000 rpm with PBS solution to remove any non-internalised bacteria and excess PMA. The cells were fixed with 2% glutaraldehyde solution in 0.1 M PBS for 2 h at room temperature. The cells were then washed three times with PBS and post-fixed with osmium tetroxide for 1 h at 4°C.