Allen, Lawrence, pp 585–587 Romanowska-Duda ZB, Grzesik M, Kalaji MH (2010) Physiological activity of energy plants fertilized with sewage sludge and usefulness of the Phytotoxkit test in practice. Environ Prot Eng 36:73–81 Luminespib molecular weight Rosenqvist E, van Kooten O (2003) Chlorophyll fluorescence: a general description and nomenclature. In: DeEll JR, Toivonen PMA (eds) Practical applications of chlorophyll fluorescence in plant biology. Kluwer, Dordrecht, pp 31–77 Ruban AV, Horton P (1999) The xanthophyll cycle modulates the kinetics of nonphotochemical energy dissipation in isolated light-harvesting complexes, intact

chloroplasts, and leaves of spinach. Plant Physiol 119:531–542PubMedCentralPubMed Rumeau D, Peltier G, Cournac L (2007) Chlororespiration and cyclic electron flow around PSI during photosynthesis

and plant stress response. Plant Cell Environ 30:1041–1051PubMed Sakai T, Kagawa T, Kasahara M, Swartz SE, Christie JM, Briggs WL, Wada M, Okada K (2001) Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc Natl Acad Sci USA 98:6969–6974PubMedCentralPubMed Samson G, Bruce D (1996) Origin of the Combretastatin A4 mw low yield of chlorophyll a fluorescence induced by single turnover flash in spinach thylakoids. Biochim Biophys Acta 1276:147–153 Samson G, Prášil O, Yaakoubd B (1999) Photochemical and thermal phases of chlorophyll a fluorescence. Photosynthetica 37:163–182 Satoh K (1981) Fluorescence induction and activity of ferredoxin-NADP+ reductase in Bryopsis chloroplasts. Biochim Biophys Acta 638:327–333 Schansker G, Strasser RJ (2005) Quantification of non-Q

B-reducing centers in buy C59 leaves using a far-red pre-illumination. Photosynth Res 84:145–151PubMed Schansker G, van Rensen JJS (1999) Performance of active photosystem II centers in photoinhibited pea leaves. Photosynth Res 62:175–184 Schansker G, Tóth SZ, Strasser RJ (2005) Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP. Biochim Biophys Acta 1706:250–261PubMed Schansker G, Tóth SZ, Strasser RJ (2006) Dark-recovery of the Chl a fluorescence transient (OJIP) after light adaptation: the qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side. Biochim Biophys Acta 1757:787–797PubMed Schansker G, Yuan Y, Strasser RJ (2008) Chl a fluorescence and 820 nm GSI-IX research buy transmission changes occurring during a dark-to-light transition in pine needles and pea leaves: a comparison. In: Allen JF, Osmond B, Golbeck JH, Gantt E (eds) Energy from the Sun. Springer, Dordrecht, pp 951–955 Schansker G, Tóth SZ, Kovács L, Holzwarth AR, Garab G (2011) Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise.

J Biol Chem 279:22866–22874PubMedCrossRef Logan BA, Baker DH, Adams WWIII, Demmig-Adams B (1997) The response of xanthophyll cycle-dependent energy dissipation in Alocasia brisbanensis to sunflecks in a subtropical rainforest. Aust buy Seliciclib J Plant Physiol 24:27–33CrossRef Matsubara S, Krause GH, Aranda

J, Virgo A, Beisel KG, Jahns P, Winter K (2009) Sun-shade patterns of leaf carotenoid composition in 86 species of neotropical forest plants. Funct Plant Biol 36:20–36CrossRef Nagel KA, Schurr U, Walter A (2006) Dynamics of root growth stimulation in Nicotiana tabacum in increasing light intensity. Plant Cell Environ 29:1936–1945PubMedCrossRef Niyogi KK, Grossman AR, Björkman O (1998) Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion.

Plant Cell 10:1121–1134PubMed Ögren E, Sundin U (1996) Photosynthetic responses to variable light: a comparison of species from contrasting habitats. Oecologia 106:18–27 Osmond CB, Grace SC (1995) Perspectives on photoinhibition and photorespiration in the field: quintessential inefficiencies of the light and dark reactions of photosynthesis? J Exp Bot 46:1351–1362 Pearcy RW (1990) Sunflecks and photosynthesis in plant canopies. Annu Rev Plant Physiol Plant Mol Biol 41:421–453CrossRef Pearcy RW, Calkin H (1983) Carbon learn more dioxide exchange of C3 and C4 tree species in the understory of a Hawaiian forest. Oecologia 58:26–32CrossRef Pfannschmidt T (2003) Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci 8:33–41PubMedCrossRef

Pons TL, Pearcy RW, Seemann MK5108 price JR (1992) Photosynthesis in flashing light in soybean leaves grown in different conditions. I. Photosynthetic induction state and regulation of ribulose-1,5-bisphosphate carboxylase activity. Plant Cell Environ 15:569–576CrossRef Schreiber U (2004) Pulse-amplitude-modulation (PAM) fluorometry and saturation pulse method: an overview. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, Dordrecht, pp 279–319 Sims DA, Pearcy RW (1993) Sunfleck frequency and duration affect growth-rate of the understorey plant, Alocasia macrorrhiza. Funct Ecol 7:683–689CrossRef Walter A, Scharr Endonuclease H, Gilmer F, Zierer R, Nagel KA, Ernst M, Wiese A, Virnich O, Christ MM, Uhlig B, Jünger S, Schurr U (2007) Dynamics of seedling growth acclimation towards altered light conditions can be quantified via GROWSCREEN: a setup and procedure designed for rapid optical phenotyping of different plant species. New Phytol 174:447–455PubMedCrossRef Walters RG (2005) Towards an understanding of photosynthetic acclimation. J Exp Bot 56:435–447PubMedCrossRef Watling JR, Ball MC, Woodrow IE (1997a) The utilization of lightflecks for growth in four Australian rain-forest species.

Eur J Clin Nutr 1996,50(11):34–740. 12. Lenon EJ, Lemann J Jr, Litzow JR: The effect of diet and stool composition on the net external acid balance of normal subjects. J Clin Invest 1996,45(10):1601–1607.CrossRef 13. Remer T: Influence of nutrition on acid-base balance-metabolic aspects. Eur J Nutr 2001,40(5):214–220.PubMedCrossRef 14. Mardon J, Habauzit V, Trzeciakiewicz A, Davicco MJ, Lebecque P, Mercier S, Tressol JC, Horcajada MN, Demigné C, Coxam V: Long-term intake of a high-protein diet with or without potassium citrate modulates acid-base metabolism, but not bone status, in male rats. J Nutr 2008,138(4):718–724.PubMed

CA4P chemical structure 15. Dawson-Hughes B, Harris SS, Rasmussen H, Song L, Dallal GE: Effect of dietary protein supplements on calcium excretion in healthy older men and women. J Clin Endocrinol Metab 2004,89(3):1169–1173.PubMedCrossRef

16. Wiederkehr M, Krapf R: Metabolic and endocrine effects of metabolic acidosis in humans. Swiss Med Wkly 2001,131(9–10):127–132.PubMed 17. Lowery LM, Devia L: Dietary protein safety and resistance exercise: what do we really know? J Int Soc Sports Nutr 2009, 6:3–9.PubMedCrossRef 18. McAllister RM: Adaptations in control of blood flow with training: splanchnic and renal blood flows. Med Sci Sports Exerc 1998,3(3):375–381. 19. Daugirda JT: Second generation logarithmic estimates of single-pool variable volume Kt/V: an analysis of error. J Am Soc Nephrol 1993,4(5):1205–1213. 17-DMAG (Alvespimycin) HCl 20. Hartman JW, Moore DR, Phillips SM: Resistance click here training reduces whole-body protein turnover and improves net protein retention in untrained young males. Appl Physiol Nutr Metab 2006,31(5):557–564.PubMedCrossRef

21. Meredith CN, Zackin MJ, Frontera WR, Evans WJ: Dietary protein requirements and body protein metabolism in endurance-trained men. J Appl Physiol 1989,66(6):2850–2856.PubMed 22. The Korean Nutrition Society: Dietary Refrerence Intakes for Koreans(KDRIs). Seoul, Korea; 2005. 23. Block KP, Soemitro S, Heywood BW, Harper AE: STI571 mouse Activation of liver branched-chain alpha-keto acid dehydrogenase in rats by excesses of dietary amino acids. J Nutr 1985,115(12):1550–61.PubMed 24. Frassetto LA, Todd KM, Morris RC Jr, Sebastian A: Estimation of net endogenous noncarbonic acid production in humans from diet potassium and protein contents. Am J Clin Nutr 1998,68(3):576–583.PubMed 25. Zaragoza R, Renau-Piqeras J, Portoles M, Hernandes-Yago J, Jorda A, Grisolia S: Rats fed prolonged high protein diets show an increases in nitrogen metabolism and liver megamitochondira. Arch Biochem Biophys 1987, 258:426–435.PubMedCrossRef 26. Kerstetter JE, Allen LH: Dietary protein increases urinary calcium. J Nutr 1990,120(1):134–136.PubMed 27. Lemann J Jr: Relationhsip between urinary calcium and net acid excretion as determined by dietary protein and potassium: a review. Nephron 1999,81(Suppl 1):18–25.PubMedCrossRef 28.

However, the recent sequencing of two strains of T. princeps from P. citri (PCIT and PCVAL) has shown that it is, in fact, the smallest (139 kb) and most simplified bacterial genome described to date [16, 19]. Functional analysis reveals that the genetic repertoire of T. princeps is unable to sustain cellular life, according to Gil et al. (2004) [20], and that it entirely Crenolanib research buy depends on M. endobia for many essential functions. Even though most of its genome is occupied by ribosomal

genes and genes involved in the biosynthesis of essential amino acids, T. princeps likely depends on its symbiotic consortium partner to build its own ribosomes and for amino acid production [16, 19]. The work published by McCutcheon and von Dohlen [16] mainly focused LY3023414 on the analysis of the T. princeps genome and detangling the amino acid biosynthetic pathways in which all three partners (T. princeps, M. endobia and the

host) appear to be involved. However, the characteristics and functionality of the M. endobia genome, as well as other possible modes of complementation between the two endosymbionts, have remained largely unexplored. In this work we present PARP inhibitor a comprehensive analysis of the predicted consortium functional capabilities and interactions, thus offering new insights into how this bacterial consortium may function internally. Additionally, we have performed a comparative analysis of both endosymbiont genomes in two P. citri strains, PCIT [16] and PCVAL ([19] and this work). Our analysis suggests that both genomes have undergone reductive evolution, albeit with some unusual genomic Interleukin-2 receptor features, probably as a consequence of their unprecedented compartmentalized organization. Results and discussion Main features and genomic variability between two

strains of P. citri nested endosymbionts The main molecular features of the genomes of T. princeps str. PCVAL [19] and PCIT [16], and M. endobia str. PCVAL (this work) and PCIT [16] are summarized in Table 1. It is worth mentioning that differences in CDS numbers and coding density between both strains are due to differences in the annotation criteria used, since the number of polymorphisms detected between the two sequenced strains of T. princeps and M. endobia is minimal (see Additional file 1 for a list of annotation differences in CDS and tRNA genes). Table 1 Main genomic features of the two strains of the P. citri endosymbiotic consortium already sequenced   T. princeps PCVAL T. princeps PCIT M. endobia PCVAL M.

Before surgery, the

animals were kept under standard laboratory conditions. In brief, a 1.5 cm side-to-side surgical EGDA was created between the first duodenal loop and the gastro-esophageal junction, about 3 cm distal to Treitz’s ligament, with accurate mucosa-to-mucosa opposition (Figure 1), so that duodenal and gastric click here contents flowed back into the esophagus. Unlike other models, this “”Kumagai-Hattori”" model PF477736 supplier preserves the animal’s normal stomach function and nutritional status [19, 21, 22]. Figure 1 Pathology findings of the esophageal cancer model. (A) Schematic illustration of the surgical intervention of the Kumagai-Hattori model (left) and representative macroscopic picture (right): unfixed esophagus, stomach and jejunum (excised en bloc) are opened through the dorsal wall (mucosal surface upward). (B-G) Histological findings observed (H&E staining): (B) anastomosis ulcer;

(C) squamous cell polypoid hyperplasia; (D) multilayered epithelium; (E) specialized columnar epithelium (intestinal metaplasia); (F) adenocarcinoma; (G) squamous cell cancer. (Original magnifications, 40×, 20× and 10×) Postoperatively, Eltanexor cost the animals had free access to water and food. No treatments with any known carcinogen were applied. Ten of the 74 rats died (mainly of respiratory complications) within 7 days after surgery and were not considered. As in already published experimental models, the animals were sacrificed

at different times after surgery (i.e. Group A [22 rats] after <10 weeks [range = 3–9.9], Group B [22 rats] after 10–30 weeks [range = 10–29.7], and Group C [20 rats] after >30 weeks [range = 31–54]) [19, 21, 22, 27, 28]. This study was approved by the Institutional Animal Care Committee of the University of Padova. All procedures were performed in accordance to the Italian law on the use of experimental animals (DL n. 116/92 art. 5) and according to the “”Guidelines on the Care and Use of Laboratory Animals”" (NIH publication 85–93, revised in 1985). Pathology Immediately after death, the thoracic and abdominal cavities were examined and the esophagus, stomach, and jejunum were excised en bloc. The esophagus was opened longitudinally through the dorsal wall. With selleck chemicals the mucosal surface uppermost, the margins of the specimen were fixed to a cork plate with pins. Gross specimens were fixed in 10% neutral-buffered formalin for 24 hours. All specimens were examined grossly (see gross pathology) and cut serially (2–3 mm thick coronal sections). The tissue samples were routinely processed. Tissue sections 4 μm thick were obtained from paraffin blocks and stained with Haematoxylin & eosin. Lung, liver, kidney and spleen tissues were also collected for histological assessment. Two experienced gastrointestinal pathologists (GI & MF) reviewed all the slides.

The azoles are antifungals commonly used to treat yeast infections [23, 24, 27, 28, 34]. Although in C. SBE-��-CD in vitro albicans the lipid biosynthesis pathways are not well documented, in S. cerevisiae these drugs operate on the biosynthesis of ergosterol at the C-14 demethylation stage [27, 28], causing a combination of ergosterol depletion and the accumulation of lanosterol, along with other 14-methylated

sterols [27, 28]. LY411575 nmr Fenpropimorph, as the other morpholines, inhibits two reactions catalyzed by Δ14 reductase (an essential enzyme) and Δ7- Δ8 isomerase [27, 28], resulting in the accumulation of 24-methylene ignosterol in the plasma membrane [27, 28]. Another group of antifungals, the polyenes, in theory interact specifically with the ergosterol present on the plasma https://www.selleckchem.com/products/epacadostat-incb024360.html membrane [26,

55], creating pores and concomitantly provoking plasma membrane physical and functional disruption, and thus cell death. In spite of the changes observed in ergosterol distribution, Cagup1Δ null mutant strain was as sensitive to polyenes as wt. Previous reports, suggest the possibility that polyenes interact also with other membrane lipids besides ergosterol [23, 24, 34]. In C. albicans the metabolism of the other lipids, namely sphingolipids and fatty acids, does not appear to be altered by the deletion of CaGUP1, as can be inferred from the susceptibly of the mutant to these lipids biosynthesis specific inhibitors (Ferreira, C., unpublished results). In a previous work, we found that the absence of ScGUP1 results in a defective cell wall composition and assembly, with a higher content in β-1,3 glucans and chitin, and lower fraction of mannoproteins [32]. By analogy, and since C. albicans Dipeptidyl peptidase and

S. cerevisiae cell walls are quite alike (with the exception of higher fraction of β-1,6 glucans on the former) [32, 56–58], one could considerer the possibility of Cagup1Δ null mutant cell wall also encompasses higher quantities of β-1,3 glucans. In C. albicans it was suggested a correlation between cell wall composition/architecture and resistance to azoles, hypha morphogenesis and virulence [59–61]. Namely, a putative role in azoles resistance on biofilm cells has been ascribed to β-1,3- glucans [61]. Nett and co-authors described cell wall architectural changes, and increased β-1,3 glucans content associated with fluconazole resistance [61]. Cell wall dynamics in C. albicans, underlie regulatory processes during the yeast-to-hyphae transition [59–63]. The ability to switch rapidly between these two forms of growth is a defining characteristic of C. albicans cells. Nevertheless, each form of growth provides critical functions required for pathogenicity/virulence [reviewed by [4] and by [5, 7]]. Namely, hyphae form is thought to facilitate host tissues invasion and escape from phagocytotic destruction [reviewed by [4] and by [5, 7, 64]].

ml-1. Table 4 Cumulative MFC Selleckchem LY2874455 profile of 65 clinical isolates of Candida spp. treated with 20-piperidin-2-yl-5α-pregnan-3β,20-diol (AZA) and 24(R,S),25-epiminolanosterol (EIL).     Cumulative MFC* (μg.ml-1) Species (no. isolates) Drugs 0.03 1 2 4 8 16 > 16 All species (65) AZA 1.52 3.04 12.16 16.72 34.96 44.08 100   EIL     6.08 15.20 30.40 51.68 100 Candida albicans (21) AZA     4.76 4.76 9.52 9.52 100   EIL       9.52 28.57 61.98 100 Candida parapsilosis (19) AZA   5.26 26.31 36.87 68.42 68.42 100   EIL     10.52 15.79 26.31 63.15 100 Candida tropicalis (14) AZA         35.71 64.28 100

  EIL     7.17 7.17 35.71 42.87 100 Candida glabrata (2) AZA     50 50 50 50 100   EIL       50 50 50 100 Candida krusei (1) AZA             100   EIL             100 Candida lusitaneae (1) AZA             100   EIL       100 100 100 100 Candida guilliermondii (3) AZA             100   EIL          

  100 Candida zeylanoides (1) AZA 100 100 100 100 100 100 100   EIL     100 100 100 100 100 Candida rugosa (1) AZA       100 100 100 100   EIL           Geneticin   100 * data is expressed in percentual of isolates. Ultrastructural effects The general morphology of untreated C. albicans was Quisinostat molecular weight observed using scanning (Figure 2a) and transmission (Figure 2b–c) electron microscopy. The shape of C. albicans varies from spherical (4.90 ± 0.49 μm diameter) to oval cells when viewed by scanning electron microscopy (Figure 2a). Transmission electron microscopy revealed the presence of normal cell walls with a thickness of 233 ± 25 nm (Figure 2b–c), including a thin electron-dense outer layer with delicate fibrillar structures clearly visible (f in Figure 2c). A continuous cytoplasmatic membrane (cm)lining

a homogeneous and electron-dense cytoplasm containing ribosomes, nucleus (n), and nucleoli Buspirone HCl (nu) could also be observed (Figure 2b–c). Treatment of C. albicans with MIC50 of AZA (0.25 μg.ml-1) and EIL (1.00 μg.ml-1) induced significant morphological changes, which ranged from discrete alterations to total destruction of the fungal cells. A common alteration observed after the treatment with AZA and EIL was a significant increase in cell size, from 5 μm to 7 μm in diameter (Figure 2d, g, j, and 2m). The number of altered cells was counted, and the morphological alterations appeared in 34.79% and 55.17% of the cells after treatment with AZA and EIL, respectively. Among the most frequently observed ultrastructural alterations were: (i) presence of small buds (asterisks in Figure 2d, g and 2j); (ii) irregular cell-wall surfaces (arrows in Fig. 2D and 2E); (iii) loss of cell-wall integrity, with an apparent shedding of cell components (Fig. 2G–J, white and black arrows); and (iv) a two- to three-fold increment of the cell wall thickness was observed after treatment with AZA and EIL, respectively (Figure 2f, i, l, and 2n).

PHA-induced proliferation of human blood mononuclear cells The isolated PBMC were distributed into 96-well flat-bottom plates in 100 µL aliquots (2 × 105 cells/well). PHA was added at a concentration of 5 µg/ml. The compounds were tested at doses of 1, 10, and 50 µg/ml. DMSO at appropriate dilutions served as control. After a four-day incubation in a cell culture incubator, the proliferative response of the cells was determined by the colorimetric MTT method (Hansen et al., 1989). The results are given in percentage inhibition as compared with appropriate

DMSO controls. Cytotoxicity of the compounds against GW3965 in vitro human blood mononuclear cells PBMC at density of 2 × 105/well, re-suspended in the culture medium, were cultured for 24 h in a cell culture incubator with the preparations at indicated concentrations. Cell survival was determined by MTT colorimetric method (Hansen et al., 1989). The results are given in percentage inhibition as compared with appropriate DMSO controls. Lipopolysaccharide-induced TNF-a production in whole blood cell culture Venous blood from a single donor was diluted 10× with RPMI-1640 medium and distributed in 1 ml aliquots in 24-well culture plates. The cultures were stimulated by QNZ supplier addition of 1 µg/ml of LPS from E. coli, O111:B4. The compounds were added to the cultures at concentrations of 5 and 25 µg/ml. Higher

concentrations of the compounds could not be used because of inhibitory effects on TNF-α production by corresponding DMSO (the solvent) dilutions. Appropriate dilutions of DMSO served as controls. After overnight incubation Selleckchem PF-3084014 in a cell culture incubator, the supernatants were harvested and frozen at −20 °C until cytokine determination by a biological assay (Espevik and Nissen-Meyer, 1986). The results are given in percentage Inositol monophosphatase 1 inhibition as compared with appropriate DMSO controls. Growth inhibition

of tumor cell lines L-1210 lymphoma and SW-948 colon tumor cell lines derived from the Collection of Cell Lines of The Institute of Immunology and Experimental Therapy, Wrocław, Poland. The lines were re-suspended in the culture medium and distributed into 96-well flat-bottom plates. L-1210 was present at 1.5 × 104 cells/well while SW-948 and at 2.5 × 104 cells/well. The preparations were added to the wells at the concentration range of 0.1–50 µg/ml. Cisplatin was used as a reference drug in the same concentrations. After 3-day incubation in a cell culture incubator, the proliferation was determined using MTT colorimetric method. The data are presented as a mean OD value from quadruplicate wells ± SE. Statistics The results are presented as mean values ± standard error (SE) or percentage inhibition = [(control value − tested value)/control value] × 100. Brown-Forsyth’s test was used to determine the homogeneity of variance between groups.

Electronic supplementary material Additional file 1: Figure S1: Using external standards to compare the sequencing

qualities between the two libraries. The identity with external standard sequence is split this website into four groups and we calculated the proportion of sequences in each sequencing batch fall into each group. Figure S2. LEfSe comparison of microbial communities between individuals B and D with different data sources. Figure S3. Alpha diversity index calculated from the V6F-V6R and V4F-V6R datasets at error rates of 0%, 0.1% and 1%. Figure S4. Procrustes analysis of datasets from the two libraries and error rates. (DOC 3 MB) References 1. Pennisi E: Human genome 10th anniversary. Tanespimycin ic50 Digging deep into the microbiome. Science 2011,331(6020):1008–1009.PubMedCrossRef 2. Heo S-M, Haase EM, Lesse AJ, Gill SR, Scannapieco FA: Genetic relationships between respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from patients in the intensive care unit undergoing mechanical ventilation. Clin Infect Dis 2008,47(12):1562–1570.PubMedCrossRef 3. Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI: The human microbiome project. Nature 2007,449(7164):804–810.PubMedCrossRef 4. Zhou HW, Li DF, Tam NF, Jiang XT, Zhang H, Sheng HF, Qin J, Liu X, Zou F: BIPES, a cost-effective high-throughput method for assessing microbial diversity.

ISME J 2011,5(4):741–749.PubMedCrossRef 5. Kuczynski J, Lauber CL, Walters WA, Parfrey LW, Clemente JC, Gevers D, Knight R: Experimental and analytical tools for studying the human microbiome. Nat Rev Genet 2012,13(1):47–58.CrossRef 6. Sogin ML, Morrison STI571 HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ: Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci USA 2006, 103:12115–12120.PubMedCrossRef 7. Huse SM, Dethlefsen L, Huber JA, Mark Welch D, Relman DA,

Sogin ML: Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genet 2008,4(11):e1000255.PubMedCrossRef 8. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R: Bacterial community variation in human body habitats across space and time. Science 2009, 326:1177486.CrossRef 9. Jumpstart Consortium Human Microbiome Project Data Generation Working Group: OSBPL9 Evaluation of 16S rDNA-based community profiling for human microbiome research. PLoS One 2012,7(6):e39315.CrossRef 10. Huse SM, Ye Y, Zhou Y, Fodor AA: A core human microbiome as viewed through 16S rRNA sequence clusters. PLoS One 2012,7(6):e34242.PubMedCrossRef 11. Junier P, Kim OS, Hadas O, Imhoff JF, Witzel KP: Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples. Appl Environ Microbiol 2008,74(16):5231–5236.PubMedCrossRef 12.

pneumoniae antibodies, Pab(rP1-I), Pab(rP1-II), Pab(rP1-III), or Pab(rP1-IV (1:500 dilutions) were added and were incubated for 1 h at 37°C. Wells were washed subsequently

and later 100 μl of secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (whole molecule, 1:100 dilutions) (Santa Cruz Biotech, USA) was added. The cells were washed twice in PBS before and after the addition of antibodies. Cells were subsequently incubated with Evans Blue diluted 1:10 for 30 min at 37°C. Finally the cells were washed with double Vistusertib cell line distilled water. M. pneumoniae adhesion inhibition assay For the adhesion inhibition assay, protocol developed by Svenstrup et al. was followed [14]. find more Briefly, the M. pneumoniae suspension (50 μl) was pre-incubated for 2 h at 37°C with 50 μl of anti-M. pneumoniae Erismodegib manufacturer antibodies, Pab (rP1-I), Pab (rP1-II), Pab (rP1-III) or Pab (rP1-IV) in different dilutions (1:50, 1:100, 1:200 and 1:500) before incubation of the HEp-2 cells. The M. pneumoniae -antibodies suspension (100 μl) was then added to the HEp-2 cells together with 1 ml of RPMI with penicillin and incubated overnight in 5% CO2 at 37°C. Fixation and addition of secondary antibodies were carried-out as described in the adhesion of M. pneumoniae. To further confirm the adhesion inhibition, the assay was performed as mentioned above except that DAPI was added at the end of the assay for further 30 min at room temperature. M. pneumoniae surface

exposure assay To detect M. pneumoniae surface protein, the primary antibodies were added before methanol fixation. Otherwise, the procedure during was the same as described for the M. pneumoniae adhesion assay. Indirect immunofluorescence

microscopy (IFM) Samples prepared for M. pneumoniae adhesion assay, M. pneumoniae adhesion inhibition assay and M. pneumoniae surface exposure assay were analyzed by IFM using Olympus BX51upright fluorescence microscope. Before microscopy analysis, a drop of anti-fade solution (p-phenyldiamine dihydrochloride 1 μg ml−1 in PBS 10% and glycerol 90%, pH 9.0) was placed between the glass cover slips and the slides. Acknowledgments This work was supported by Indian Council of Medical Research, New Delhi for financial grant (File No. 5/3/3/9/2003-ECD-I) and Senior Research Fellowship to Bishwanath Kumar Chourasia (ICMR File No. 80/576/2007-ECD-1). We thank Mr. Promod Kumar for his assistance in M. pneumoniae culture. Electronic supplementary material Additional file 1: Immune response of P1 protein fragment rP1-I in rabbits. Bar diagram showing immune responses in four different White New Zealand rabbits immunized with purified recombinant protein fragment, rP1-I with complete/incomplete Freund’s adjuvant. Control rabbits were injected with complete/incomplete Freund’s adjuvant in normal saline according to the immunization schedule. (TIFF 29 KB) Additional file 2: Western blot analysis of recombinant P1 protein fragments with rabbits pre-bleed sera.