The microarray technique is thus analogous to performing many PCR

The microarray technique is thus analogous to performing many PCR reactions and hybridization reactions at the same time and has the advantage of being versatile [16]. The aim of this study was to develop a diagnostic microarray for the identification of single strains of food-borne fungi that are most prevalent in South African #see more randurls[1|1|,|CHEM1|]# food commodities, and to detect the ability of these fungi to produce

mycotoxins in laboratory and food samples. A total of 40 food-borne fungi isolated from different foods that belong to the genera Alternaria, Aspergillus, Bipolaris, Claviceps, Curvularia, Diplodia, Drechslera, Eurotium, Fusarium, Penicillium and Pithomyces, were used. For fungal discrimination, the polymorphisms of the internal transcribed spacer (ITS) regions and the elongation factor 1- alpha (EF-1 α) gene were exploited for the design of the oligonucleotide probes. The specificity of a probe was increased in some instances by substituting an oligonucleotide with a high affinity DNA analogue known as locked nucleic acid (LNA). A locked nucleic acid nucleotide analogue consists of a 2′-O,4′-C methylene bridge and locks the LNA structure into a rigid bicyclic formation and displays unprecedented hybridization affinity towards complementary DNA and RNA [17]. It is most disruptive, and thus gives a better signal, in a centre position. For the detection

of fungi that can produce mycotoxins, oligonucleotide probes for the genes leading to mycotoxin production were selected

from public databases and included in the oligonucleotide array. The combination of ITS, EF-1 PF-02341066 cell line α and mycotoxin genes on the same array was evaluated for the potential of the array to identify the forty fungal isolates and the genes involved in pathways leading to toxin production. Results Probe design A 96-probe oligonucleotide microarray was constructed for the simultaneous Sodium butyrate detection and identification of potentially mycotoxigenic fungi. Probes for the array were designed by exploiting the polymorphisms of the internal transcribed spacer (ITS) regions of the rRNA complex. Amplification of fungal DNA with the universal fungal primers ITS1 and ITS4 and subsequent sequence analysis allowed the differentiation of most of the fungal species studied. Several unique polymorphisms (sequence data can be found in GenBank with accession numbers [GenBank:FJ864706, GenBank:FJ864709, GenBank:FJ864710, GenBank:FJ864708, GenBank:FJ864711, GenBank:FJ864703, GenBank:FJ864704, GenBank:FJ864705, GenBank:FJ864707, and GenBank:FJ864712]) could be identified within the PCR products generated for each fungal species. However, amplification of the Fusarium species showed no significant differences between the sequences of the PCR products generated with the ITS primers. Therefore, the elongation factor 1-alpha (EF-1 α) gene was used for the identification of polymorphisms in Fusarium species and for the design of unique species- or genus-specific probes.

However, PCR products of strains

However, PCR products of strains LM27553stx1 and LM27553stx2 were larger than expected, indicating insertion of foreign DNA into or closely to the tia gene [15] (Table 1). Following this, the structure of the subAB 2 operon and AG-881 adjacent DNA was analyzed using the primer pair tia_lo/ SubAB2-3′tia targeting the region of the tia gene, an intergenic region (linker), subAB 2, as well as 316 bp of the downstream region (Figure 2B). This should reveal a PCR product of 3174 bp. In these PCRs, 6 STEC strains were positive (see Figure 3A, lanes 3, 5–9), indicating the presence of subAB 2 linked

to the tia gene (Table 1). However, one of AZD5363 datasheet these PCRs with strain LM27553stx1 as a template, revealed a PCR product of approximately 4500 bp (Figure 3A, lane 3). Since the open reading frames of subA 2-1 and subB 2-1 in this strain were of the correct size, insertion of foreign DNA between subA 2-1 and tia is assumed. PCR of STEC strains LM14603/08, LM16092/08 and LM27553stx2 with the same primers was negative (Figure 3A, lanes 1, 2, and 4), and therefore direct association of subAB 2 with the tia gene could not be demonstrated. Weak

bands Selleck Copanlisib in Figure 3A, lanes 1, 2, and 4 reflect unspecific amplification products. Figure 3 Agarose gel electrophoresis of PCR products of subAB 2 alleles with primers tia_lo/subAB2-3′tia targeting the SE-PAI (A), and subAB5′-OEP/subA_out targeting the OEP-locus locus (B). Gene Ruler 1 kb DNA ladder (M), (Fermentas) LM14603/08 (1), LM16092/08 (2), LM27553stx1 (3), LM27553stx2 (4), LM27564 (5), LM27558stx2 (6), LM27555 (7), LM14960 (8), LM27558stx1 (9), with identical order of strains on both agarose gels. Strain LM27564 was used as positive control. Due to these negative results, the subAB 2 reference

sequence of STEC strain ED32 (GenBank Acc. No. JQ994271) was searched with BLAST against the NCBI nucleotide database to evaluate the possibility of further subAB gene loci in Cediranib (AZD2171) these strains. Interestingly, a further subAB operon with different flanking regions was detected in Escherichia coli strain 1.2264 in contig 3905 (Acc. No. AEZO02000020.1) and in Escherichia coli strain 9.0111 in contig 1125855384441 (Acc. No. AEZZ02000028.1), which in addition carry the SE-PAI described by Michelacci et al. [16]. The new gene locus carries genes hypothetically encoding parts of a type 1 secretion system (T1SS), and an outer membrane efflux protein (OEP), which are located upstream of subAB 2 and are linked to the latter by a 1496 bp sequence (for a scheme see Figure 2C). Downstream of subAB 2 , the nanR gene hypothetically encoding the transcriptional regulator of the nan-operon was present in a 1400 bp distance in strain E. coli 1.2264 and 3842 bp in E. coli 9.011 where additional putative transposases are inserted (data not shown). In the following, this new gene region is termed OEP-locus.

PubMedCrossRef 82 Jackson SR, Dryden M, Gillett P, Kearney P, We

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Umansky SR, Tomei LD: Transrenal DNA testing: progress and perspectives. Expert review of molecular diagnostics 2006,6(2):153–163.PubMedCrossRef 85. Price LB, Liu CM, Johnson KE, Aziz M, Lau MK, Bowers J, Ravel J, Keim PS, Serwadda D, Wawer MJ, et al.: The effects of circumcision on the penis microbiome. PLoS ONE 2010,5(1):e8422.PubMedCrossRef 86. Nelson KE, Weinstock GM, Highlander SK, Worley KC, Creasy HH, Wortman JR, Rusch DB, Mitreva M, Sodergren E, Chinwalla

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from 16S rRNA sequences produced by highly parallel pyrosequencers. Nucleic Acids Res 2008,36(18):e120.PubMedCrossRef 92. Lazarevic V, Whiteson K, Hernandez D, Francois P, Schrenzel J: Study of inter- and intra-individual variations in the salivary microbiota. BMC genomics 2010, 11:523.PubMedCrossRef 93. Foxman B: The epidemiology of urinary tract infection. Nat Rev Urol 2010,7(12):653–660.PubMedCrossRef 94. Fredricks DN, Fiedler TL, Marrazzo JM: Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 2005,353(18):1899–1911.PubMedCrossRef 95. Menard JP, Mazouni C, Salem-Cherif I, Fenollar F, Raoult D, Boubli L, Gamerre M, Bretelle F: High vaginal concentrations of Atopobium vaginae and Gardnerella vaginalis in women undergoing preterm labor. Obstet Gynecol 2010,115(1):134–140.PubMedCrossRef 96.

Haplotypes one position away from the founding haplotype on the e

Haplotypes one position away from the founding haplotype on the eBurst diagrams differed in one trait from LESB58, and isolates two positions away from the founding haplotype on the eBurst diagram differed in two traits. This method of analysing P. aeruginosa haplotypes has been published previously by Mowat et al.[9]. Statistical analysis A generalised linear model with a negative binomial

error distribution was used to test whether the number of novel haplotypes was differed between ASM and ASM plus antibiotic treatments, Vemurafenib price with significance assessed using a likelihood ratio test. Haplotype diversity was calculated as the probability of two randomly picked clones being the same haplotype based on the haplotype frequencies selleck products within a sample (equivalent to the Simpson’s Index) and analysed in a linear model following a logistic transform. Hierarchical analysis of variance was performed using the ade4 package in R [62] in order to estimate the population differentiation between treatment groups, between populations within treatment groups and between clones within populations. Acknowledgements This work was supported by The

Dr Hadwen Trust for Humane Research, the UK’s leading medical research charity funding exclusively non-animal research techniques to replace animal experiments, and the Wellcome Trust (093306/Z/10/Z). References 1. Teichgraber V, Ulrich M, Endlich N, Riethmuller J, Wilker B, De Oliveira-Munding CC, van Heeckeren AM, Barr selleck inhibitor ML, von Kürthy G, Schmid KW, Weller M, Tümmler B, Lang F, Grassme H, Döring G, Gulbins E: Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat Med 2008, 14:382–391.PubMedCrossRef 2. Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL: Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002, 34:91–100.PubMedCrossRef Amylase 3. Hart CA, Winstanley C: Persistent and aggressive bacteria in the lungs of cystic fibrosis children. Br Med Bull 2002, 61:81–96.PubMedCrossRef

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Book Kre-alkalyn® supplementation has no beneficial effect on cre

Book Kre-alkalyn® supplementation has no beneficial effect on creatine-to-creatinine conversion rates 2007. 16. Greenwood M, Kreider RB, Rasmussen C, Almada AL, Earnest INCB018424 cell line CP: D-Pinitol augments

whole body creatine retention in man. J Exerc Physiol Online 2001, 4:41–47. 17. Green AL, Hultman E, Macdonald IA, Sewell DA, Greenhaff PL: Carbohydrate ingestion augments skeletal muscle creatine accumulation during creatine supplementation in humans. Am J Physiol 1996, 271:E821–826.PubMed 18. Steenge GR, Simpson EJ, Greenhaff PL: Protein- and carbohydrate-induced augmentation of whole body creatine retention in humans. J Appl Physiol 2000, 89:1165–1171.PubMed 19. Kreider RB: Effects of creatine supplementation on performance and training

adaptations. Mol Cell Biochem 2003, 244:89–94.PubMedCrossRef 20. Jager R, Kendrick I, Purpura M, Harris R, Ribnicky , Pischel I: The effect of Russian Tarragon (artemisia dracunculus L.) on the plasma creatine concentration with creatine monohydrate administration. Book The effect of Russian Tarragon (artemisia dracunculus L.) on the plasma creatine concentration with creatine monohydrate administration 2008, 5:4. 21. Wang ZQ, Ribnicky D, Zhang XH, Raskin I, Yu Y, Cefalu WT: Bioactives of Artemisia dracunculus L enhance cellular insulin signaling in primary human skeletal muscle culture. Metabolism 2008, 57:S58–64.PubMedCentralPubMedCrossRef 22. Harris RC, Hultman E, Nordesjo LO: Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. PD332991 Scand J Clin Lab Invest 1974, 33:109–120.PubMedCrossRef 23. Evans WJ, Phinney SD, Young VR: Suction applied to a muscle biopsy maximizes sample-size. Med Sci Sports Exerc 1982, 14:101–102.PubMed HA-1077 chemical structure 24. Soderlund K, Hultman E: Effects of delayed freezing on content of phosphagens in human skeletal muscle

biopsy samples. J Appl Physiol 1986, 61:832–835.PubMed 25. SBI-0206965 price Tarnopolsky MA, Parise G: Direct measurement of high-energy phosphate compounds in patients with neuromuscular disease. Muscle Nerve 1999, 22:1228–1233.PubMedCrossRef 26. Bloomer RJ, Canale I, Pischel I: Effect of an aqueous Russian tarragon extract on glucose tolerance in response to an oral dextrose load in non-diabetic men. Nutr Diet Suppl 2011, 3:43–49.CrossRef 27. Ribnicky DM, Poulev A, Watford M, Cefalu WT, Raskin I: Antihyperglycemic activity of Tarralin, an athanolic extract of Artemisia dracunculus L . Phytomedicine 2006, 13:550–557.PubMedCrossRef 28. Steenge GR, Lambourne J, Casey A, Macdonald IA, Greenhaff PL: Stimulatory effect of insulin on creatine accumulation in human skeletal muscle. Am J Phys 1998, 275:E974-E979. 29. Pischel I, Burkard N, Kauschka M, Butterweck V, Bloomer RJ: Potential application of Russian tarragon (artemisia dracunculus l.) in health and sports.

, Austin, TX, USA), loaded into the SRNIL equipment, and leveled

, Austin, TX, USA), loaded into the SRNIL equipment, and leveled against a patterned quartz template/mould. For each target imprint area, QNZ datasheet nanoliter droplets of UV-curable, low-viscosity acrylate resist (MonoMat from Molecular Imprints, Inc.) were dispensed onto it and the quartz mould was brought into close proximity with the substrate, thus displacing the resist. This induced the resist to spread across the imprint field and fill up the mould relief via capillary action. A short exposure to UV light caused the polymerization of the monomers in the resist, after which the mould was separated from the substrate, leaving behind an inverse replica

of the mould pattern. This UV nanoimprint process was optimized for full pattern transfer while minimizing the residual material at the base of the recessed features and maintaining its uniformity across selleck chemicals llc the field. The optimized nanoimprint process was step-and-repeated over the surface of the wafer Panobinostat clinical trial to achieve wafer-scale

nanopatterning. The residual layer and underlying planarization layer were then removed by an oxygen reactive ion etching (RIE) process, thus exposing the underlying Si in these regions. Figure 1 Schematic diagram illustrating steps involved in step-and-repeat nanoimprint lithography (SRNIL) to produce pillar- or pore-patterned nanoimprinted wafers. In this work, three different pore-patterned quartz moulds were employed, allowing the corresponding inverse patterns to be defined. The replicated patterns consist of (a) 300-nm period hexagonal array of 180-nm (facet-to-facet dimension) hexagonal pillars/studs, (b) 300-nm period square array of 200 nm × 100-nm rectangular pillars, and (c) 150-nm period hexagonal array of 50-nm diameter circular studs. By incorporating some degree of lateral etching in RIE after NIL to remove the residual material in the recessed regions, NIL pillars/studs can be narrowed, thereby providing some

tunability in the dimensions of the NIL features. The patterns are shown in Figure 2a,b,c. Figure 2 SEM images of the nanoimprinted samples after RIE. Inset shows the respective Coproporphyrinogen III oxidase cross-sections. (a) 300-nm period hexagonal array of 180-nm (facet-to-facet) hexagonal pillars/studs, (b) 300-nm period square array of 200-nm × 100-nm rectangular pillars, and (c) 150-nm period hexagonal array of 50-nm diameter circular studs. The patterned area in each 300-nm period mould is 10 mm × 10 mm, while that for the 150-nm period mould is 5 mm × 5 mm, enabling equal-sized imprints to be replicated over a wafer surface. An instance of wafer-level nanoimprinting by SRNIL is shown in Figure 3. In this case, 32 nanoimprinted fields were generated over the surface of a 4″ Si wafer.

It seems that intraperitoneal inoculation is more efficient in di

It seems that intraperitoneal inoculation is more efficient in disseminating the infectious agents than intranasal inoculation and also that the peritoneal route would make it easier for infectious agents to reach the adventitia, which may be the main entrance for infectious agents. The ultrastructural study, which was performed only in one case for group, confirmed the presence of mycoplasma cells in the plaque, and of CP elementary bodies in the myocardial fibers, as well as of mycoplasma in the myocardial extracellular matrix. These data suggested that the studied infectious agents reached the circulation check details and many organs. The aggravation

of atherosclerosis is ARRY-438162 research buy probably caused by elements derived from infectious agents such as heat shock proteins or lipoproteins and not by direct presence of these agents in the lesion [22]. The mice fed with cholesterol enriched diet since the age of 8 weeks were infected at the age of 32 weeks and sacrificed at 40 weeks of age. This quite late period for inoculation increases the possibility that bacteria may be present in the atheroma plaques only as innocent bystanders, as they get a good breeding ground. However herein, the experimentally infected mice groups

showed increased severity and different morphologies in atherosclerotic plaques than non infected animals. The presented results strongly point to that the studied infectious agents have a relevant role in atherosclerosis Selleckchem VS-4718 aggravation inducing injury directly by their presence in the plaques and/or indirectly by immune system activation. All the infected groups showed low titers of serum antibodies to CP and MP. This is an expected result, since chlamydia and mycoplasma infections usually do not progress with high levels of antibodies probably due to the microbe escape

mechanisms from the immune response [23, 24]. Due to the small amount of blood collected from each animal, an individual antibody serum analysis could not be performed. The atherosclerosis was correlated more with the cholesterol levels than the antibodies to CP [25, 26]. For this reason, the lack of individual animal antibody titers to CP or MP may be not so relevant for the interpretation ID-8 of the studied infection. The progression of atherosclerosis may be influenced by repeated microbe infections. Periods of increase and decrease of atherosclerotic lesions are seen by angiographic studies [27, 28]. Bacterial lipopolysaccharides and endotoxins, autoimmunity due to molecular mimetization between the infectious agents, endovascular proteins such as Heat Shock Proteins and the activation of toll-like receptors by lipoproteins of the infectious agents are some of the mechanisms attributed to the development of inflammation and endothelial dysfunction in atherogenesis [29, 30].

Figure 3 LIBS spectra and emission lines (a) LIBS spectra of In0

Figure 3 LIBS spectra and emission lines. (a) LIBS spectra of In02PbTe for selected range from 300 to 466 nm. (b) LIBS indium emission lines at 410 nm for samples PbTe-2 (blue), In01PbTe (green), and In02PbTe (red), respectively. (c) LIBS indium emission lines at 325 nm for samples PbTe-2 (blue), In01PbTe (green), and In02PbTe (red), respectively. Figure  4 shows the SEM images of the PbTe

samples S63845 research buy prepared at 140°C and 200°C with different solvents, respectively. Figure  4a is the SEM image of the sample prepared with ethanol as the solvent at 140°C for 24 h which shows particles with appreciably uniform shape and average particle size of about 200 nm. However, with ethanol at 200°C for 24 h (Figure  4b), particles grow larger to an average size of about 300 nm. For comparison, Dorsomorphin in vivo the synthesis of PbTe samples was attempted with water as Doramapimod order the solvent. Figure  4c,d presents the SEM images of the PbTe samples synthesized with water as the solvent at 140°C and 200°C for 24 h, respectively.

From the images, it is clear that the PbTe samples formed with water as the solvent have chunks with various shapes and sizes. The PbTe sample prepared at 200°C for 24 h with water as solvent (Figure  4d) shows nano- to micron-sized spherical particles along with irregularly shaped particles. This result indicates that water alone is not sufficient for the formation of uniform small-sized PbTe nanoparticles. Figure  4e is the SEM image of the PbTe sample formed with water/glycerol (3:1 volume ratio) at 140°C for 24 h. It

shows clearly the fine particles with similar shape and a size in the range of 70 to 200 nm. The SEM image of the sample prepared with water/glycerol at 200°C for 24 h (Figure  4f) shows larger particles in the range of 200 to 500 nm in various shapes. The SEM results indicate that the particle size increases with the increase in the synthesis temperature when water/glycerol is used as solvent. From the SEM images, it can also be concluded that the combination of water and glycerol gives rise to nanoparticles with similar shape and small size compared to the use of alcohol or water alone as solvents. The use of ethanol or a water/glycerol mixture as solvent yields PbTe nanoparticles with uniform shape and size as compared with the PbTe particles prepared with only water. A report all by Zhu et al. [17] also suggests that solvothermal route of synthesis is more favorable than the hydrothermal one due to the strong polarity of the organic material in the solvothermal route which accelerates the dissolution of Te in the reaction process. Figure 4 SEM images of the PbTe samples prepared at 140°C and 200°C with different solvents. SEM images of undoped PbTe nanoparticles prepared without surfactants for 24 h in ethanol at (a) 140°C and (b) 200°C, in water at (c) 140°C and (d) 200°C, and in water/glycerol solution at (e) 140°C and (f) 200°C.

J Clin Oncol 2006, 24:2137–2150 PubMedCrossRef 3 Fuchs CS, Mayer

J Clin Oncol 2006, 24:2137–2150.PubMedCrossRef 3. Fuchs CS, Mayer RJ: Gastric carcinoma. N Engl J Med 1995, 333:32–41.PubMedCrossRef 4. Jatzko GR, Lisborg LY294002 PH, Denk H, Klimpfinger M, Stettner HM: A 10-year experience with Japanese-type radical lymph node dissection for gastric cancer outside of Japan. Cancer 1995, 76:1302–1312.PubMedCrossRef

5. Bremers AJ, Rutgers EJ, van de Velde CJ: Cancer surgery: the last 25 years. Cancer Treat Rev 1999, 25:333–353.PubMedCrossRef 6. Guo HQ, Guan P, Shi HL, Zhang X, Zhou BS, Yuan Y: Prospective cohort study of comprehensive prevention to gastric cancer. World J Gastroenterol 2003, 9:432–436.PubMed 7. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin 2011, 61:69–90.PubMedCrossRef 8. Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Rüschoff J, Kang YK, ToGA Trial Investigators: Trastuzumab

in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010, 376:687–697.PubMedCrossRef 9. Adachi M, Fukuda M, Nishida E: Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J 1999, 18:5347–5358.PubMedCrossRef 10. Zeng L, Imamoto A, Rosner MR: Raf kinase inhibitory protein (RKIP): a physiological regulator and future therapeutic target. Expert Opin CUDC-907 new Ther Targets 2008, 12:1275–1287.PubMedCrossRef 11. Keller ET, Fu Z, Brennan M: The role of Raf kinase inhibitor protein (RKIP) in health and disease. Biochem Pharmacol 2004, 68:1049–1053.PubMedCrossRef 12. Trakul N, Rosner MR: Modulation of the MAP

kinase signaling cascade by Raf kinase inhibitory protein. Cell Res 2005, 15:19–23.PubMedCrossRef 13. Yeung K, Seitz T, Li S, Janosch P, McFerran B, Kaiser C, Fee F, Katsanakis KD, Rose DW, Mischak H, Sedivy JM, Kolch W: Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 1999, 401:173–177.PubMedCrossRef 14. Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, Sedivy JM, Kolch W: Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase buy TH-302 pathway by the raf kinase inhibitor protein. Mol Cell Biol 2000, 20:3079–3085.PubMedCrossRef 15. World Medical Association: World Medical Association Declaration of Helsinki: Ethical Principales for Medical Research involving Human Subjects. [http://​www.​wma.​net/​en/​30publications/​10policies/​b3/​17c.​pdf] 16. Al-Mulla F, Hagan S, Al-Ali W, Jacob SP, Behbehani AI, Bitar MS, Dallol A, Kolch W: Raf kinase inhibitor protein: mechanism of loss of expression and association with genomic instability. J Clin Pathol 2008, 61:524–529.PubMedCrossRef 17.

Table 3 Experimental design of S titanus transmission trials No

Table 3 Experimental design of S. titanus transmission trials. No. of individuals (donors + receivers) Transmission type Acquisition time Destination 20 (10 + 10) Co-feeding with Asaia 24 hours q-PCR 38 (19 + 19)   48 hours   28 (14 + 14)   72 hours   20 (10 + 10)   96 hours   8 (4 + 4)   48 hours FISH Tot. co-feeders: 114 (57 + 57)       10 (5 + 5) Asaia ABT-263 Venereal transfer (male to female) 24 hours q-PCR 10 (5 + 5)   48 hours   10 (5 + 5)   72 hours   14 (7 + 7)  

96 hours   10 (5 + 5)   48 hours FISH 10 (5 + 5) Asaia Venereal transfer (female to male) 24 hours q-PCR 14 (7 + 7)   48 hours   10 (5 + 5)   72 hours   12 (6 + 6)   96 hours   8 (4 + 4)   48 hours FISH Tot. mated: 108 (54 + 54)       6 (3 + 3) 3-Methyladenine concentration Co-housing control trial (males with males) 24 hours   6 (3 + 3)   48 hours   6 (3 + 3)   72 hours   6 (3 + 3)   96 hours   10 (5 + 5) Co-housing control trial (females with females) 24 hours

q-PCR 6 (3 + 3)   48 hours   6 (3 + 3)   72 hours   6 (3 + 3)   96 hours   Tot. co-housed: 52 (26 + 26)       20 (10 + 10) Negative control for Co-feeding 24 hours q-PCR 22 (11 + 11)   48 hours   28 (14 + 14)   72 hours   32 (16 selleck chemicals + 16)   96 hours   10 (5 + 5)   48 hours FISH Tot. co-feeders: 112 (56 + 56)       16 (8 + 8) Negative control for venereal transfer (male to female) 24 hours q-PCR 10 (5 + 5)   48 hours   8 (4 + 4)   72 hours   14 (7 + 7)   96 hours   10 (5 + 5)   48 hours FISH 8 (4 + 4) Negative control for venereal transfer (female to male) 24 hours q-PCR 14 (7 + 7)   48 hours   12 (6 + 6)   72 hours   10 (5 + 5)   96 hours   10 (5 + 5)   48 hours FISH Tot. mated: 112 (56 + 56)       Number of insect specimens used for each trial. The duration of the acquisition period, as well as the type of analysis carried out, are indicated both for samples submitted P-type ATPase to experiments performed with Gfp-tagged Asaia and for negative controls. Venereal transmission trials When Gfp-tagged Asaia-infected

males were mated with uninfected females, transfer of Gfp-tagged symbiotic cells was observed, although a longer period was required to reach infection rates similar to those of the co-feeding trials. After a 24 hour incubation time subsequent to mating, only 20% of females (1 out of 5 individuals) were gfp gene-positive, with 40% (2 out of 5) positive after 48 hour, 60% (3 out of 5 individuals) at 72 hours, with 4 out of 7 individuals infected at 96 hours (Figure 1B). The average concentration of the marked symbiont in the body of S. titanus also increased with longer incubation periods, even though it remained significantly lower than that of donor individuals (df= 18; F= 11.663; P<0.05) (Figure 1E).