Cancer cell assays MDA-MB-231 cells were grown in DMEM/F12 supple

Cancer cell assays MDA-MB-231 cells were grown in DMEM/F12 supplemented with 5% fetal

bovine serum and 5 μg/ml insulin. For the LysoTracker red assay, cells grown on coverslips were incubated with 100 nM LysoTracker red (Molecular Probes) for 25 min before addition of chemicals for 35 min. Cells were fixed with 3.7% paraformaldehyde in PBS, washed and DNA was stained with Hoechst 33342. For EGF internalization assays, cells grown on coverslips were incubated at 4°C for 1 h with 0.4 μg/ml FITC-EGF (Molecular Probes) in cell culture medium supplemented with buy Alisertib 2 mg/ml bovine serum albumin. Cells were then washed twice with cold medium before adding chemicals in cell culture medium at 37°C. After different times at 37°C, cells were SB273005 fixed with 3.7% paraformaldehyde in PBS, washed twice and mounted on slides for selleckchem microscopy. For EGFR immunostaining, cells grown on coverslips were fixed with 3.7% paraformaldehyde in PBS, permeabilized with 0.6% Triton X-100 in PBS, blocked with PBS containing 10% fetal bovine serum and 2% bovine serum albumin, incubated with 3 μg/ml monoclonal anti-EGFR antibody (Merck), washed and further incubated with CY3-conjugated goat anti-mouse IgG, F(ab’) fragment-specific antibody (Jackson Laboratory). Acknowledgements We thank Hilary Anderson for fruitful discussions, Martha Cyert for the genomic library, Raymond

Andersen and David Williams for motuporamines and Philip Hieter for the cyc3Δ yeast deletion strain. CN, GG and SH thank Ron Davis for providing the environment that allowed the development of the assays they contributed to this study. This work was supported by grants from the Canadian Institute of Health to GG (MOP-81340) and CN (MOP-84305), and by a Canadian Cancer Society grant through the National Cancer Institute of Canada to MR (017392). References

1. Sturgeon CM, Kemmer D, Anderson HJ, Roberge M: Yeast as a tool to uncover the cellular targets of drugs. Biotechnol J 2006,1(3):289–298.CrossRefPubMed 2. Simon JA, Bedalov A: Yeast as a model system for anticancer drug discovery. Nat Rev Cancer 2004,4(6):481–492.CrossRefPubMed 3. Luesch H, Wu TY, Ren P, Gray NS, Schultz PG, Supek F: A genome-wide Montelukast Sodium overexpression screen in yeast for small-molecule target identification. Chem Biol 2005,12(1):55–63.CrossRefPubMed 4. Giaever G, Shoemaker DD, Jones TW, Liang H, Winzeler EA, Astromoff A, Davis RW: Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat Genet 1999,21(3):278–283.CrossRefPubMed 5. Lum PY, Armour CD, Stepaniants SB, Cavet G, Wolf MK, Butler JS, Hinshaw JC, Garnier P, Prestwich GD, Leonardson A, Garrett-Engele P, Rush CM, Bard M, Schimmack G, Phillips JW, Roberts CJ, Shoemaker DD: Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell 2004,116(1):121–137.CrossRefPubMed 6.

To assess for differences between outcomes in the intervention an

To assess for differences between outcomes in the intervention and IWR-1 mw control groups, multi-level hierarchical modelling using the General Estimating Equation (GEE) approach was used to account for clustering to estimate the treatment effect as an odds ratio and test for significance [33, 34]. First-order interaction terms (specifically: sex by intervention status) were evaluated. The 95% confidence intervals and p values were calculated using the sandwich estimator of variance.

The analysis was carried out using R: A Language and Environment for Statistical Computing version 2.10.1 [35, 36]. The GEE models were fit using the R package geepack Screening Library supplier version 1.0-17. Results Study flow Of the 54 eligible hospitals, 36 agreed to participate and

were randomly assigned to intervention or control group (18 in each group). We obtained 801 records for fracture patients within 3 months of their admission to the ED; 139 were received 3 months after fracture. Of these, 443 were excluded: 298 were unable to reach, 51 had died or were in long-term care, 43 lived outside of the hospital catchment area, 21 refused, 18 had previously been screened by a fracture clinic coordinator and 12 had significant cognitive or hearing impairment, resulting in 358 enrolled subjects (Fig. 1). Fig. 1 Flow of patients through the trial Cluster size was comparable between the groups with ten (range, 3–16) Afatinib find more in the intervention and ten (range, 4–18) in the control hospitals. Of those randomized, 52 from the intervention hospitals and 39 from the control hospitals were lost to follow-up

leaving a total of 267 subjects with complete data for analysis. The primary analysis is a ‘complete case’ and includes only those whose outcome is known [37]. A secondary analysis was the strict intention to treat analysis in which all randomized subjects were included. Baseline characteristics The mean age of the study participants was 66.0 years in the intervention and 65.4 in the control group; about two thirds were female and married. Twenty-seven percent had a history of a previous fracture since the age of 40 years, 20% were current smokers and 23% had fallen in the previous 12 months. Thirty-one percent had a BMD test in the previous 12 months, 25% self-reported a diagnosis of osteoporosis and 19% were currently taking osteoporosis medications. The most common fracture type was wrist (34%), followed by ankle (16%), rib (12%), shoulder (12%) and hip (8%). There was no significant difference in demographic and clinical characteristics among patients in the intervention and control groups (Table 1).

The μ of a given species under equilibrium conditions is equal in

The μ of a given species under equilibrium conditions is equal in all phases that are in contact [22]. Therefore, we can obtain (3) In addition, ABT-888 cell line C Mg is limited by the formation of Mg3N2 to substitute Mg for Ga or Al as an acceptor [10]. This limitation meets the relation

(4) By substituting Selleck THZ1 Equations 3 and 4 into Equation 2, we can obtain (5) which, aside from ΔE, depends only on μ N , since the μ AlN/GaN and are constants [25]. μ N should be limited between μ N (Al/Ga-rich) ≤ μ N  ≤ μ N (N-rich) [11], namely, , to drive the source materials to form Al x Ga1 – x N alloys instead of the undesirable phases (bulk Ga, Al, and N2). Our calculated ΔHGaN value of -1.01 eV is higher than the ΔHAlN value of -2.97 eV, which are consistent with the experimental values of -1.08 and -3.13 eV [25]. Therefore, as the growth condition varies from Ga-rich to N-rich conditions, μ N changes from MGCD0103 ic50 to . Thus, ΔH f varies over a range corresponding to 1/3ΔH GaN of 0.337 eV, as shown in Figure 2a. This variation

indicates that the N-rich growth atmosphere favor the Mg incorporation effectively in AlGaN. Generally, the N-rich condition is modulated by increasing the V/III ratio. However, for a fixed III flow, the Al x Ga1 – x N growth has an optimal V/III ratio for the best crystal quality [13–16]. Nonetheless, the max flow limitation of the MOVPE system does not allow the V flow to be increased infinitely. Accounting for these limitations, an inspiration can be obtained from Figure 1c, in which the protecting atmosphere with NH3 flow just provides an ultimate V/III ratio condition (extremely N-rich) for C Mg enhancement when the epitaxy ends with the III flow becoming zero. Simultaneously,

the stopped growth avoids the formation of low-quality Al x Ga1 – x N crystal. If this special condition 17-DMAG (Alvespimycin) HCl is introduced as an intentional interruption during the continuous p-Al x Ga1 – x N growth, then the overall Mg incorporation could be improved. Figure 2 Formation enthalpy difference of Mg Ga /Mg Al and C Mg profile of Al 0.49 Ga 0.51 N film. (a) Formation enthalpy difference of MgGa and MgAl between Ga-rich and N-rich condition. (b) C Mg profile of Al0.49Ga0.51N film with three different Cp2Mg flows grown by the MSE technique. The inset in (b) illustrates the source supply sequence of the MSE technique, an ultimate V/III ratio condition is shortly produced during the interruption. To validate this hypothesis, a growth interruption experiment was designed, as shown schematically in the inset of Figure 2b. We closed the metal flows (TMAl, TMGa, and Cp2Mg flows) three times. In these three periods (35 nm thick), different Cp2Mg flows (0.45, 0.81, and 0.99 nmol/min) were applied to investigate the interruption effect systematically. Figure 2b shows the SIMS C Mg profile of Al0.49Ga0.51N film across three periods.

Optical lithography and e-beam lithography have been widely used

Optical lithography and e-beam lithography have been widely used in the formation of microelectronic devices, and these two technologies combined with ion implantation have been already applied to fabricate FET. Hayden et al. [37] utilized optical lithography and ion implantation to produce an n-type/intrinsic/n-type junction in the silicon nanowires. With the n-doped substrate under the silicon oxide layer as the global back gate, metal oxide semiconductor FET was finished by ion implantation and optical lithography (details in Figure 8). Colli et al. [2] implanted P or B ions into

silicon nanowires that have a thick oxide shell surrounding the silicon core and then evaporated Ni on the silicon nanowires as the electrode through e-beam lithography. Throughout 3-Methyladenine order the entire

experimental process, it is the crucial step to choose the appropriate implantation energy. It must be ensured that the dopants were stopped within the core of nanowires. The incident ion energy and implantation fluences may impact the quality of the FETs. Jang selleck chemical et al. [38] reported that the CNT-FET exhibited p-type behaviors after oxygen implantation at low doses and metallic behaviors at high doses. Zinc oxide nanowires have been widely applied in the fabrication of FETs; Liao et al. [39] utilized Ga+ ion implantation to improve the performance of nanowire-based FETs. The improvement of the performance is attributed to a reduced surface effect after ion implantation. There are many other semiconductors used to produce FET, but there is still little for doping through ion implantation. Figure 8 Preparation process of nanowire devices. (a–c) Schematic representation of the NWFET fabrication. (d) SEM micrograph of a nanowire device with top contacts. Reprinted with permission from Hayden click here et al. [37]. Optical properties Owing to the desirable optical properties of semiconductor

nanomaterials, many nanomaterials were used to fabricate light-emitting diodes [40–42] and nanowire lasers [43]. However, there are still some imperfections of these nanodevices; doping with optically activated impurities (like transition metals and rare earth elements) through ion implantation may improve the properties of these nanodevices [44]. Transition metals (TM) are interesting doping elements for semiconductor nanowires because of its enormous optical influences to semiconductor nanowires. Doping with rare earth elements is another significant research direction, as rare earth elements have a special outermost electron structure [45]. Silica nanowires are significant nanomaterials for integrated photonics and biosensing because silica nanowires are suitable hosts for optically active impurities, are chemically inert, and are excellently biocompatible. Elliman et al. [46] reported silica nanowire doping with erbium by ion implantation, and they found that PFT�� ic50 luminous intensity and lifetime have a very obvious enhancement.

To overcome these obstacles and limitations in our current techni

To overcome these obstacles and limitations in our current techniques for genetic analysis of Histoplasma, we have developed a procedure for isolating chromosomally-located gene mutants without reliance on homologous recombination. We employed random mutagenesis to create collections of mutants. One approach to identify the desired gene disruption would be to characterize the mutation of each isolate in the collection of random mutants. However, this requires many resources, substantial time and effort, and thus is not well suited for studies targeting a particular gene. In forward genetics, random mutagenesis is successful because the desired mutant selleck chemicals llc can be typically isolated

or identified out of the much larger collection of mutants by growth phenotype or morphological changes. In reverse genetics, the mutant phenotype is the very aspect under MAPK Inhibitor Library supplier investigation and thus mutants can not be identified by predicted changes. To enable reverse genetics following random mutagenesis in Histoplasma, we adapted PCR-based procedures employed for large scale screening in Arabidopsis and C. elegans [28–30]. We optimized a mutant pooling strategy and utilized PCR to efficiently identify mutant pools

which contain the strain with the disrupted gene. To extract the strain with the targeted mutation, the pool is subsequently subdivided and individual clones addressed and screened by PCR. We demonstrate the effectiveness of this method by employing it to isolate a cbp1 mutant in the NAm 2 Histoplasma strain background.

Results and Discussion Insertion mutant screening To generate insertion see more mutations in the Histoplasma genome, we used Agrobacterium tumefaciens-mediated transformation. This mutagen was selected because Agrobacterium-mediated Progesterone transfer of T-DNA is efficient in producing random insertional mutations in Histoplasma yeast cells [23, 31]. The majority of T-DNA insertions are single integration events [31] and thus the chance of secondary background mutations is minimized. Other mutagens such as UV or chemical agents result in multiple changes to the genome, and while these background mutations can be removed by repeated backcrossing of mutants to wild type, no reliable techniques for crossing laboratory strains have been developed for Histoplasma [32–34]. Additionally, insertional mutagens provide a molecular tag with known sequence (e.g. the T-DNA element) which we can exploit in PCR-based screening for mutations in particular chromosomal loci using a T-DNA specific primer in conjunction with a primer specific for the targeted gene (Figure 1A). The molecular weight of the PCR amplicon provides an estimate of the distance from the gene specific primer to the T-DNA insertion, and this distance can be used to determine whether the T-DNA element disrupts the targeted gene.

Oyster gill microbiota, on the other hand, harboured a substantia

Oyster gill microbiota, on the other hand, harboured a substantial amount of variation between individuals (Figures 2 and 3). The between individual variation in microbial community composition correlated with genetic relatedness of the oysters, suggesting that microbial communities might assemble according to individual hosts or even host genotypes. Stable host associations have been reported for several gut microbiota in a variety of host species [48–51]. The human gut bacterial community, for example, is considered to be stable

over extended periods Mocetinostat molecular weight of time, but is also unique for each individual [51] and similar between related individuals [52]. Similarly, stable associations have been reported from insects [50] and crustaceans [49] and have also been observed in oyster species in the Mediterranean where associations were stable even after invasion from the Red Sea [18]. Such stable associations harbour an environmental component PXD101 in vitro depending on food [49] but also genetic components as NVP-HSP990 molecular weight suggested by similar communities found within mother-twin triplets [53]. The fact that the similarity in microbial communities correlated with the genetic relatedness

of the Pacific oyster demonstrated here, further suggests that bacterial communities are not only unique

to individuals but can also assemble according to host genotypes. In combination with the lack of significant differentiation of community structure between oyster beds this suggests that larger scale environmental differences between beds may play a limited role selleck compound when compared to host genotype. Furthermore, correlations between genetic microbial community distances depended to a large degree on OTUs only occurring rarely in the communities (Figure 6). This suggests that while abundant taxa may lead a generalist life style and are found in the majority of host genotypes, rare specialists within the community assemble according to host genotypes. An alternative explanation for the formation of genotype specific microbiome associations is vertical inheritance [54, 55]. While we cannot rule out this possibility for Pacific oysters, the transient nature of the genotype specific associations suggests that previously encountered disturbance events should also have led to the loss of the inherited genotype-specific microbiota. A recovery of genotype specific associations prior to our experiment therefore rather suggests an uptake from the environment.

It is possible

It is possible

RG7420 price that the large proteolytic fragment of LigB remaining with the ligB transformants retains the fibronectin-binding region but has lost sequences mediating the interaction of LigB with a different and distinct renal cell receptor. Further studies with lig transformants could include analyzing lig-mediated host cell adhesion by using additional cell lines representing different species and cell types. Conclusion In conclusion, by using L. biflexa as a surrogate host, we have shown that Lig proteins are factors involved in the attachment to fibronectin, fibrinogen, and laminin and to host cells and can act as microbial surface components recognizing host extracellular matrix proteins. Although important advances in the genetic system of EVP4593 the pathogen L. interrogans have been made in the last years [5, 7], this Dorsomorphin molecular weight bacterium remains poorly transformable and few mutants have been fully characterized [3]. We believe that L. biflexa can serve as a model bacterium for investigating the function of additional leptospiral pathogenesis mechanisms. Genetic studies in L. biflexa should provide information about the roles of

key components in the pathogenesis of leptospirosis. Methods Bacterial strains and culture conditions Leptospires were cultivated in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium [47, 48] or on 1% agar plates at 30°C and counted in a Petroff-Hausser counting chamber (Fisher Scientific). The saprophyte Leptospira biflexa serovar Patoc strain Patoc I and the pathogen L. interrogans serovar Copenhageni strain Fiocruz L1-130 were used in this study. E. coli was grown in Luria-Bertani (LB) medium. When appropriate, spectinomycin or kanamycin was added to culture medium at the final concentration of 40 μg/ml. Plasmid constructions The Borrelia burgdorferi flgB promoter was amplified with PflgA (5′-TAATACCCGAGCTTCAAGGAAG-3′) PR-171 cell line and PflgB (5′-AACATATGGAAACCTCCCTC-3′) and cloned into pCR2.1 (Invitrogen) to generate plasmid

pCRPromFlgB. The ligA and ligB genes were amplified with flanking NdeI and XhoI sites, using primer pairs LANF (5′-GGGAATTCCATATGAAGAAAATATTTTGTATTTCG-3′) – LAXR (5′ CGGCTCGAGTTATTATGGCTCCGTTTTAATAGAGG-5′) and LBNF (5′-GGGAATTCCATATGAAGAAAATATTTTGTATTTCG-5′) – LBXR (5′-CGGCTCGAGTTATTATTGATTCTGTTGTCTGT-3′), respectively, from genomic DNA of L. interrogans serovar Copenhageni strain Fiocruz L1-130. Amplified lig genes were then digested with NdeI and XhoI restriction enzymes, purified, and inserted between the corresponding restriction sites of pCRPromFlgB to generate pCRPflgBLigA and pCRPflgBLigB, respectively. The DNA fragment containing Prom flgB ligA (4183 bp) and Prom flgB ligB (6188 bp) were released from plasmids pCRPflgBLigA and pCRPflgBLigB by SpeI and XbaI digestion, then blunt-ended, and cloned into the PvuII restriction site of the E. coli-L. biflexa shuttle vector pSLe94 [49] to generate pSLePFligA and pSLePFligB (Figure 1). Plasmid constructs were verified by nucleotide sequencing.

J Appl Ecol 45:141–150CrossRef Brewer KRW, Hayes D (2011) Underst

J Appl Ecol 45:141–150CrossRef Brewer KRW, Hayes D (2011) Understanding and using Fisher’s p. Part 1: countering the p-statistic fallacy. Math Sci 36:117–125 Bunker DE, De Clerck F, Bradford JC, Colwell RK, Perfecto I, Phillips OL, Sankaran M, Naeem S (2005) Species loss and aboveground carbon storage in a tropical forest. Science 310:1029–1031PubMedCrossRef Chazdon RL, Peres CA, Dent D, Sheil

D, Lugo AE, Lamb D, Stork NE, Miller S (2009) The potential for species conservation in tropical secondary forests. Conserv Biol 23:1406–1417PubMedCrossRef Condit R, Engelbrecht BMJ, Pino D, Pérez R, Turner BL (2013) Species distributions in response to individual soil nutrients and seasonal drought across a community of tropical trees. Proc Natl Acad Sci USA EVP4593 molecular weight 110:5064–5068PubMedCrossRef Cornelissen JHC, Lavorel S, Garnier E, Diaz S, Buchmann N, Gurvich DE, Reich PB, ter Steege H, Morgan HD, van der Heijden MGA, Pausas JG, Poorter H (2003) A handbook of protocols for standardised and easy measurement of plant functional

traits worldwide. Aust J Bot 51:335–380CrossRef Dallmeier F, Comiskey JA (1996) From the Dorsomorphin in vitro forest to the user: a methodology update. In: Wilson D, Sandoval 3-MA manufacturer A (eds) The biodiversity of southeastern Peru. Smithsonian Institution Press, Washington, DC, pp 41–56 Delbaere B (2002) Biodiversity indicators and monitoring. European Centre for Nature Conservation, Tilburg Duckworth JC, Kent M, Ramsay PM (2000) Plant functional types: an alternative to taxonomic plant community description

in biogeography? Progr Phys Geogr 24:515–542 Dudley N, Baldock D, Nasi R, Stolton S (2005) Measuring biodiversity and sustainable management in forests and agricultural landscapes. Philos Trans R Soc B 360:457–470CrossRef Dufrêne M, Legendre P (1997) Species assemblages and indicator species: the need for a flexible asymmetrical approach. Coproporphyrinogen III oxidase Ecol Monogr 67:345–366 Duraiappah AK, Naeem S (2005) Ecosystems and human well-being: biodiversity synthesis. A report of the millennium ecosystem assessment. World Resources Institute, Washington DC Eggleton P, Bignell DE, Sands WA, Waite B, Wood TG, Lawton JH (1995) The species richness of termites (Isoptera) under differing levels of forest disturbance in the Mbalmayo Forest Reserve, Southern Cameroon. J Trop Ecol 11:85–98CrossRef European Academies’ Science Advisory Council (ESAC) (2004) A users’ guide to biodiversity indicators. http://​www.​easac.​eu/​fileadmin/​PDF_​s/​reports_​statements/​A.​pdf. Accessed 10 May 2012 Folke C, Holling CS, Perrings C (1996) Biological diversity, ecosystems and the human scale.

Infect Agents Dis 1993,2(4):255–258 PubMed 33 Liu Y, Shepherd EG

Infect Agents Dis 1993,2(4):255–258.PubMed 33. Liu Y, Shepherd EG, Nelin LD: MAPK phosphatases – regulating the immune response. Nat Rev Immunol 2007,7(3):202–212.PubMedCrossRef 34. Li H, Xu H, Zhou Y, Zhang J, Long C, Li S, Chen S, Zhou JM, Shao F: The phosphothreonine lyase activity of a bacterial type III Linsitinib chemical structure effector family. Science 2007,315(5814):1000–1003.PubMedCrossRef 35. Lin SL, Le TX, Cowen DS: SptP, a Salmonella typhimurium type III-secreted

protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation. Cell Microbiol 2003,5(4):267–275.PubMedCrossRef 36. Orth K, Xu Z, Mudgett MB, Bao ZQ, Palmer LE, Bliska JB, Mangel WF, Staskawicz B, Dixon JE: Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like find more protein C59 wnt order protease. Science 2000,290(5496):1594–1597.PubMedCrossRef 37. Yarbrough ML, Li Y, Kinch LN, Grishin NV, Ball HL, Orth K: AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 2009,323(5911):269–272.PubMedCrossRef 38. Bhattacharjee RN, Park KS, Chen X, Iida T, Honda T, Takeuchi O, Akira S: Translocation of VP1686 upregulates

RhoB and accelerates phagocytic activity of macrophage through actin remodeling. J Microbiol Biotechnol 2008,18(1):171–175.PubMed 39. Hobbie S, Chen LM, Davis RJ, Galan JE: Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells. J Immunol 1997,159(11):5550–5559.PubMed

40. Satchell KJ: Activation and suppression of the proinflammatory immune response by Vibrio cholerae toxins. Microbes Infect 2003,5(13):1241–1247.PubMedCrossRef 41. Yu Y, Zeng H, Lyons S, Carlson A, Merlin D, Neish AS, Gewirtz AT: TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via posttranscriptional mechanism. Am J Physiol Gastrointest Liver Physiol 2003,285(2):G282–290.PubMed 42. Reissinger A, Skinner JA, Yuk MH: Downregulation of mitogen-activated protein kinases by the Bordetella bronchiseptica Type III secretion system leads to attenuated nonclassical macrophage activation. Infect Immun 2005,73(1):308–316.PubMedCrossRef 43. Kramer RW, Slagowski NL, Eze NA, Giddings KS, Morrison MF, Siggers KA, Starnbach MN, Lesser CF: Yeast functional genomic screens lead to identification of a role for GBA3 a bacterial effector in innate immunity regulation. PLoS Pathog 2007,3(2):e21.PubMedCrossRef 44. Hii CS, Sun GW, Goh JW, Lu J, Stevens MP, Gan YH: Interleukin-8 induction by Burkholderia pseudomallei can occur without Toll-like receptor signaling but requires a functional type III secretion system. J Infect Dis 2008,197(11):1537–1547.PubMedCrossRef 45. Kim WH, Goo SY, Shin MH, Chun SJ, Lee H, Lee KH, Park SJ: Vibrio vulnificus -induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species.

3b) The #

3b). The selleck chemical Wolbachia-free G. m. morsitans line contained only the

smaller 453 bp version of the fbpA gene, suggesting again that this gene fragment is the result of a horizontal gene transfer event to the host chromosome. Figure 3 Overview of deleted fragments in two Wolbachia genes A) PCR amplified products from G. m. morsitans (GmmY and Gtet) of the 16S rRNA and fbpA genes were resolved on 2.5% agarose gels stained with ethidium bromide. A 100-bp ladder was used as size standard. The input of the negative (neg) control was water. B) 16S rRNA and fbpA fragments from tsetse flies Wolbachia strains aligned with the corresponding regions of strain wMel. Red dashes represent the deletion region, the numbers show the positions before and after the deletions in respect to the wMel genome. The blue arrows

represent the corresponding wMel genes. Deleted fragments were detected in G. m. morsitans samples (Gmormor: GmmY, 12.3A, 24.4A, 30.9D, 32.3D and Gtet). The right-left red arrows below the number indicate the size of deletion in base pairs. Tissue specific detection of cytoplasmic and nuclear Wolbachia markers The tissue specific distribution of the Wolbachia markers in G. m. morsitans were tested in ovary, salivary gland, midgut and find more carcass in normal and tetracycline-treated (Wolbachia-cured) flies. Two 16S rRNA PCR products (438 and 296 bp as described in Figure 3, corresponding to cytoplasmic and nuclear Wolbachia markers) could be amplified from ovary and testes tissues of uncured flies, while only the truncated 296 bp product that corresponds to the nuclear Wolbachia marker was amplified from all of the tissues (Figure 4). In contrast, the fragment that corresponds to the cytoplasmic 16S rRNA marker could not be amplified from any of the

tissues of Wolbachia cured tetracycline-treated flies, including the reproductive organs (ovary and testes) (Fig. 4). The ACP-196 price amplification of the larger product that also corresponds to the cytoplasmic Wolbachia only from testes and ovary tissues of adults suggests that Wolbachia is restricted to the gonadal tissues in this species. Unlike for the 16S rRNA, a single wsp PCR product was observed in all tissues of Wolbachia infected and cured adults (Fig. 4). While it was not possible to differentiate between amplifications of cytoplasmic and nuclear Wolbachia, amplification from tetracycline treated adults suggests a horizontal transfer event also for the wsp gene. The size heterogeneity was also observed for fbpA. The larger 509 bp amplification which corresponds to the cytoplasmic marker was restricted to the reproductive tissues of the tsetse flies while the smaller derived 453 bp product corresponding to the nuclear marker was present in all tissues of infected and cured adults, suggesting horizontal transfer of fbpA to the G. m. morsitans genome (Fig. 4). Figure 4 Tissue tropism of Wolbachia infections in G. m. morsitans. G. m.