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2004, 126:1620–1633.PubMedCrossRef 14. Thomas CM, Versalovic J: Probiotics-host communication: modulation of signaling pathways in the intestine. Gut Microbes 2010, 1:1–16.CrossRef 15. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, Lochs H: Mucosal flora in inflammatory bowel disease. Gastroenterology 2002, 122:44–54.PubMedCrossRef 16. Collado MC, Calabuig M, Sanz Y: Differences between the fecal microbiota of celiac infants and healthy controls. Curr Issues Intest Microbiol 2007, 8:9–14.PubMed 17. Medina M, De Palma https://www.selleckchem.com/products/apo866-fk866.html G, Ribes-Koninckx C, Calabuig M, Sanz Y: Bifidobacterium strains suppress in vitro the pro-inflammatory milieu

triggered by the large intestinal microbiota of coeliac patients. J Inflamm 2008, 5:19.CrossRef 18. De Angelis M, Rizzello CG, Fasano A, Clemente MG, De Simone C, Silano M, De-Vincenzi M, Losito I, Gobbetti M: VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for celiac sprue. BBA – Mol Basis Dis 2005, 1762:80–89. 19. Lyton A, McKay L, Williams D, Garrett V, Gentry R, Sayler G: Development Bumetanide of Bacteroides 16S rRNA gene TaqMan-based Real-Time PCR assays for estimation of total, human, and bovine fecal pollution in water. Appl Environ Microbiol 2006, 72:4214–4224.CrossRef 20. Kopečný J, Mrázek J, Fliegerová K, Kott T: Effect of gluten-free diet on microbes in the colon. Folia Microbiol 2006, 51:287–290.CrossRef 21. Kopečný J, Mrázek J, Fliegerová

K, Frühauf P, Tučková L: The intestinal microflora of childhood patients with indicated celiac disease. Folia Microbiol 2008, 53:214–216.CrossRef 22. Bertini I, Calabro A, De Carli V, Luchinat C, Nepi S, Porfirio B, Renzi D, Saccenti E, Tenori L: The metabonomic signature of celiac disease. J Proteome Res 2009, 8:170–177.PubMedCrossRef 23. De Palma G, Nadal I, Medina M, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y: Intestinal dysbiosis and reduced immunoglobulin-coated bacteria associated with coeliac disease in children. BMC Microbiol 2010, 10:63.PubMedCrossRef 24. Walter J, Hertel C, Tannock GW, Lis CM, Munro K, Hammes WP: Detection of Lactobacillus , Pediococcus , Leuconostoc , and Weisella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl Environ Microbiol 2001, 67:2578–2585.PubMedCrossRef 25.

Lin AE, Krastel K, Hobb RI, Thompson SA, Cvitkovitch DG, Gaynor EC: Atypical roles for Campylobacter jejuni amino acid ATP binding cassette transporter components PaqP and PaqQ in bacterial stress tolerance and pathogen-host cell dynamics. Infect Immun 2009, 77:4912–4924.PubMedCrossRef 31. Van Deun K, Pasmans F, Ducatelle R, Flahou B, Vissenberg K, Martel A, Van den Broeck Dasatinib supplier W, Van Immerseel F, Haesebrouck F: Colonization strategy of Campylobacter jejuni results in persistent infection of the chicken gut. Vet Microbiol 2008, 130:285–297.PubMedCrossRef 32. Eucker TP, Konkel ME: The cooperative action of bacterial fibronectin-binding

proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling. Cell Microbiol 2012, 14:226–238.PubMedCrossRef 33. Bernhardt

TG, de Boer PA: The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway. Mol Microbiol 2003, 48:1171–1182.PubMedCrossRef 34. Kassem II, Zhang Q, Rajashekara G: The twin-arginine translocation system: contributions to the pathobiology of Campylobacter jejuni. Future Microbiol 2011, 6:1315–1327.PubMedCrossRef 35. Frirdich E, Biboy J, Adams C, Lee J, Ellermeier J, Gielda LD, Dirita VJ, Girardin SE, Vollmer W, Gaynor EC: Peptidoglycan-modifying enzyme Pgp1 is required for helical cell shape and pathogenicity traits in Campylobacter jejuni. PLoS Pathog 2012, 8:e1002602.PubMedCrossRef 36. Taveirne ME, Sikes ML, Olson JW: Molybdenum and tungsten in Campylobacter jejuni: their physiological role and identification Staurosporine concentration of separate transporters regulated by a single ModE-like protein. Mol Microbiol 2009, 74:758–771.PubMedCrossRef 37. Wilson DL, Bell JA, Young VB, Wilder

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3%, 0.4%, and 0.5% agar at 18°C and 28°C. (B) Motility assays in semisolid KB media containing 0.3% (left) and 0.5% (right) agar. (C) The results obtained using the stab technique in M9 and KB media. Low temperature induces oxidative stress and iron metabolism Another group of genes differentially expressed at 18°C correspond to genes related to iron metabolism (Cluster 6). Iron fulfills a vital role in virtually all organisms because of its participation in several cellular processes. Because iron is in short supply in many habitats, bacteria secrete siderophores, compounds that are specific iron chelators, to mobilize inside

the cell through membrane receptor molecules [44]. Two genes, PSPPH_3753 that encodes a protein related to siderophore synthesis and PSPPH_1923 buy AZD2014 that is involved in pyoverdine synthesis (a major siderophore of the fluorescent Pseudomonas sp.), were induced at 18°C relative buy LY2835219 to 28°C [45]. Likewise, the gene encoding sigma factor protein PvdS, which is required for expression of pyoverdine synthesis genes, was induced under these conditions [46]. The induction of this PvdS protein was validated by RT-PCR analysis (Figure 3). One gene encoding the regulatory protein FecR (PSPPH_2117) and proteins involved in iron transport were also included in this group. It is known that in P. aeruginosa, the Fur protein is the master regulator of iron homeostasis. It represses

pyoverdine synthesis via negative regulation of the pvdS gene under high iron concentrations. However, in iron-limiting conditions, Fur repression is released and transcription can occur [47]. It has been reported that PvdS sigmulon is conserved among the fluorescent pseudomonads, including the P. syringae group [46]. Although the fur gene was not printed on our microarray, the functional status of Fur protein can be inferred as inactive because the genes regulated by this protein are induced in the conditions evaluated. This expression profile

simulates conditions of iron deficiency. To phenotypically evaluate changes in the expression of siderophores synthesis genes in function of temperature, we performed quantitative analyses of siderophores at 18°C and 28°C. The results of these assays showed that at 18°C, the amount of siderophores in the culture very supernatant was higher (58.6 ± 0.39 μM) compared to when the bacterium is grown at 28°C (20.53 ± 0.844 μM). Thus, the results demonstrate that low temperature induces siderophores production by the bacterium. Additionally, it has been reported that in several bacteria, the Fur protein positively regulates the expression of genes involved in various pathways in response to large iron amounts, such as oxidative stress genes (e.g. catalases) [47]. In our microarray, the PSPPH_3274 gene (encoding the catalase KatB) was induced at 18°C, which would be inconsistent with our hypothesis about an inactive status for the Fur protein at low temperatures.

Bacteriophages can influence the level of virulence of BGB324 mouse bacterial pathogens [16] and can change the phenotypic properties of closely related strains of bacteria. In Wolbachia-infected Drosophila, Culex, Nasonia and other insects, WO prophages appear to be temperate, that is, they have an integrated prophage form and can also generate virions which result in bacterial lysis [6, 11, 15, 17] and [18]. In the parasitoid wasp, N. vitripennis, Bordenstein et al used a quantitative PCR

assay to demonstrate that Wolbachia titer, which correlates with CI intensity, is inversely related to copy number of temperate WOVitA [15]. This relationship, known as the Phage Density Model, predicts that low CI strains of Wolbachia will have a high number of phage particles, and, conversely, high CI strains of Wolbachia

will have low titers this website of phage particles [15, 19]. In Drosophila, however, it is not known which of the diverse prophage elements give rise to lytic viruses, how their lytic properties are regulated, or the effect of lysis on host phenotype. Although most tailed bacteriophages have evolved a temperate lifestyle, it is not yet known if the prophage elements in wRi are functional, defective, satellite phages, or agents of gene transfer [20]. Typically, mature WO phage particles are detected using primers specific to the open reading frame encoding a putative minor capsid protein C (ORF7) [5]. In wRi of D. simulans, however, ORF7 is present in all four prophage insertions [WRi_005560], [WRi_007170], [WRi_010220], and [WRi_012630] and so the presence of ORF7 is not a specific indicator of which phage is active. In this paper we measure the relative copy number of mature, active WORiC phage particles in whole flies and tissues

of D. simulans and determine variations in Wolbachia and WO copy number between individual larval hosts by quantitative PCR. A comparison of the genome architecture of known active phages WOVitA1 and WOCauB2 to WORiC identifies modules for head assembly and DNA packaging as well as tail morphogenesis that are conserved in all known active WO phages. Methods Strains Fenbendazole and media D. simulans (Riverside) (DSR) stocks were maintained at room temperature on a standard diet of cornmeal, dextrose and yeast. Stocks were stably infected with a single Wolbachia strain (wRi) and have been maintained at the University of Alberta laboratory for approximately 6 years. The presence of Wolbachia was confirmed at regular intervals using 81F and 691R wsp primer pairs [21]. DNA extractions DNA from whole flies and gonads was extracted from animals that were less than 5 days post eclosion. Newly eclosed flies were separated by sex and allowed to develop to the appropriate age. Gonad DNA samples were obtained from 4 groups of 100-150 testes each and 4 groups of 75-150 ovaries each. Whole fly DNA was taken from 4 groups of 15 flies each.

Despite the economic and environmental damages caused by the RPW in all the areas where it is endemic and where it has been accidentally

introduced, little is known about its gut microbiota. The bacterial community that is embedded in the frass produced inside the tunnels of the palm Phoenix canariensis Chabaud by the RPW larvae is dominated by Enterobacteriaceae with a facultative fermentative metabolism [2]. The purpose of this study was to analyse the diversity of the gut microbiota of the R. ferrugineus larvae, that represent the development selleck chemicals stage responsible for damages to palms. Field-caught larvae were sampled from its favourite host P. canariensis in different seasons and sites in Sicily (Italy), and analysed for the diversity of their gut microbiota. The analysis of the bacterial community was carried out by culture-independent methods using temporal thermal gradient gel electrophoresis (TTGE) and FLX454 pyrosequencing Doxorubicin ic50 of PCR-generated amplicons from the 16S rRNA gene. Results Total diversity of the gut microbiota of field caught RPW larvae Bacterial TTGE profiles were generated using PCR-amplified bacterial 16S rRNA gene fragments from the content of pooled RPW larval guts collected from the trunks of infested P. canariensis palms in three different seasons and two areas in Sicily (Italy). TTGE

band profiles indicate the presence of an average of 25 bands per sample, that correspond to putative bacterial phylotypes in RPW larval guts. An example of TTGE gel is shown in Figure 1, where three different pooled guts collected in December 2010 and April 2011 in Palermo (lanes 1 and 2, respectively), and in April 2011 in San Vito lo Capo (Trapani, lane 3) were analysed. All samples shared 16 bands, while 4, 2 and 4 bands were unique for samples 1, 2, 3, respectively. Similar profiles were obtained

from larvae collected in October both in Palermo and Trapani (data not shown). Random sequencing of TTGE bands identified the presence of uncultured Gammaproteobacteria (of the genera Pantoea and Enterobacter) and Firmicutes (of genera Megasphaera and Clostridium) clonidine (Figure 1). Figure 1 Temporal Thermal Gradient gel Electrophoresis (TTGE) profiles of PCR-amplified 16S gene fragments derived from field collected larvae of Rhynchophorus ferrugineus . Lane 1: TTGE profile of a pool of three larvae (average weight: 3.25 g; SD: 0.55) collected in December 2010 in a palm tree in the urban area of Palermo (Italy). Lane 2: TTGE profile of a pool of three larvae collected in April 2011 (average weight: 3.86 g; SD: 0.64) in the urban area of Palermo (Italy). Lane 3: TTGE profile of a pool of three larvae collected in April 2011 (average weight 3.60 g; SD: 0.53) in San Vito lo Capo (Trapani, Italy).

Subsequently values from the predefined timepoints

were analyzed with the pre inoculation (P.I.) values using paired t-test (Y to Z). Ethics statement To reduce the numbers of experimental animals used, we combined the earlier published influenza pathogenesis study [21] with the current study addressing questions related to activation of coagulation and tissue fibrin deposition during influenza virus infection. Animal housing and experiments were all in compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997) as documented previously [21]. The study protocol was approved by the independent animal experimentation ethical review committee of the Netherlands Vaccine Institute (permit number 200900201). Animal welfare was observed on a daily basis, and

animal handling was performed under light anesthesia C59 wnt molecular weight using a mixture of ketamine and medetomidine. After handling, atipamezole was administered to antagonize the effect of medetomidine. Coagulation assays Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured CT99021 using a BCS-XP coagulation analyzer (Siemens Healthcare Diagnostics) according to the instructions of the manufacturer. Clotting was initiated with Thromborel S (PT) and Pathrombin SL (APTT). VWF ristocetin cofactor activity was also determined on the BCS-XP with reagents of the manufacturer, and was expressed as percentage of normal pooled human plasma. Thrombin-antithrombin complexes (TAT, Siemens Healthcare Diagnostics) and D-dimer levels (Asserachrom, Roche, The Netherlands) were measured using enzyme-linked immunosorbent assay. All these assays were carried out within the BSL-3 setting after careful calibration and validation. Pathology and fibrin staining Gross pathology

and histopathology were evaluated as previously described [21]. Relative lung weight was used as a validated measure of gross pathology and lung inflammation [47]. Phosphatidylinositol diacylglycerol-lyase For detection of fibrin, tissues were stained with the Lendrum staining according manufacturers’ protocol (MSB RRSK2-100 stain kit, Atom scientific). On each slide a small piece of human placenta was added as a positive control. Semi-quantitative assessment of fibrin expression in the lungs was performed as follows: for the alveoli, 25 arbitrarily chosen, 20x objective, fields of lung parenchyma of one lung section were examined by light microscopy for the presence of fibrin, without the knowledge of the identity of the animals. The scores (+ or -) were multiplied by 4 and presented as percentage. Virology The presence of virus and virus replication in the respiratory tract were measured by determining infectious virus titers at different sites of the upper respiratory tract (URT) and lower respiratory tract (LRT).

Acknowledgements This work was supported by grants from Fundação Carlos Chagas MAPK inhibitor Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). References 1. Galle PC: Clinical presentation and diagnosis of endometriosis. Obstet Gynecol Clin North Am 1989, 16:29–42.PubMed 2. Wheeler JM: Epidemiology and prevalence of endometriosis. Infertil Reprod Med Clin North Am 1992, 3:545–549. 3. Giudice LC, Kao LC: Endometriosis. Lancet 2004, 364:1789–1799.PubMedCrossRef 4. Groothuis PG, Nap

AW, Winterhager E, Grümmer R: Vascular development in endometriosis. Angiogenesis 2005, 8:147–156.PubMedCrossRef learn more 5. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995,1(1):27–31.PubMedCrossRef 6. Taylor RN, Lebovic DI, Mueller MD: Angiogenic factors in endometriosis. Ann N Y Acad Sci 2002, 955:89–100.PubMedCrossRef 7. Olson JE, Cerhan

JR, Janney CA, Anderson KE, Vachon CM, Sellers TA: Postmenopasual cancer risk after self-reported endometriosis diagnosis in the Iowa women’s health study. Cancer 2002, 94:1612–1618.PubMedCrossRef 8. Melin A, Sparen P, Bergqvist A: Endometriosis and the risk of cancer with special emphasis on ovarian cancer. Hum Reprod 2006, 21:1237–1242.PubMedCrossRef 9. Patan S: Vasculogenesis and angiogenesis. Cancer Treat Res 2004, 117:3–32.PubMed 10. Chen QH, Qu JU, Xu YY, Qiu NX, Zhuang YZ, Zhong S, Fang QQ: Expressions of matrix metalloproteinase-9 and

tissue inhibitor of metalloproteinase-1 in ectopic and eutopic endometrium. Zhonghua Fu Chan Ke Za Zhi 2004, 39:809–812.PubMed 11. Risau W: Mechanisms of angiogenesis. Nature 1997,386(6626):671–674.PubMedCrossRef 12. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Müller KH, Sharkey AM, et al.: Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996, 98:482–489.PubMedCrossRef 13. Pupo-Nogueira A, de Oliveira Staurosporine in vivo RM, Petta CA, Podgaec S, Dias JÁ, Abrão MS: Vascular Endothelial Growth Factor concentrations in the serum and peritoneal fluid of women with endometriosis. Int J Gynecol Obstet 2007,99(1):33–37.CrossRef 14. Ferrara N: Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol 2001, 280:1358–1366. 15. Bourlev V, Volkov N, Pavlovitch S, Lets N, Larsson A, Olovsson M: The relationship between microvessel density, proliferative activity and expression of vascular endothelial growth factor-A and its receptors in eutopic endometrium and endometriotic lesions. Reproduction 2006,132(3):501–509.PubMedCrossRef 16.

Shewanella oneidensis is a Gram-negative γ-Proteobacterium that is a facultative anaerobe found in a wide range of environments. S. oneidensis is a member of a class of bacteria known as the dissimilatory metal-reducing bacteria (DMRB). Under anaerobic conditions, S. oneidensis has the ability to utilize an impressively wide range of both organic and metallic SAR245409 research buy terminal electron acceptors. These metallic terminal electron acceptors include Cr(VI), Fe(III), Mn(III) and (IV), and U(VI) [9, 10]. The ability to mitigate the toxicity of soluble Cr(VI) and U(VI) by reduction

to insoluble oxides of Cr(III) and U(IV), respectively, makes Shewanella an attractive potential bioremediating organism. In addition, the ability to deliver electrons to the extracellular environment allows Shewanella to generate electrical current in microbial fuel cells [11]. Because the transition between aerobic and anaerobic metabolism is likely to occur frequently in nature, it is probable that sRNAs play a role in the transition between these metabolic states in S. oneidensis. To gain insight into the functions of Hfq in S. oneidensis, we have constructed and characterized a null allele of the hfq gene. The hfq∆

mutation in S. oneidensis is pleiotropic, resulting in defects in aerobic growth and greatly reduced recovery of colony forming units (CFU) from stationary phase cultures. In addition, loss of hfq results in compromised anaerobic growth on fumarate and diminished capacity to AZD1152-HQPA mw Oxalosuccinic acid reduce Cr(VI). Finally, we have found that the S. oneidensis hfq∆ mutant is highly sensitive to oxidative stress. Importantly, each of the hfq mutant phenotypes we have described is complemented by a plasmid-borne copy

of the wild type S. oneidensis hfq gene, strongly suggesting that the mutant phenotypes we have observed are the result of the loss of hfq and not due to disruption of another gene. Our results suggest that Hfq in S. oneidensis is involved in both common and organism-specific regulatory processes. To our knowledge, this is the first characterization of an hfq mutant in a dissimilatory metal reducing bacterium. Methods Media and growth conditions Aerobic cultures were grown in either LB (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl) or a modified version of the original M1 medium [9] with 30mM lactate as the electron donor. The modified M1 medium used in this study contains buffer/salts (3mM PIPES buffer, pH 7.0, 28mM NH4Cl, 1.34mM KCl, 4.4mM NaH2PO4, 125mM NaCl), vitamins [81.8nM D-biotin (vitamin B7), 45.3nM folic acid (vitamin B9), 486.4nM pyridoxine HCl (vitamin B6), 132.8nM riboflavin (vitamin B2), 133.6nM thiamine HCl (vitamin B1), 406.2nM nicotinic acid (vitamin B3), 209.8nM D-pantothenic acid, 0.74nM vitamin B12, 364.6nM p-aminobenzoic acid, 242.4nM lipoic acid], minerals [78.5μM nitriloacetic acid (trisodium salt), 249.1μM MgSO4 · 7 H2O, 29.6μM MnSO4 · 1 H2O, 171.1μM NaCl, 3.6μM FeSO4 · 7 H2O, 6.8μM CaCl2 · 2 H2O, 4.2μM CoCl2 · 6 H2O, 9.

A dynA ezrA double deletion leads to a strongly exacerbated phenotype in cell division, suggesting that like EzrA, a regulator

of FtsZ ring formation, B. subtilis dynamin affects an early stage in cell division. However, the combination of a dynA deletion with a divIB deletion also leads to a synthetic effect on cell division. DivIB affects a state in division clearly later than the formation of the Z ring, indicating that the function of DynA in division cannot be correlated with a defined stage in division. In any event, the accumulation of dynamin at the Z ring underlines the idea that dynamin confers a function during division. Expression of DynA in a eukaryotic cell system showed that the protein has intrinsic affinity to the cell membrane DAPT and can assemble into tubulated Erastin research buy structures. However,

these pointed outwards of the cells, while the assumed function of dynamin in the bacterial cell would either be an inward bending of the membrane during cell division, or the fusion of membranes as the last step during division. It is likely that DynA needs cofactors for its appropriate function in the bacterium. Interestingly, the combination of a dynA deletion with the deletion of a gene encoding for a flotillin-like protein, FloT, also leads to a synthetic defect in cell division. Flotillin proteins are implicated in lipid raft formation in eukaryotic and in prokaryotic cells. Although our experiments do not allow Regorafenib clinical trial us to make any clear conclusion as to the detailed function of dynamin or flotillin, they show that bacterial dynamin and flotillin proteins play non-redundant functions in membrane dynamics. This is supported

by our findings that each mutation does not affect the localization of the other protein. We suggest that dynamin is important for the generation of cell curvature, possibly via its putative mechanochemical activity, and likewise flotillin proteins, which may be important to recruit lipids that favour membrane bending. Indeed, there appears to be a link between flotillin in B. subtilis and membrane fluidity [37]. This idea is supported by our finding that DynA can distort the cell membrane in a heterologous cell system, suggesting that DynA may facilitate membrane invagination and/or couple Z-ring formation with membrane invagination. Alternatively, flotillin may be important to facilitate the recruitment of cell division proteins to the Z ring. In any event, the role of dynamin and flotillins in cell division is not redundant, because of the synthetic effect, and because of their different localization patterns.

ELISA To identify immunopositive phage clones, the ELISA plates were coated overnight at 4°C with 100 μl mAb 4D10(100 μg/ml) and blocked 2h at 4°C. Phage clones were added to the wells (1.5 × 1011 pfu in 100 Nutlin-3a solubility dmso μl per well) and incubated with agitation for 2h at room temperature. The plates were then washed with washing buffer, and 1:5000-diluted horseradish-peroxidase (HRP)-conjugated anti-M13

antibody (Pharmacia) in blocking buffer was added. The plates were incubated at room temperature for 1 h with agitation and washed with washing buffer. HRP/substrate solution was added to each well and incubated at room temperature. The reaction was stopped with 2 N H2SO4 and the plates were read using a microplate reader Selleckchem Ibrutinib set at 450 nm. For antibody-binding assay, ELISA plates were coated with 100 μl per well of individual synthetic peptides at a concentration of 10 μg/ml. For the sensitivity binding assay, 2-fold serial peptide

antigens (concentrations ranging from 20 to 0.31 μg/ml) were coated to the plates. Anti-prM mAb diluted in 1:200 was added to each well. Subsequently, the wells were incubated with corresponding HRP-conjugated anti-mouse IgG, then the same steps as above were followed and absorbance was measured. DNA sequencing and computer analysis The DNA sequences of ELISA-positive phage clones were sequenced with the 96 gIII sequencing primer: 5’-TGAGCGGATAACAATTTCAC-3’, based on phage cloning vector (GenBank: L08821), as described by the manufacturer’s instructions (New England BioLabs Inc.). Sequences of DNA inserted into target phage clones were translated into amino acid sequences and aligned with that of prM protein of DENV2 using Standard protein–protein BLAST [blast] and ClustalW Multiple Sequence Alignment [clustal] public software. Bioinformatics analysis of DENV2 prM B-cell epitopes Using DNASTAR software and ExPaSy multiple bioinformation

software, we performed general evaluation of DENV prM B-cell epitopes including Silibinin Hopp &Wood hydrophilicity; Granthan polarity; Jameson & Wolf antigenicity; Bhaskaran & Ponnuswamy flexibility; Emini accessibility; Deleage & Roux alpha-helix regions; Deleage & Roux beta-turn regions [46–51]. Considering the results of phage biopanning together, one predominant epitope peptide PL10 (13IVSRQEKGKS22) (GenBank: AAC59275), control peptides PH10 (3LTTRGGEP HM12) (GenBank: AAC59275) and PM10 (SQNPPHRHQS) (Ph.D.-12™ Phage Display Peptide Library Kit, New BioLabs Inc.) were synthesized (purity >95%, China Peptides Co., Ltd). Competitive-inhibition assay In competitive-inhibition experiments, coating with anti-prM mAb, blocking, and washing were performed. Synthetic peptide PL10 was added 0.1 μg per well and corresponding phage clones were added simultaneously. Then the same steps as described in “ELISA” were followed.