We determined the effect of silencing MDR1 expression by ultrasound microbubble-mediated siRNA delivery on multidrug resistance of yolk sac carcinamo cells. P-glycoprotein encoded

by MDR1 gene is in charge of decreasing drug accumulation in multidrug-resistant cells, including tumor cells. Daunorubicin is used in cancer chemotherapy and its subcellular distribution is related to multidrug resistance. Daunorubicin produces red fluorescence with laser excitation at 488 nm, which is readily detected in drug-treated tissues or cells. Thus, Daunorubicin accumulation assay was https://www.selleckchem.com/products/pf-04929113.html performed to detect P-glycoprotein activity. Our results indicated that ultrasound microbubble-mediated delivery effectively transferred siMDR1 into L2-RYC cells and led to an increased Daunorubicin accumulation. Chemotherapeutic drugs are means to combat cancers clinically. However, drug-resistance of tumor cells severely limits therapeutic

outcomes. Drug sensitivity can be estimated by tumor cell viability treated with anti-cancer drug. Vincristine and Dactinomycin both of which are most commonly used chemo drugs and also known as substrates of P-glycoprotein. Thus, MTT assay was carried out to detect cell viability find more at different learn more concentrations of Vincristine and Dactinomycin and to determine the IC50 ratios of two drugs in each group. Our results revealed that the L2-RYC cells treated with ultrasound microbubble-mediated siMDR1

delivery became more sensitive to anti-cancer drugs. Conceivably, silencing MDR1 should achieve excellent therapeutic efficacy at lower drug dosages so that chemotherapy-associated side effects can be alleviated to certain extends. Conclusions In this study, we constructed plasmids expressing siMDR1 and confirmed their silencing efficiency in L2-RYC cells. Ultrasound microbubble-mediated delivery can effectively transfer siMDR1 into L2-RYC cells and lead to inhibition Endonuclease of MDR1 expression and function of P-glycoprotein. Drug sensitivity was also improved by silencing MDR1. Thus, ultrasound microbubble-mediated delivery approach is a safe and effective gene transfection method and targeted inhibition method. Our results strongly suggested that combined gene silencing and chemotherapy may be further explored as a novel and potentially efficacious treatment of yolk sac carcinoma. Acknowledgements We thank the editors and reviewers for their valuable comments and suggestions which are helpful for improving this manuscript. This work was supported by a research grant from the National Natural Science Foundation of China (No.81001030). Electronic supplementary material Additional file 1: Supplementary Figure 1. Map of pSEB-HUS vector and schematic diagram of recombination. (JPEG 487 KB) Additional file 2: Supplemental table 1. siRNA targeting MDR1 and PCR primer oligonucleotide sequence. (DOC 34 KB) References 1.

, Anyang, Korea). An isotonic phosphate buffer (25 mM sodium phosphate, 100 mM NaCl; pH = 7.4) was used as mobile phase at a flow rate of 1.0 ml/min. The examination was carried out by UV monitoring at 214 nm. The BSA, GM-CSF, and G-CSF were also dissolved in distilled water and then dispersed in dichloromethane to get controlled water-in-oil (W/O) emulsion. The controlled emulsion and standard protein solutions were also subject to SEC-HPLC for comparing with dextran nanoparticles loaded with proteins. Selleck AZD5363 Bioactivity assay of proteins during

the formulation steps The GM-CSF, G-CSF, and β-galactosidase were selected as model proteins to examine the bioactivity during the process. The bioactivity of the GM-CSF recovered during the steps was determined by the proliferation effect induced on TF-1 cell line. The TF-1 cells were grown in a PRMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The cultures were maintained in plastic flasks and incubated in CO2/air (5:95, v/v) at 37°C in a humidified incubator. The bioactivity of the G-CSF recovered Brigatinib clinical trial was determined by the proliferation effect induced on an NSF-60 cell line. The NFS-60 cells were grown in a PRMI 1640 medium supplemented with 10% FBS. The cultures were maintained in plastic flasks and incubated in CO2/air (5:95, v/v) at 37°C in a humidified incubator. The catalysis bioactivity

of the β-galactosidase on o-nitrophenol recovered was determined by the ortho-nitrophenyl-β-galactoside (ONPG) assay. The assay

was carried out according to a protocol from Sigma. Protein activity was determined by the absorbance of the reaction product of ONPG at 420 nm. The β-galactosidase and GM-CSF were also dissolved in distilled water and then were dispersed in dichloromethane to get the controlled W/O emulsion. The controlled emulsion and standard protein solutions were also subject to bioactivity assay for comparing with dextran nanoparticles loaded with proteins. Ability of dextran nanoparticle to overcome acidic microenvironment LysoSensor™ Yellow/Blue dextran (Life Technologies Corporation, Grand Island, NY, USA) was loaded into the dextran nanoparticle to evaluate the ability to attenuate the local acidic microenvironment in the PLGA MTMR9 microsphere during the in vitro release period. The dextran nanoparticles were encapsulated into composite PLGA microsphere by the solid-in-oil-in-water method [15]. Accordingly, the LysoSensor™ Yellow/Blue dextran solution was encapsulated into the PLGA matrix to act as the controlled sample by the traditional water-in-oil-in-water (W/O/W) double emulsion method [9]. To monitor the change in pH within PLGA LY3039478 microspheres vs. time, 10 mg of dried PLGA microspheres loaded with the LysoSensor™ Yellow/Blue dextran were incubated in tubes containing 1 ml of 20-mM PBS buffer at 37°C under 90 rpm continuously for 12 days.

Figure this website 4 Expression profiles of five known genes of T. harzianum determined by click here Northern blot hybridization. The fungus was cultured in MS basal medium alone

or in the presence of tomato plants (MS-P), 2% glucose (MS-G), or 1% chitin (MS-Ch), as described in Methods. Fungal 18S rDNA was used as a loading control. Identification of T. harzianum genes expressed in response to tomato plants Since we were interested in identifying the genes induced in T. harzianum CECT 2413 by the presence of tomato plants, we selected the 257 probe sets affording significant differential expression in MS-P vs. MS (fold-change greater than 2.0 and FDR = 0.23; see additional file 3), and the corresponding transcript sequences were annotated according to the GO classification and the hierarchical structure using the Blast2GO suite [27]. GO categories were assigned to 85 of the 257 sequences examined (see additional file 4) whereas another 57 had no results after mapping or annotation processes (many of them were hypothetical proteins), and the remaining 115 sequences did not yield significant hits in the databases. As summarized in additional file 5, the annotated sequences represented a total of 46 different genes. Additionally, three sequences without Blast2GO annotation (T34C26, T34C242 and L10T34P112R10010)

but corresponding to three portions of the known protein QID74 [Prot: O74567] of T. harzianum CECT 2413 were also included in additional file 5. Within the genes identified as showing up-regulation in MS-P vs. MS, about 45% were

genes encoding homologues of proteins involved in metabolic pathways, mainly enzymes for carbohydrate, SGC-CBP30 solubility dmso lipid and amino acid metabolism, but also enzymes for vitamin and cofactor biosynthesis, and energy- LY294002 and detoxification- related processes. Interestingly, some of these up-regulated genes (encoding O-glycosyl hydrolase family 2, aldose 1-epimerase, dihydroxyacetone kinase, acid sphingomyelin phosphodiesterase, GTP cyclohydrolase I, glutathione-dependent formaldehyde-activating enzyme, plus two hypothetical proteins) were classified according to Blast2GO in the functional category “”growth or development of symbiont on or near host surface”" since their homologues in Magnaporte grisea were differentially expressed during appresorium formation [28]. Proteins related to carbohydrate metabolism included several enzymes of the glycolysis/gluconeogenesis pathways plus a phosphoketolase of the pentose phosphate pathway, and a 1,3-beta-glucan synthase involved in cell wall biosynthesis. The three up-regulated genes with homologues in lipid metabolism corresponded to a phosphatidylserine synthase participating in phospholipid biosynthesis; a dihydroxyacetone kinase involved in glycerolipid metabolism, and an acid sphingomyelin phosphodiesterase, responsible for breaking sphingomyelin down into phosphocholine and ceramide.

One derivative

containing an RDD triplet in the receptor-binding site was obtained from the serotype Asia 1 field isolate after a single cattle-to-pig transmission and subsequent BHK-21 in vitro passage. Sequence analysis of 10 biological clones of the VP1 encoding region of this population demonstrated that RDD viruses instead of the original RGD virus became predominant at an early phase of Asia1/JS/CHA/05 quasispecies evolution. Unexpectedly, however, both RGD and RSD viruses were obtained from the Asia1/JSM4 population that were generated after four serial passages of the Asia1/JS/CHA/05 field isolate in suckling mice, via intraperitoneal inoculation. The population equilibrium of RSD mutant and ancestor viruses CHIR-99021 in vivo was maintained after 20 passages of the Asia1/JSM6 population in BHK-21 cells. Although RDD- or RSD-containing FMDV are unusual, they were genetically stable upon extended replication in cell culture. Our results suggest that, in the context of the capsid proteins of Asia1/JS/CHA/05, a highly conserved RGD motif is not essential for replication in vitro and in vivo, suggesting functional flexibility of FMDV to enter cells

in response to environmental modifications. Like other RNA viruses, FMDV exists as closely related but non-identical genomes, termed viral quasispecies [30, 31]. Genetic diversity is an intrinsic property of the quasispecies, which arise due to the lack of proofreading check details activity during viral genome replication, a short replication cycle, and other environmental selective pressures [32, 33]. Our observations Copanlisib chemical structure showed that evolution of FMDV population exhibited receptor binding motif diversity (genetic diversity) subjected to short-term passage of field isolate in different environments. From the standpoint of RNA virus population evolution, one possible scenario could explain this observation. The early interactions between viruses and host cells exert major selective force on virus populations, thus, the L-NAME HCl variants (RSD- and RDD-containing viruses) may already be

present at low frequency in the natural population that are possibly more fit in new environments and become dominant strains. While this presumption is contrary to the view that the RGD triplet is highly conserved among natural isolates of FMDV, there is direct evidence that an RDD containing field virus was isolated from pigs during a type Asia 1 FMD outbreak in China. RDD-containing FMDV VP1 genes were amplified from sheep oesophageal-pharyngeal fluids (OP-fluids) collected during 2006 from a sheep herd in the region of China that had endemic Asia 1 serotype FMDV [34, 35]. The emergence of these non-RGD mutants in nature is likely to be influenced by specific epidemiological and immunological aspects of host-virus interaction as well as the quasispecies composition of the viral population [36–39].

However, based on this final statement, our failure to include a true control group not receiving CR supplementation but undergoing a progressive decrease in rest interval length does not allow us to make such a statement with absolute confidence, regarding

the ability of selleckchem CR to off-set any additional decrease in training volume that may have been apparent. This is indeed a limitation of the present work and should be a focus of future research. A previous study from our research group [15] compared the effect of 8-weeks of resistance training using CI and DI between sets and exercises on strength and CBL0137 ic50 hypertrophy. Recreationally resistance training subjects were randomly assigned to either a CI or DI training group. The results indicated no significant differences between the CI and DI training protocols for CSA, 1RM and isokinetic peak torque. Similar to the current study, these results [15] indicated that a training protocol with DI was as effective as a CI protocol over short training periods (8-weeks) for increasing

maximal strength and muscle CSA. Muscle mass is important for health and survival through the lifespan [7]. Resistance training has been recognized as an essential component of a comprehensive fitness program for individuals

with diverse fitness goals [19]. Manipulation of training variables (e.g. load, volume, rest interval between sets) is dependent on the specific training selleck antibody inhibitor goals of the individual and the GW786034 nature of the physical activities performed during daily life [20, 21]. The length of rest interval must be sufficient to recover energy sources (e.g., adenosine triphosphate [ATP] and PCR), buffer and clear fatigue producing substances (e.g., H+ ions), and restore force production [22]. Certain ergogenic substances have been shown to augment resistance training adaptations beyond that which may occur through resistance training alone. With regard to the function of the Phosphagen energy system, the ergogenic value of CR supplementation has been examined extensively with significant benefits reported in strength/power, sprint performance, and/or work performed during multiple sets of maximal effort muscle contractions [1, 2, 23–25]. The improvement in exercise capacity has been attributed to increased total creatine (TCR) and PCR content, thus resulting in greater resynthesis of PCR, improved metabolic efficiency and/or an enhanced quality of training; thus promoting greater neuromuscular adaptations.

Already in 1986, Allegrantte and Sloan discussed how workplace health promotion may pose ethical problems. In 1987, Gordon presented her doubts on health promotion at the workplace and described that trust is an essential ingredient for successful health promotion. The debate still continues to what extent employers are entitled to interfere with the lifestyle and health of their workers. Where does undue interference begin? In this context, little information is available on the opinion of employees regarding WHP.

Within the framework of a WHP program, we have investigated moral considerations among workers in relation to WHP offered by their employer. Methods Study design A-1155463 mw and population The study is embedded in a larger study in which we investigated the effectiveness of a WHP program consisting of a physical health check with subsequent advice, and a website with general information, individualized advice and for the intervention group possibilities to ask questions and to monitor their own behavior. An extensive description of the study protocol is published elsewhere (Robroek et al. 2007). Employees working in six companies from different branches were invited to participate in the study. Participants received a questionnaire asking for individual characteristics, lifestyle,

and health. A sample of 860 non-participants in the health care organizations selleckchem (n = 2) and all non-participants in the commercial services organizations (n = 2) and in the executive branch of government (n = 1) received an abbreviated version of the questionnaire. In the other organization in the executive branch of government (n = 1), non-respondents were not invited to fill in the questionnaire because the program was initiated in the holiday period and communicated in a very limited way, and only 200 workers were allowed to participate. Therefore, most workers in that organization were unaware of the program. Montelukast Sodium Due

to privacy regulations, the questionnaire was send out only once without any reminders. In total, 213 employees out of 860 non-participants responded (24.8%). Moral considerations Non-participants were asked why they did not participate, with multiple responses possible. In addition, both participants and non-participants were asked to indicate on a 5-point scale ranging from “totally disagree” to “totally agree” to what extent they agree with five statements SNX-5422 order addressing their opinion on WHP (Table 1). Table 1 Answers of participants (P) and non-participants (NP) on five statements addressing their opinion on WHP Statement Disagree (%) Neutral (%) Agree (%) P NP P NP P NP 1. A healthy lifestyle is important for me 2.1 1.0 8.0 7.7 89.9 91.3 2. My lifestyle is a personal matter 13.1 11.7 16.4 23.4 70.6 64.9 3.

Livak KJ, Schmittgen TD: Analysis of relative gene expression buy VS-4718 data using real-time quantitative PCR and 2-ΔΔCt method. Methods 2001, 25:402–408.PubMedCrossRef 38. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248–254.PubMedCrossRef 39. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR: Site-directed mutagenesis by overlap extension using the polymerase

chain reaction. Gene 1989, 77:51–59.PubMedCrossRef 40. Bobrov AG, Kirillina O, Perry RD: The phosphodiesterase activity of the HmsP EAL domain is required for negative regulation of biofilm formation in Yersinia pestis. FEMS Microbiol Lett 2005, 247:123–130.PubMedCrossRef 41. Kuchma SL, Brothers KM, Merritt JH, Liberati NT, Ausubel FM, O’Toole GA: BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseodomonas aeruginosa PA14. J Bacteriol CA4P purchase 2007, 189:8165–8178.PubMedCrossRef Authors’ contributions The authors have no competing interests.

K.-C. Wang drafted the manuscript and performed mutant strain construction and PDE assay. Y.-H. Hsu helped the experimental design and data analysis. Y.-N. Huang assisted molecular cloning and site-directed mutagenesis, protein purification experiments. K.-S. Yeh conceived and coordinated this study and also helped to draft the manuscript. All authors read and approved the final manuscript.”
“Background Pneumonia is the most common cause of death related to infectious diseases. Even after aggressive antimicrobial treatment pneumococcal pneumonia causes mortalities of up to 10% [1]. Children

and young adults are susceptible to lower respiratory tract infection typically caused by Staphylococcus aureus Haemophilus influenzae and Pseudomonas aeruginosa[2]. Moreover, P. aeruginosa colonization of the lung is frequently found in cystic fibrosis patients, which worsens the prognosis of this disease [3]. Pneumonia signifcantly increases the average duration of intensive care unit (ICU) stays and mortality [4]. The diagnosis of nosocomial pneumonia often requires invasive and time consuming methods (e.g. bronchoscopy) [5]. Therefore, it is of utmost interest to develop a non-invasive method CYTH4 for the early diagnosis of this disease, preferably allowing the identification of the specific pathogens. Attempts on screening of volatile bacterial metabolites for detection and classification of virulent bacteria was already undertaken in the past. However, the vast majority of studies on volatile organic compounds (VOCs) released from bacteria included qualitative analyses only [6–10]. Also direct mass spectrometric methods were used for the investigation of VOC release, comprising selected ion flow tube mass spectrometry (SIFT-MS) [11, 12] and proton transfer reaction mass spectrometry (Selleckchem Idasanutlin PTR-MS) [13–15].

The chromosomal toxR-lacZ transcriptional fusion was constructed by cloning the 5′ toxR region into the suicide vector pVIK112, which also contains a promoterless lacZ gene [31]. The resulting see more plasmid was then integrated into the chromosomes of V. cholerae lacZ – strains by homologous recombination to create a single-copy toxR-lacZ and an intact copy of toxR. P BAD -controlled aphA and aphB plasmids were constructed by cloning aphA and aphB coding sequences into the pBAD24 vector [32]. pBAD-tcpPH plasmid construct was

described in [8]. In-frame deletions of toxR, toxS, tcpP, tcpA, toxT, aphA, and aphB were either described previously [15] or constructed by cloning the PCI-34051 cost regions flanking target genes into the suicide vector pWM91 containing a sacB counter-selectable marker [33]. The resulting plasmids were introduced into V. cholerae by conjugation and deletion mutants were selected for double homologous recombination events. Lux activity assays Bacteria were grown at 37°C or 22°C under conditions indicated. At different time points, cultures were

withdrawn and luminescence was measured by using a Bio-Tek Synergy HT spectrophotometer. Lux expression is calculated as light units/OD600. Western blotting and SDS-PAGE electrophoresis Whole-cell lysates were prepared from bacteria overnight cultures in LB Sapanisertib research buy conditions at 37°C and samples were normalized to the amount of total protein as assayed by the Biorad protein assay (Biorad). The isolation of outer membrane (OM) proteins from V. cholerae was performed using the method described by Miller and Mekalanos [34]. Whole-cell lysates or OM preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel and stained with Coomassie brilliant blue for visualization. SDS-PAGE gels were transferred to nitrocellulose membrane Staurosporine nmr for Western blot analysis using polyclonal rabbit anti-ToxR antibody. Gel retardation assays MBP-AphB protein was purified through amylose columns according to the manufacturer’s instructions (New England Biolabs). PCR products of the different lengths of toxR promoter

regions were digested with EcoRI and end-labeled using [α-32P]dATP and the Klenow fragment of DNA polymerase I. Binding reactions contained 0.1 ng of DNA and MBP-AphB proteins in a buffer consisting of 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 60 mM KCl, and 30 mg of calf thymus DNA/ml. After 20 minutes of incubation at 25°C, samples were size-fractionated using 5% polyacrylamide gels in 1× TAE buffer (40 mM Tris-acetate, 2 mM EDTA; pH 8.5). The radioactivity of free DNA and AphB-DNA complexes was visualized by using a Typhoon 9410 PhosphorImager (Molecular Dynamics). Acknowledgements This study was supported by the NIH/NIAID R01 (AI072479) (to J.Z.), and a NSFC key project (30830008) (to B.K.). References 1.

J Phys Chem A 2003, 107:3372–3378.CrossRef 32. Kuncicky DM, Prevo BG, Velev OD: Controlled assembly of

SERS substrates templated by colloidal crystal films. J Mater Chem 2006, 16:1207–1211.CrossRef 33. Khlebtsov BN, Khanadeev VA, Panfilova EV, Minaeva SA, Tsvetkov MY, Bagratashvili VN, Khlebtsov NG: Surface-enhanced Raman scattering platforms on the basis of assembled gold nanorods. Nanotechnologies in Russia 2012, 7:359–369.CrossRef 34. Farcau C, Potara M, Leordean C, Boca S, Syk inhibitor Astilean S: Reliable plasmonic substrates for bioanalytical SERS applications easily prepared by convective assembly of gold nanocolloids. Analyst 2013, 138:546–552.CrossRef 35. Gabudean AM, Focsan M, Astilean S: Gold nanorods performing as dual-modal nanoprobes

via metal-enhanced Selleckchem A-1155463 fluorescence (MEF) and surface-enhanced Raman scattering (SERS). AZD5363 J Phys Chem C 2012, 116:12240–12249.CrossRef 36. Le Ru EC, Blackie E, Meyer M, Etchegoin PG: Surface enhanced Raman scattering enhancement factors: a comprehensive study. J Phys Chem C 2007, 111:13794–13803.CrossRef 37. Blaber MG, Schatz GC: Extending SERS into the infrared with gold nanosphere dimers. Chem Commun 2011, 47:3769–3771.CrossRef 38. Wustholz KL, Henry AI, McMahon JM, Freeman RG, Valley N, Piotti ME, Natan MJ, Schatz GC, Van Duyne RP: Structure-activity relationships in gold nanoparticle dimers and trimers for surface-enhanced Raman spectroscopy. J Am Chem Soc 2010, 132:10903–10910.CrossRef 39. Fang Y, Seong NH, Dlott DD: Measurement Histamine H2 receptor of the distribution of site enhancements in surface-enhanced Raman scattering. Science 2008, 321:388–392.CrossRef 40. Natan MJ: Concluding remarks. Surface enhanced Raman scattering. Faraday Discuss 2006, 132:321–328.CrossRef 41. Greeneltch NG, Blaber MG, Schatz GC, Van Duyne RP: Plasmon-sampled surface-enhanced Raman excitation spectroscopy on silver immobilized nanorod assemblies and optimization for near infrared (λ ex = 1064 nm) studies. J Phys Chem C 2013, 117:2554–2558.CrossRef 42. Greeneltch NG, Blaber

MG, Henry A-I, Schatz GC, Van Duyne RP: Immobilized nanorod assemblies: fabrication and understanding of large area surface-enhanced Raman spectroscopy substrates. Anal Chem 2013, 85:2297–2303.CrossRef 43. Zhurikhina VV, Brunkov PN, Melehin VG, Kaplas T, Svirko Y, Rutckaia VV, Lipovskii AA: Self-assembled silver nanoislands formed on glass surface via out-diffusion for multiple usages in SERS applications. Nanoscale Res Lett 2012, 7:676.CrossRef 44. Zhu SQ, Zhang T, Guo XL, Wang QL, Liu X, Zhang XY: Gold nanoparticle thin films fabricated by electrophoretic deposition method for highly sensitive SERS application. Nanoscale Res Lett 2012, 7:613.CrossRef 45. Dykman L, Khlebtsov N: Gold nanoparticles in biomedical applications: recent advances and perspectives. Chem Soc Rev 2012, 41:2256–2282.CrossRef 46. Zhao P, Li N, Astruc D: State of the art in gold nanoparticle synthesis. Coord Chem Rev 2013, 257:638–665.CrossRef 47.

This study EGD-e D EGD-eΔinlA with inlA locus recreated containing SDM changes T164A, K301I and G303E in the chromosome. This study EGD-e InlA m * ::pIMC3ery EGD-e InlA m * with the IPTG inducible expression of erythromycin integrated in the tRNAARG locus, Cmr. This study EGD-e::pIMC3kan EGD-e with the IPTG inducible expression of kanamycin integrated

in the tRNAARG locus, Cmr. [18] EGD-e A::pIMC3kan EGD-e A with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr This study EGD-e B::pIMC3kan EGD-e B with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr This study EGD-e C::pIMC3kan EGD-e C with the IPTG inducible expression of kanamycin integrated in the tRNAARG locus, Cmr This study EGD-e #check details randurls[1|1|,|CHEM1|]# D::pIMC3kan EGD-e D with the IPTG SAHA HDAC supplier inducible expression of kanamycin integrated in the tRNAARG locus, Cmr This study NZ9700 Nisin producer, progeny of NIZO B8 and MG1363 (Rifr and Strpr) conjugation. [26] Plasmids     pNZB Nisin inducible plasmid with

heterologous gene expressed from the nisA promoter. BglII site upstream of nisA removed. This study pNZBinlA WT Internalin A from EGD-e containing the entire gene including signal sequence. Cloned into NcoI/PstI of pNZB. This study pNZBinlA m * Internalin A containing S192N and Y369 S in pNZB. This study pNZBinlA Bank-iii Error Prone PCR with low level of mutation 0-4.5 nt per kb. This study pNZBinlA Bank-iv Error Prone PCR with medium level of mutation 4.5-9 nt per kb. This study pNZBinlA Bank-v Error Prone PCR with high level of mutation 9-16 nt per kb. This study pNZBinlA Bank-vi Error Prone PCR with very high level of mutation 9-16 nt per kb. This study pORI280 RepA negative gene replacement vector, constitutive lacZ, 5.3 kb, Emr. [40] pORI280inlA(SDM) PCR amplified mutated inlA m * into pORI280 as NcoI/PstI fragment. Contains wild type inlA promoter. This study pORI280inlA(A) PCR amplified

mutated inlA (from bank v clone 6 containing heptaminol N259Y) into pORI280 as NcoI/PstI fragment. Contains Wt inlA promoter. This study pORI280inlA(B) PCR amplified mutated inlA (from bank iii clone 3 containing Q190L) into pORI280 as NcoI/PstI fragment. Contains Wt inlA promoter. This study pORI280inlA(C) PCR amplified mutated inlA (from bank v clone 6 containing S173I, L185F, L188F) into pORI280 as NcoI/PstI fragment. Contains Wt inlA promoter. This study pORI280inlA(D) PCR amplified mutated inlA (from bank v clone 8 containing T164A, K301I, G303E) into pORI280 as NcoI/PstI fragment. Contains Wt inlA promoter. This study pVE6007 Temperature-sensitive helper plasmid, supplies RepA in trans. Cmr.