Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications.Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases.Our proprietary M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced  PHAs for future high value applications in various fields.This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The  Polyhydroxyalkanoates with functional groups  have attracted increasing attention in the last ten years or so -9. It haAlthough many types of functionalized PHAs have been described in the literature, generally speaking, the quantities produced have been very modest and barely sufficient to obtain a basic characterization of the physico-chemical properties of the new biomaterials . SeveralPseudomonas species have been most widely used for producing polyhydroxyalkanoates with double bonds in their side chains since they generate functionalized PHAs at comparably high rates of productivity. The incorporation of functional groups in PHAs by pseudomonads occurs by oxidizing functionality-related substrates via the β-oxidation pathway. The most commonly used substrate to obtain unsaturated polymeric side chains has been 10-undecenoic acid (C11=), likely owing to its superior availability compared to other alkenoic acids. The β-oxidation cycle is accompanied by a partial C2 reduction so that unsaturated monomers resulting from C11= exhibit a length of nine and, respectively, seven carbon atoms. The production of purely unsaturated polyhydroxyalkanoates was reported [co-3-hydroxyalkenoate)s with defined monomeric compositions [A commonly applied route for obtaining polyhydroxyalkanoates with desirable functionalities is to produce PHAs with terminal double bonds followed by chemical modification steps. Carbon double bonds are comparatively inert but can be easily transformed into reactive functional groups under mild reaction conditions. Following this approach, polyhydroxyalkanoates with carboxyl, hydroxyl, epoxy and halogenic groups have been produced  and medium-chain-length  monomers and belong to a novel class of PHAs that will likely gain much attention due to their potential for high value applications in various fields including tissue engineering. One reason for choosing a methylotrophic microorganism for such purpose was that an important portion of the production process would use methanol, a simple, inexpensive, very abundant, and non-food substrate [M. extorquens as a new cell factory, as strongly advocated in a recent review [In this study, we present data showing that some newly developed recombinant ubstrate -26. Anott review .Methylobacterium extorquens ATCC 55366 was used throughout this study [M. extorquens strains were similarly stored except that medium contained tetracycline at 15 mg/L to maintain selective pressure.The proprietary, wildtype strain is study . Fresh pTwo media were used, CHOI4 medium  or CHOI5M. extorquens strains. The medium used to grow the recombinant strains also contained tetracycline at 15 mg/L.Starting from cultures maintained on CHOI4 plates at 4°C, 250-300 mL-shake flasks containing 50 mL of medium were inoculated and incubated at 30°C, 260 rpm, for approximately 48 h. The medium contained 0.2 vol% methanol as sole carbon source. Incubation was extended to 72-96 h in the case of the recombinant phaC genes, cumate (4-isopropylbenzoic acid) was added to a final concentration of 20 mg/L. Whenever applicable, addition of the secondary substrate (carboxylic acids) was usually done after 48 or 72 h, or as specifically indicated in the Results and Discussion section. 1 M KOH was used for manual pH control.Most experiments were conducted using 2 L-shake flasks containing 500 mL of either CHOI4 medium or CHOI5 medium. The media contained an initial concentration of 1 vol% methanol. Media used with the recombinant strains contained tetracycline at 15 mg/L. For induction of the recombinant phaC2 was induced by adding 4-isopropylbenzoic acid to reach a final concentration of 20 mg/L. Microbial growth was monitored by spectrophotometry at 600 nm.Fermentation was performed in a 20 L-bioreactor  that was inoculated with 10 vol% of a 72 h pre-culture grown in shake flasks. Upon oxygen depletion, methanol  was manually added with a syringe. The co-substrate 7-octenoic acid (C8=) was supplied by the same means. Shortly prior to co-substrate addition, expression of the PHA synthase gene phaC1 and phaC2 from Pseudomonas fluorescens GK13, the genomic DNA of P. fluorescens GK13 was isolated and subjected to PCR using, for phaC1, the primers PhaC1FNhe (5' -CGC TAG CAT GAG CAA CAA GAA CAA TGA AGA CCT GCA GCG C- 3') (the NheI site is underlined), PhaC1RMFE (5' -GCA ATT GTC AAC GTT CGT GGA CAT AGG TCC CTG G- 3') (the MfeI site is underlined) and, for phaC2, the primers PhaC2FNhe (5' -CGC TAG CAT GCG AGA GAA ACA GGT GTC GGG AGC CTT G- 3') (the NheI site is underlined), PhaC2RMFE (5' - GCA ATT GTC AGC GCA CGT GCA CGT AGG TGC CGG G- 3') (the MfeI site is underlined), thus, yielding 1680-bρ and 1683-bρ PCR products, respectively. The PCR products were digested with NheI and Mfel, and cloned into pAll-gfp  digested with the same restriction enzymes to generate pAll-phaC1 and pAll-phaC2, respectively  synthase genes phaC -minus mutant of M. extorquens via gene knock-out of the native phaC gene using the TargeTron™Gene Knockout system from Sigma-Aldrich . A good number of mutants were obtained and PCR detection of the integrated intron in 9 randomly selected mutants showed the presence of the insert in all of them and its absence in the wildtype M. extorquens strain. However, all the mutants grew very poorly on methanol and, therefore, they could not be employed for further genetic engineering work.Attempts were made to engineer a M. extorquens cells were prepared as previously described [phaC1 or pAll-phaC2 ) and the tubes placed on ice for 20 min. The mixtures were transferred to an ice-cold electroporation cuvette and treated in a Bio-Rad electroporator . Immediately thereafter, 1 mL of CHOI medium [μL of culture was spread on selective plates (CHOI agar with 35 μg of tetracycline per mL). The plates were incubated at 30°C for 48 h until colonies appeared. Typically, about 300-500 transformants per plate were obtained.Competent escribed ,29. CompM. extorquens cultures were prepared using a French press and protein concentration was estimated by the method of Bradford [μg per well on a 4-12% NuPAGE Novex gel . Gels were stained with Coomassie Brilliant Blue R-250.Crude extracts of recombinant Bradford  using thCulture samples were centrifuged and the resulting pellets were washed in distilled water, methanol and, again, in distilled water followed by re-centrifugation after each washing step. Finally, the biomass was resuspended in water and freeze-dried . Lyophilized biomass was leached in approximately 20× volume (v/w) of chloroform overnight in a rotary shaker at 30°C. The soaked cells were then filtered through Whatman #4 paper  and the filtrate purged with nitrogen. Resulting polymer gels were re-dissolved in chloroform and subjected to precipitation overnight in 10× volume (v/v) methanol at 4°C. Finally, precipitates were filtered through one sheet of Fisher Scientific P5 paper  and air-dried.Freeze-dried biomass resulting from fermentation was leached in 20× volume (v/w) of chloroform or acetone for at least 6 h in a Branson model 5200 sonicator  at 60°C. The soaked cells were then vacuum-filtered through Whatman #4 paper  and the filtrate boiled down in a Rotavapor . Resulting polymer gels were re-dissolved in chloroform and subjected to precipitation overnight in 10× volume (v/v) methanol at 4°C. Finally, precipitates were vacuum-filtered through two sheets of Fisher Scientific P5 paper  and air-dried. Chloroform-extracted PHAs were fractioned by hot acetone in a Soxhlet apparatus as previously described .GC-FID analysis was carried out as follows. Biomass samples were centrifuged in 50 mL conical Sarstedt tubes  and the pellets lyophilized. Methanolysis was used for analyzing the intracellular PHA content. Briefly, the dry biomass was treated with acidified methanol in the presence of benzoic acid as internal standard at 100°C for 3 h to convert 3-hydroxyalkanoate monomers to the corresponding methyl esters. The methyl esters were extracted in chloroform for subsequent analysis in a gas chromatographic system . PHBV from Sigma-Aldrich  and PHBHx from Procter & Gamble  were used as PHA standards. Purified biopolyesters were analyzed by the same method.1H 1D spectra were acquired with a sweep width of 12000 Hz and 128 transients. Assignment of 1H and 13C resonances were achieved via 1H-13C correlated spectra detecting naturally abundant 13C. The 1H-13C Heteronuclear Single Quantun Coherence (HSQC) [2) × 200(t1) complex points and 1H and 13C sweep widths of 10000 and 27145 Hz, respectively. Broadband 13C decoupling was achieved during acquisition using WURST2 decoupling over 140 ppm. Typically, 128 scans were acquired for each t1 value. HMBC   and multelation, ) and H2Belation, ) experim nmrPipe  and analtrans-2-pentenoic acid (t - C5=), 98% purity; hexanoic acid (C6), 99% purity.Pentanoic acid (C5) was obtained from A & C American Chemicals Ltd. , 99% purity; 5-hexenoic acid (C6=) was obtained from either Sigma-Aldrich or TCI America , both at 98% purity; 7-octenoic acid (C8=) was from Richman Chemical Inc. , 98% purity. The following carboxylic acids were all from Sigma-Aldrich: 10-undecenoic acid (C11=), 98% purity; 4-pentenoic acid (C5=), 97% purity; M. extorquens ATCC 55366 was tested to utilize various fatty acids including fatty acids with C-C double bonds. The assays were performed in shake flasks using medium CHOI4 or medium CHOI5. Nearly 100 shake flask assays were conducted. The M. extorquens cultures were grown first on methanol as sole carbon and energy source and the fatty acid of interest was added at some point to the growing cultures, generally at a final concentration in the 0.1-0.3% range. A summary of the substrates tested and of the results obtained is presented in Table M. extorquens ATCC 55366 strain was unable to accumulate functionalized PHAs containing C-C double bonds. As a consequence, the next step consisted in developing M. extorquens strains harboring heterologous PHA synthase genes allowing for biosynthesis of the wanted functionalized PHAs.The potential of the pink facultative methylotroph M. extorquens strain to yield the desired PHAs was assumed to be due to a too narrow substrate specificity of the wildtype PHA synthase enzyme. As done by several research groups [M. extorquens strains harboring different PHA synthase (phaC) genes known or assumed to code for PHA synthases of broader substrate specificity. After careful review of the literature, the phaC1 and phaC2 genes present in the P. fluorescens GK13 strain were selected because the PHA synthesis machinery of this bacterium is able to also produce longer PHA chains [phaC1 and phaC2 genes were successfully isolated using PCR and the isolated genes were identical to the reported sequences [phaC genes were cloned into the pAll plasmid  and of the phaC2 gene (lane 6). Both protein bands indicated a molecular mass of approximately 62 kDa. Interestingly, a strong band, with an apparent molecular mass of 40 kDA, was present in the samples for noninduced C1 M. ex-phacells (lane 3) or C2 M. ex-phacells (lane 5). The same band was quasi inexistent in induced cells of the same recombinant strains. This additional band is certainly not related to the insert (phaC gene) since it has been observed with other recombinant M. extorquens strains harboring different and various inserts. Ideally, the two recombinant phaC genes should have been introduced into a phaC -minus mutant of M. extorquens. Unfortunately, although we were able to obtain such mutants, the mutants grew very poorly in the culture media containing methanol as sole carbon source. A similar observation on the growth of strain AMI of M. extorquens on C1 and C2 compounds was also reported by Korotkova and Lidstrom [The two recombinant Lidstrom . ConsequM. ex-phaC2 strain appeared to grow better than the M. ex-phaC1 strain under the conditions used. Phase contrast microscopy of the cultures showed the presence of granules, tentatively identified as PHB (or PHA) granules, in cells from the three M. extorquens strains and the granules increased in size over time. The appearance of the granules occurred between 68 h and 74 h and it coincided with the addition of the methanol + C11= mixture  and in Figure C2M. ex-pha). The results were similar to those obtained with 5-hexenoic acid (C6=) except that the OD600nm profiles varied significantly more between flasks. As with 5-hexenoic acid, the pH profile varied with each flask but pH values were always between 6.2 and 7.6. With 10-undecenoic acid (C11=), however, growth appeared even more "erratic" as there was significant variation in the OD600nm profile between flasks . Maximal OD600nm values varied between only 4 and almost 10  to further identify which unsaturated components might be present. The results are summarized in Table M. ex-phaC2 on methanol and 7-octenoic acid will be presented in more detail:Consequently, the representative PHA samples, including a PHA sample produced by the wildtype 1H 1D spectrum indicated that 3HB was the major component comprising over 96% of the total signal (spectrum not shown). Using natural abundance 2D 1H-13C correlation spectra  and 13C (115 ppm and 135 ppm) resonances indicative of double bonds. These were assigned to the 3HHx= and 3HO= monomers. Further analysis of the NMR observations indicated that the double bond for these two monomers must be at the end of the alkyl chain;2. A notable feature of the 2D HSQC spectrum was the presence of high frequency 3. Using the C3 resonances in the HSQC, we obtained a ratio of 3HB:3HO= (1:0.06), 3HB:3HHx= (1:0.05), 3HB:3HP (1:0.02), 3HB:3HHx , 3HB:3HO .Unfortunately, 2D NMR could not be performed with every sample due to cost and equipment availability. However for GC-FID analysis, it was now obvious that 3-hydroxyalkanoate monomers derived from 5-hexenoic acid would result in peaks for 3HHx= and 3HHx, while peaks for 3HP, 3HHx=, 3HHx, 3HO= and 3HO could be expected from metabolism with 7-octenoic acid. Consequently, GC-FID was routinely used thereafter to determine the composition of the PHAs produced and to look for the presence of C-C double bonds. Peaks for 3HB, 3HP and 3HHx were identified from corresponding methanolized PHA standards . Since methyl esters resulting from 3-hydroxyalkenoates have a lower molecular weight compared to their saturated analogs, the peak that was recorded shortly prior to 3HHx was identified as 3HHx=. In addition to thereby related peaks for 3HB, 3HP, 3HHx= and 3HHx, GC chromatograms of PHA samples derived from methanol and 7-octenoic acid metabolism showed two more peaks that eluted at later time. From 2D NMR analysis it was concluded that these remaining peaks had to correspond to 3HO= and, eluted immediately thereafter, to 3HO. Peak areas were used to calculate the proportional monomeric compositions.M. extorquens strains capable of accumulating functionalized PHAs harboring C-C double bonds within the side chains. As done by others, the production of such PHAs requires feeding an unsaturated carboxylic acid in the hope that a portion of the acid will be incorporated into the PHA molecule by the recombinant PhaC1 or PhaC2 enzyme, thus, leading to the potential presence of C-C double bonds in the PHA side chains.From the start, our intention was to develop C1 M. ex-phastrain was able to produce PHA containing a small percentage, 0.75% +/- 0.57%, of C6:5 bond-containing material starting from C6=. The same strain, however, was very poor at incorporating C-C double bonds when either C8= or C11= was fed; only trace amounts of C-C double bonds were detected. Interestingly, the C1 M. ex-phastrain was able to incorporate significant quantities of a C5 unit starting from C8=. In this regard, C8= appeared much superior as chemical donor. Due to the high toxicity of the carboxylic acids used, cell densities were always low, generally between 1 and 2 g/L, on a dry weight basis.Numerous shake flask studies were conducted using various unsaturated carboxylic acids, various feeding regimes and process conditions in the hope of identifying initial conditions that could lead to maximization of functionalized PHA production. Resulting PHA samples were generated and analyzed according to NMR and/or GC-FID analysis. As Table C2 M. ex-phastrain appeared better than the C1 M. ex-phastrain at incorporating C6:5 bonds into PHA (C-C double bond at position 5 of C6 monomer chain length). An average of 4.76% +/- 2.94% of the PHA was made up of C6:5 bond-containing units upon feeding of C6=. The same strain appeared also better at incorporating a C6 monomer from either C6= or C8=. When C8= was fed, four of the five samples showed the obvious presence of C8:7 bond-containing units. The concentrations of C6:5 material and those of a C6 monomer were also significantly higher. A C5 monomer was detected in the samples derived from C6= but it was much more prevalent when C8= was fed, an observation valid for both recombinant strains. Feeding of C11=, as with the first strain, did not lead to production of PHA containing significant percentages of C-C double bonds. Cell densities, again, were quite low. Table C2 M. ex- phastrain could generate both C6:5 material and C8:7 material when fed C8=.As illustrated in Table C2 M. ex-phawas better at producing PHA material containing C-C double bonds; (2) the same strain was better at producing PHA material containing C-C bonds from C8=; (3) both strains gave very poor results with C11=. Several groups have observed or hypothesized that PhaC2 synthases have a lower substrate specificity than PhaC1 synthases [M. extorquens strains gave poor results with C11= and, therefore, only C6= or C8= proved to be adequate secondary substrates.The results listed in Table ynthases ,38,40. Oynthases ,44,48. CM. ex-phaC2 and production of PHAs in shake flasks were successfully validated in a 20 L-bioreactor (14 L as working volume). A total of 11 mL of 7-octenoic acid was added throughout the fermentation in four individual additions starting at 29 h of running time. After 96 h the final dry cell weight (DCW) reached 5 g/L with a 37% PHA content on a dry cell weight basis (data not shown). Polymers were extracted from biomass with chloroform and subjected to routine structural analysis. The monomeric composition reflected the results from shake flask experiments when 7-octenoic acid was fed  (PHB) is only soluble in acetone when it is amorphous [M. ex-phaC2 produced blends made up of SCL-PHAs and SCL/MCL-PHAs. Since the native PhaC enzyme is not capable of incorporating monomers other than SCL-3HA, the production of SCL/MCL-PHAs must have resulted from the action of the recombinant PhaC2 enzyme. Random abundance of the different monomers is likely the reason why some MCL-3HA units remained in the acetone-insoluble fraction. It is suggested here that the MCL-3HA content was very small in some copolymeric chains so that they could not dissolve in acetone.Growth of recombinant morphous ,50. PHB phaC genes in our recombinant strains, we conclude that a major portion of the materials consisted of PHB or PHBV resulting from the native PHA synthase. Since the PHA synthase coded by the phaC2 gene of P. fluorescens GK13 was shown to have a broad substrate specificity [Our experimental PHA samples from shake flask and fermentation experiments exhibited mainly SCL-3HA monomers . Due to the presence of two unrelated cificity , we conccificity . For thicificity  and refeM. extorquens ATCC 55366 strain could not produce functionalized PHAs starting from a methanol + unsaturated carboxylic acid mixture. As a consequence, a metabolic engineering approach was used to convert the wildtype strain into a "cell factory" capable of producing functionalized PHAs containing C-C double bonds. The presence of the C-C double bonds in the PHA side chains was confirmed by NMR. It was also found that the M. extorquens cell factory harboring the phaC2 gene appeared superior at utilizing unsaturated carboxylic acids and at incorporating C-C double bonds into PHA starting from either C6= or C8=. Our two M. extorquens cell factories were able to produce functionalized short-chain-length/mediumchain-length PHAs (SCL/MCL-PHAs), which combine desirable functionality of some MCL-PHAs with, reportedly, superior thermo-mechanical properties of inert SCL/MCL-PHAs. Double bonds may be used to induce side chain crosslinking and to covalently bind small or macromolecules upon chemical or enzymatic modifications. To date, the advantage of exhibiting functionality has been exclusively reserved for MCL- and LCL-PHAs.It was found that the wildtype t-C5=: trans-2-pentenoic acid; C6: hexanoic acid; C6=: 5-hexenoic acid; C8:= 7-octenoic acid; C11=: 10-undecenoic acid; CX:Y: double bond at position Y of monomeric chain length X; DCW: dry cell weight; GC-FID: gas chromatography-flame ionization detector; H2BC: Heteronuclear 2-Bond Correlation; HMBC: Heteronuclear Multiple Bond Correlation; HMQC: Heteronuclear Multiple Quantum Coherence; HSQC: Heteronuclear Single Quantum Coherence; KOH: potassium hydroxide; LCL: long-chain-length; MCL: medium-chain-length; MeOH: methanol; NMR: Nuclear Magnetic Resonance spectroscopy; OD: optical density; PHB: poly(3-hydroxybutyrate); PHA: polyhydroxyalkanaote; PHBV: poly; PHBHx: poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); PPM: parts per million; SCL: short-chain-length; TMS: tetramethylsilane.3HA: 3-hydroxyalkanoate; 3HB (C4:0): 3-hydroxybutyrate; 3HHx (C6:0): 3-hydroxyhexanoate; 3HHx= (C6:5): 3-hydroxyhex-5-enonate; 3HO (C8:0): 3-hydroxyoctanoate; 3HO= (C8:7): 3-hydroxyoct-7-enoate; 3HP (C5:0): 3-hydroxypentanoate; 3HV (C5:0): 3-hydroxyvalerate; C5: pentanoic acid; C5=: 4-pentenoic acid; The authors declare that they have no competing interests.M. extorquens, performed the initial bioreactor studies, participated into some of the molecular biology work and analyzed all results from GC-FID. YJC and CBM used an emerging expression cassette developed by themselves and colleagues for cloning the two PHA synthase genes and for delivering a number of recombinant strains. MO performed the NMR work and was responsible for data analysis thereof. PV as PhD director and DG as co-director coordinated the study and offered general and expert supervision. All authors read and approved the final manuscript.PH as PhD student carried out most of the experimental work and co-wrote the manuscript. PH also selected the two PHA synthase genes that were cloned into 1H 1D spectrum indicated that 3HB was the major component comprising over 96% of the total signal (spectrum not shown). Using natural abundance 2D 1H-13C correlation spectra , Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear 2-Bond Correlation(H2BC) 2D spectra  and HMBC (31.8 ppm) cross-peaks and comparison with previously published data [1H methyl resonance originated from a longer chained 3-hydroxyalkanoate. We have assigned this to 3HO  although we cannot exclusively rule out other longer chained monomers. Finally, at very low contour levels, we were able to detect and assign resonances at the terminus of polymer  and 13C (115 ppm and 135 ppm) resonances indicative of double bonds. These were assigned to the 3HHx= and 3HO= monomers based on the following reasoning. Firstly, the sign of these resonances in the multiplicity-edited HSQC (data not shown)showed the higher frequency 1H peaks (5.7 ppm) tobe CH resonances  and the peaks at 5.0 ppm to arise from CH groups . The H2BC spectrum , 3HB:3HHx= (1:0.05), 3HB:3HP (1:0.02), 3HB:3HHx , 3HB:3HO .A notable feature of the 2D HSQC spectrum was the presence of high frequency


Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown.Prostate cancer is the second leading cause of cancer mortality among US men. Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1α,25 dihydroxyvitamin D2D  on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH)2D . Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60% up), 48 h, 3566 (38% up). 288 transcripts were regulated similarly at all time points  and many of the promoters for these transcripts contained putative vitamin D response elements. Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling. Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways). There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h.We examined the effect of 1,25(OH)Our data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention. In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis. The hview see ). In pron factor . However2D-regulated gene targets have been definitively identified in the context of prostate cancer, much less normal prostate biology. For example, 1,25(OH)2D directly induces transcription of the cyclin dependent kinase inhibitor gene p21 in U937 leukemia cells [2D mediated accumulation of p21 mRNA appears to be indirect [2D analog EB1089 up-regulates expression of TGFβ1 and β2 mRNA [2D-mediated transcriptional repression [2D modulates expression of transcripts encoding extracellular matrix proteins, cell adhesion proteins, DNA repair enzymes, and factors controlling oxidative stress. These data suggest that the cancer preventive impact of 1,25(OH)2D may utilize unique mechanisms in different tissues or that 1,25(OH)2D impacts multiple pathways involved in carcinogenesis.Many vitamin D target genes have been identified and characterized in the context of vitamin D's traditional actions in the control of calcium metabolism . In contindirect  through indirect . A numbe β2 mRNA  and down β2 mRNA , while ipression ,15. In apression ,17 shows2D [2D induced changes in the transcriptome of the phenotypically normal, immortalized human prostate epithelial cell line RWPE1. These findings provide new insight into the mechanisms that may be used by vitamin D to prevent the development of human prostate cancer.cDNA microarray analysis has been used on both human primary prostate epithelial cells and prostate cancer cells to identify potential target genes of 1,25(OH)2D -22. Howe2D the number of differentially expressed transcripts was increased over time from 1571 at 6 h to 3566 at 48 h. At 6 and 24 h, the transcripts were predominantly up-regulated (60.7% and 59.6%) while at the 48 h time point the transcripts were predominantly down-regulated (62.3%). 1,25(OH)2D treatment significantly altered the expression of 288 transcripts at all three time points; 262 of these changed in the same direction and 63.2% were up-regulated. Although many of our transcript-level changes were greater than 1.5-fold, our use of sample replicates, quality controls, and careful statistical analysis allowed us to see more subtle changes that may have biological relevance. A detailed list of all significantly differentially expressed transcripts is available in Additional File Using a 5% FDR cut-off, we identified 5435 transcripts as significantly differentially expressed in at least one time point Table . Followi2D induced were selected for PCR validation. These were selected based upon three criterion: they are classic vitamin D target genes , they had been identified in other studies , and they spanned a wide range of expression levels 2D-mediated expression of these in other prostate cell lines to determine if their regulation was a generalizable response. A similar effect of 1,25(OH)2D on these transcripts was observed cultures of primary human prostate epithelial cells, but a blunted response to 1,25(OH)2D treatment was seen in LNCaP cells 2D-treated RWPE1 cells  and indirect (i.e. no binding in a ChIP assay).Potential VDR binding sites were identified in the genes for nine 1,25(OH)2D-regulated expression were determined using Self Organizing Maps (SOM) Between 6 and 48 h the total number of significantly enriched genesets  was increased and the proportion of motif and curated genesets that were up-regulated fell Table . In cont2D at all three time points. Two other genesets were induced at all three time points: "induced during differentiation"  and "suppressed by JNK". Genesets suppressed at all time points were for VEGF target genes (i.e. suggesting an anti-angiogenic profile) and cytokine pathways (i.e. an anti-inflammatory profile). Genesets altered by 1,25(OH)2D treatment at 6 h only include one induced for "p53 and BRCA1 target genes", suggesting an early pro-apoptosis programming, and suppression of genesets containing WNT-, Notch-, and IGF1- target genes, suggesting the disruption of signals that promote cell proliferation. The shift to a less proliferative, more differentiated cell was supported by the suppression of genesets at 24 and 48 h that contain transcripts whose protein products promote proliferation and suppress differentiation .Genesets that were significantly changed at each time point were grouped according to related function Table . As expe2D treatment was increased over time, due to an increase in the number of suppressed maps. The detailed results from our GenMapp analysis of local maps and GO terms are available in Additional files The total number of local maps, GO terms, or Metacore maps influenced by 1,25(OH)An additional GenMAPP analysis was conducted to determine the functional characteristics of each of the five groups from the cluster analysis , and maps for oxidative stress  were induced at 6 h and at later time points. Similarly, Metacore maps related to apoptosis, keratins, and "cell adhesion through extracellular modeling" were up-regulated when all time points were evaluated together.We also looked at the functional categories that were regulated by 1,25(OH)The primary suppressed function that is consistent with a hypothesized role for vitamin D in cancer prevention was cell proliferation. At the later time points a clear reduction in specific functional groups and maps related to "cell cycle regulation" was observed. Consistent with this, the local map and Metacore maps for Wnt signaling were suppressed at 6 h and in the Metacore analysis, Notch signaling maps were significantly suppressed at 6 h and for all time points combined. Several other interesting processes that were down-regulated significantly at later time points include: angiogenesis , androgen receptor signaling, and various aspects of the cytokine signaling .2D influences many pathways relevant to prostate carcinogenesis and they underscore the critical role of this molecule in the maintenance of prostate epithelial development, function, and turnover.Optimal vitamin D status been proposed to prevent prostate carcinogenesis  and this2D treatment causes cell cycle arrest and growth suppression of primary prostate epithelial cell lines or prostate cancer cell lines. While this has been viewed as the major anticancer effect for 1,25(OH)2D, the mechanism accounting for this effect is not known with certainty. Liu et al. showed that the cyclin-dependent kinase (CDK) inhibitor p21 was strongly induced by 1,25(OH)2D treatment in the monocytic cell line HL-60 and they identified a functional Vitamin D Response Element (VDRE) in the p21 promoter [2D does not increase p21 transcript level in LNCaP cells [2D on p21 mRNA level is modest (1.32-fold at 6 h). Another CDK inhibitor, Wee1, is induced by 1,25(OH)2D in cultured keratinocytes leading to G2/M arrest [2D in RWPE1 cells (1.4-fold at 6 h). We examined other transcripts related to cell cycle control in our study but most of these were suppressed only at the later time points: e.g. GAS6, ETS1, CDK6, cyclin B2, cyclin A, CDC25C, and CDC27. This suggests that they are not primary effects of 1,25(OH)2D action.Several studies have shown that 1,25(OH)promoter . HoweveraP cells  and our M arrest  and this2D-mediated growth arrest. A number of Wnt pathways and genesets were reduced by 1,25(OH)2D treatment by 6 h, e.g. the geneset containing genes with LEF1/TCF4 binding motifs in their promoters that includes classical Wnt target genes like c-myc, cyclin D, PPARδ 2D treatment in RWPE1 cells (each reduced by -2 fold at 6 or 24 h). NOTCH1 and JAG1 are proposed as markers of normal prostate stem cells and they are necessary for fate determination of the proliferating stem cell [2D could be associated with cancer protection.Our data also show that transcripts for the Notch ligands, JAG1, JAG2, and DLL1 were suppressed by 1,25(OH)tem cell . In additem cell  and prostem cell ,28. Coll2D on VEGF gene regulation has been confusing. In mouse embryo fibroblasts and human vascular smooth muscle cells 1,25(OH)2D induces VEGFA expression through a VDRE in its promoter [2D can also suppress VEGF-induced vasculogenesis in cultured endothelial cells and in nude mice implanted with MCF-7 breast cancer cells [2D treatment suppressed VEGFC mRNA levels at all time points (-2.1 to -1.6 fold). Higher expression of VEGFC occurs after NKX3.1 loss in prostate cancer and is correlated with lymph node metastasis of prostate cancer [2D significantly suppressed KDR and NRP1 expression. Suppressing the activation of these receptors reduces tumor angiogenesis and promotion in the Dunning cell carcinoma model [2D induced expression of several semaphorin isoforms including SEMA3B, 3F, and 6D . Collectively our array data show that 1,25(OH)2D induces an anti-angiogenic transcript profile in RWPE1 cells.In addition to modulating cell grown, vitamin D has been proposed to inhibit the development of the tumor vasculature that is required for the progression of solid tumors -32 by supromoter , yet 1,2er cells . We foune cancer . VEGF prma model . Finallyma model , and modma model . In our 2D influenced the expression of genes related to these events  is an enzyme involved in maintaining cellular glutathione levels and its mRNA was significantly induced at all time points following 1,25(OH)2D treatment in RWPE1 cells (3.4-6.8 fold). Recently, Bao et al. [2D in prostate epithelial cells through a VDRE located in the first intron of the gene and that the induction of G6PD by 1,25(OH)2D protected RWPE1 cells against H2O2-induced apoptosis. It is also possible that vitamin D-mediated protection from pro-oxidant stress is indirect due to the induction of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), a transcription factor that controls expression of genes for many antioxidant enzyme systems [2D treatment, e.g. GPX3, HMOX1, AKR1C2, and TXNRD1.While our discussion has focused on the modulation of classical anti-cancer effects, our array analysis also revealed other potential mechanisms for vitamin D mediated cancer prevention. For example, oxidative stress-induced damage of DNA and other cellular components are implicated in cancer . These eo et al.  showed t systems . NFE2L2  systems . Consist2D promotes immunotolerance and immunosuppression by altering the differentiation and function of tolerogenic dendritic cells [2D up-regulated transcripts in RWPE1 cells. Prostate epithelial cells are thought to be early sensors of infection and CD14 and toll-like receptor 4 (TLR4) production in these cells contributes to protection from Chlamydia infection [Several studies have shown that vitamin D is anti-inflammatory and our data are consistent with these findings. In the immune system 1,25(OH)ic cells , suppresic cells , and incic cells . These aic cells  and promic cells . Howevernfection . While anfection .2D suppressed several components of JAK-STAT signaling in RWPE1 cells including JAK1, STAT1, n-myc and STAT interactor (NMI), STAT2, and STAT3. JAK-STAT signaling is required for the pro-proliferative effects of many cytokines including the pro-inflammatory cytokines IL6, IL12 and IFNγ [2D-treatment  [2D treatment; i.e. upregulation of IkB (NFKBIA) expression and suppression of RELB mRNA levels. These observations are consistent with data showing that 1,25(OH)2D suppressed secretion of IL-8 by interfering with NFκB signaling in RWPE1 cells [Another point where vitamin D may inhibit inflammatory processes is through suppression of cytokine signaling and production. Consistent with this, we found that 1,25(OH)and IFNγ . In addi T cells  and Th1  T cells . In addinalysis) . FinallyE1 cells  and thatE1 cells .2D in LNCaP cells. Moreno et al. [2D reduced the mRNA levels for two prostaglandin receptors  in prostate cancer cells. Since the prostanoid pathway is a critical component in acute inflammation that may contribute to the development of prostate cancer [2D in RWPE1 cells (6.3-fold at 6 h) and neither 15-PGDH nor prostaglandin receptor mRNA levels were altered.Prostaglandin signaling is a final pro-inflammatory pathway that has been identified as vitamin D regulated by others. Krishnan et al.  found tho et al.  subsequee cancer , this su2D reveals how the biology of prostate epithelial cells is changed by the hormone but microarray studies cannot differentiate between transcripts that are differentially regulated due to direct, VDR-mediated transcriptional activation and those that are secondary effects following the primary transcriptional events. As such, we can only infer the direct VDR gene targets based upon our data and their relationship to other studies. Unfortunately, there is very little information regarding the effect of 1,25(OH)2D on the prostate epithelial cell transcriptome to draw upon from earlier studies. Using a spotted cDNA microarray with no sample replicates, Peehl et al. [2D regulated in primary normal prostate epithelial cells and 52 transcripts in primary cultures of prostate cancer cells. Twenty-one of the differentially regulated transcripts from the normal prostate epithelial cells and 28 of the transcripts from the primary prostate cancer cells were also differentially regulated by 1,25(OH)2D in RWPE1 cells. Still, the only overlap between the three lists was CYP24, DUSP10, AKAP12, P2RY2, BMP6, TGFB2, and TXNRD1. In contrast, of the 22 transcripts identified by Krishnan et al. [2D-treated LNCaP cells only IGFBP3, ABCA1 and FKBP5 were regulated in the same direction in RWPE1 cells. This suggests that the response of RWPE1 cells is more similar to that of primary cultures of human prostate epithelial cells.The functional analysis of transcript-levels changes induced by 1,25(OH)l et al. ,22 identn et al. ,22 as di2D. Our ChIP examination of the AKAP12 and CYP26B1 promoters revealed significant VDR binding to putative VDREs in those promoters too, but given the large number of transcript level changes we observed, we expect that many more primary VDR target genes exist in the prostate epithelial cells. Before our analysis, the most comprehensive array-based analysis of 1,25(OH)2D action and putative VDR target genes was conducted by Wang et al. [2D at 6 h in RWPE1 cells based on the assumption that the early time point is less likely to contain transcripts that are regulated as a secondary consequence of primary vitamin D-mediated transcription events. This analysis identified 414 transcripts that were vitamin D regulated in both cell lines. 267 of these had a putative VDRE g et al. . Using aal cells . This sual cells  will be 2D-induced changes in the transcript profile of non-tumorigenic prostate epithelial cells. As such, it provides new insight into the mechanisms used by 1,25(OH)2D to prevent the early stages of prostate cancer. By using several independent procedures we identified multiple 1,25(OH)2D-regulated pathways and mechanisms that may disrupt the promotion of carcinogenesis in vivo. This includes anticancer mechanisms that have been traditionally attributed to 1,25(OH)2D action, e.g. suppression of cell proliferation and angiogenesis, as well as several potential new mechanisms including gene-protective and immunosuppressive effects. Further research is necessary to determine whether the genes identified in our array study are direct VDR target genes as well as to determine whether these transcripts are regulated by vitamin D signaling in vivo.This study is the most comprehensive functional analysis of 1,25(OH)2D was purchased from Biomol International .Unless otherwise noted, all chemicals were obtained from Sigma . Defined Keratinocyte-Serum Free medium (SFM) and RPMI medium 1640 were obtained from Invitrogen  and cell culture plasticware was from Corning-Costar . 1,25(OH)RWPE1 cells  were obt6 cells per flask) and grown until cells reached 60% confluence. At this point, cells were treated with medium containing 100 nM of 1,25(OH)2D or vehicle (0.1% ethanol) for 6, 24 or 48 h . For the 48 h time point, media was replaced at 24 h prior to cell harvest. Total RNA was isolated from the cells using TriReagent  in accordance with the manufacturer's instructions. Isolated total RNA was further purified using the RNeasy kit . The quality of the isolated RNA was confirmed using agarose gel electrophoresis.RWPE1 cells were plated in T75 flasks . RNA labeling, chip hybridization and chip scanning was carried out at the Ohio State University Comprehensive Cancer Center by  using standard Affymetrix protocols . Chips were scanned and raw data was saved into CEL files or analyzed using the Affymetrix Microarray Suite (MAS) 5.0 software. Microarray data may be accessed at the NCBI Gene Expression Omnibus (GEO) database (accession # GSE15947).http://www.Bioconductor.org[The quality of microarray images from individual chips was examined using methods from the affyPLM package of Bioconductor uctor.org. The quaMicroarray data from CEL files for all 24 chips was normalized simultaneously and expression levels were generated using the gcRMA package in Bioconductor . The nor2D treated) using the Significance Analysis of Microarray program  [2D treatment was determined at the 5% FDR at each time point.Differentially expressed transcripts were identified at each time point on 3.02) . Data foon 3.02) . Signifihttp://www.genome.wi.mit.edu/MPR/. Only data from transcripts identified by SAM as significantly differentially expressed in at least one time point (FDR<5%), were used for the cluster analysis. Prior to the clustering, the data was normalized to mean = 0 and variance = 1. For this analysis we used a 4 × 3 matrix and the default settings of the software .Patterns of differentially expressed genes were determined by cluster analysis using Self-Organizing Maps in GeneCluster http://www-stat.stanford.edu/~tibs/GSA/ and geneset databases from the Molecular Signatures Database http://www.broad.mit.edu/gsea/msigdb/index.jsp: c2-curated, c3-motif, and c4-cancer neighborhood. Analysis for each time point was done using a two class, unpaired analysis with T-statistics, 1000 permutations, automatic estimation of s0 factor for denomination, K-nearest Neighbors imputer and a random number seed.Gene Set Analysis (GSA) was conducted using the GSA function in SAM 2D treatment: GeneMAPP http://www.genmapp.org[Two methods were used to identify and visualize biological processes and pathways that were enriched due to 1,25(OH)nmapp.org and MetaFor the Metacore analysis, transcripts significantly differentially induced or suppressed  were mapped to Metacore maps representing functional pathways individually for each time point or for all time points combined. Enrichment of Metacore maps was determined by the p-value of hypergeometric distribution representing probability that a transcript would be matched to a map by chance. This analysis takes into account the sizes of the dataset, map or ontology group. Metacore maps with p-value < 0.05 were considered significant. Networks were generated de novo from the differentially expressed transcripts at each time point by using Metacore. In addition, the "Analyze network (transcription factors)" program was used to create networks centered around specific transcription factors of interest.2D treatment on the accumulation of these 11 transcripts was also examined in RNA from 1,25(OH)2D-treated LNCaP cells and in primary normal human prostate epithelial cells. 80% confluent LNCaP cells were treated with medium containing 100 nM of 1,25(OH)2D or vehicle (0.01% ethanol) for 6 h. RNA from 1,25(OH)2D-treated primary human prostate epithelial cells was a provided by Dr. Scott Cramer . Three human primary prostate epithelial cell preparations were treated with growth medium containing 10 nM of 1,25(OH)2D or vehicle (0.01% ethanol) for 8 h. Cells were harvested in TriReagent and RNA isolated. cDNA was prepared from RNA samples as we have described previously [For the validation of microarray data, differential expression of 11 genes was examined in cDNA prepared from the RNA for the microarray study by Real Time Polymerase Chain Reaction (RT-PCR). The impact of 1,25(OH)eviously .-ΔΔCt described elsewhere [cDNA samples were analyzed by RT-PCR analysis using the BioRad MyiQ Real-Time PCR system and the BioRad SYBR Green supermix . Expression levels were determined from the threshold cycle (Ct) value using the method of 2lsewhere  and usin2D at 6 h, were analyzed for presence of VDR binding sites using a bioinformatics approach. The -10 kb to +10 kb region  of candidate genes was downloaded from the GenBank database at the NCBI. We then screened the conserved regions of the promoters utilizing an in silico approach by using CONSITE http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite/ and the following settings: window size = 50 bp; conservation cut-off = 70%, Transcription Factor score threshold = 65%. Putative VDR binding sites with similarity score over 3.5 were accepted as candidates for ChIP validation. Ten potential VDR binding sites located within conserved regions were selected for further testing within the following gene promoters: SEMA3B, CD14, P2RY2, AKAP12, SERPINB1, HBEGF, TXNRD1, CYP26B1, MTSS1 and LOX. The location of these sites is provided in Additional file The promoters of the genes encoding the top 50 transcripts significantly up-regulated by 1,25(OH)2D or vehicle for 3 h. ChIP assays for VDR association to DNA were done as we have previously described [http://frodo.wi.mit.edu to amplify a fragment containing the predicted VDR binding sites. Primers used for analysis of CD14, P2RY2, AKAP12, SERPINB, HBEGF, TXNRD1, CYP26B1, MTSS1 and LOX are provided in Additional file RWPE1 cells were cultured to 60% confluence and treated with 10 nM 1,25(OH)escribed . ChIP asescribed ,68. For The analysis for treatment effects in the RT-PCR data was conducted using one-way ANOVA using the SAS statistical software package . Pairwise comparisons were conducted when appropriate using Fisher's Protected LSD. Values are expressed as the means ± SEM. Differences between means were considered significant at p < 0.05.15-PGDH: hydroxyprostaglandin dehydrogenase 15-(NAD); AKAP12: a kinase (prka) anchor protein 12; AKR1C2: aldo-keto reductase family 1, member C1; APCDD1: adenomatosis polyposis coli down-regulated 1; BMP6: bone morphogenetic protein 6; BRCA1: breast cancer 1; CALML3: calmodulin-like 3; CD14: CD14 antigen; CDC25C: cell division cycle 25 homolog C (S. pombe); CDC27: cell division cycle 27 homolog (S. cerevisiae); CDK: cyclin-dependent kinase; CDK6: cyclin-dependent kinase 6; ChIP: chromatin immunoprecipitation; COX2: prostaglandin-endoperoxide synthase 2; CYP24: cytochrome p450, family 24, subfamily A, polypeptide 1; CYP26B1: cytochrome p450, family 26, subfamily b, polypeptide 1; DLL1: delta-like 1 (Drosophila); DR3: direct repeat with 3 bp spacing; DUSP10: dual specificity phosphatase 10; EGFR: epidermal growth factor receptor; ETS1: v-ets erythroblastosis virus E26 oncogene homolog 1 (avian); FDR: false discovery rate; FLT1: fms-related tyrosine kinase 1; G6PD: glucose-6-phosphate dehydrogenase; GADD45a: growth arrest and DNA-damage-inducible, alpha; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAS6: growth arrest specific 6; GCRMA: GC Robust Multi-array Average; GO: gene ontology; GPX3: glutathione peroxidase 3; GSA: gene set analysis; HBEGF: heparin-binding egf-like growth factor; HMOX1: heme oxygenase (decycling) 1; IGF1: insulin-like growth factor 1; IGFBP3: insulin-like growth factor binding protein 3; IL-17: interleukin 17; IL-27: interleukin 27; IL-6: interleukin 6; JAG1: jagged 1; JAG2: jagged 2; KDR: kinase insert domain receptor; LEF1: lymphoid enhancer binding factor 1; LOX: lysyl oxidase; MTSS1: metastasis suppressor 1; NFE2L2: nuclear factor (erythroid-derived 2)-like 2; NFKBIA: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; NOTCH1: Notch homolog 1, translocation-associated (Drosophila); NRP1: neuropilin 1; NUSE: normalized and the unscaled standard error; P2RY2: purinergic receptor p2y, G-protein coupled 2; PCR: polymerase chain rreaction; PPARδ: peroxisome proliferator-activated receptor delta; RAD9A: RAD9 homolog A (S. pombe); RELB: v-rel reticuloendotheliosis viral oncogene homolog B; RLE: relative log expression; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SAM: significance analysis of microarray; SEM: standard error of the mean; SEMA3B: semaphorin 3B; SEMA3F: semaphorin 3F; SERPINB1: serpin peptidase inhibitor, clade b , member 1; SEM: standard error of the mean; SFM: serum free medium; SULT1A3: sulfotransferase family, cytosolic, 1A, phenol-preferring, member 3; TCF4: transcription factor 4; TGFβ1: transforming growth factor, beta 1; TLR4: toll-like receptor 4; TSS: transcription start site; TXNRD1: thioredoxin reductase 1; VAV3: vav 3 oncogene; VDR: vitamin D receptor; VDRE: vitamin D response element; VEGF: vascular endothelial growth factor; VEGFA: vascular endothelial growth factor A; VEGFC: vascular endothelial growth factor C; WNT: Wingless-type MMTV integration site family.The authors declare that they have no competing interests.PLK conducted the RWPE1 experiment, performed statistical, functional and cluster analysis of microarray data, conducted validation of target genes using QRT-PCR and prepared the manuscript. ZZ carried out preliminary studies and participated in the RWPE1 experiment and sample preparation. MC conducted ChIP experiments and bioinformatic identification of VDREs in the promoter regions of genes. SKC was responsible for microarray analysis and provided interpretation of the data. JCF was responsible for the overall design of the project, data interpretation and final paper editing. All authors of the paper read and contributed to the final manuscript.Significantly differentially expressed transcripts. Significantly changed transcripts at any time point from SAM analyzed microarray data on RWPE1 cells treated 100 nm 1,25(OH)2D vs. vehicle.Click here for fileGSA currated gene sets c2. GSA curated genesets (c2) significantly enriched in differentially expressed transcripts.Click here for fileGSA motif genesets c3. GSA motif genesets (c3) significantly enriched in differentially expressed transcripts.Click here for fileGSA cancer computational genesets (c4). GSA cancer computational genesets (c4) significantly enriched in differentially expressed transcripts.Click here for fileGenMAPP local map results. Table of GenMAPP analysis of local maps for enrichment .Click here for fileGenMAPP GO group results. GenMAPP Gene Ontology terms significantly changed .Click here for fileMetacore summary. Metacore Maps enriched in significantly changed genes .Click here for fileGeneMAPP SOM groups Local maps results. Table showing GenMAPP analysis on SOM groups using Local maps.Click here for fileGeneMAPP SOM groups GO group results. Table of GeneMAPP GO map analysis for SOM groups.Click here for file2D on Wnt and Notch signaling at 6 hEffect of 1,25(OH). Figure showing the effect of 1,25(OH)2D  on transcripts controlling Wnt and Notch signaling in RWPE1 cells. Differentially expressed transcripts  were examined by time point for functional changes using GenMAPP and GSA. The GeneMapp local map for Wnt signaling  was identified as significantly down regulated at 6 h. In addition, a GSA motif geneset (c3 #162) for genes containing Lef1 domains in their promoters (a Wnt pathway targeted transcription factor) was significantly down-regulated.Click here for file2D on c-Myc transcriptional activity at 6 hEffect of 1,25(OH). Image representing effect of vitamin D induced supression of c-Myc on the mRNA level of c-myc target genes. Significantly differentially expressed transcripts at 6 h  were analyzed by using Metacore Network analysis (Transcription factor). Up--regulated genes are marked with red circles; down--regulated with blue circles. Arrows are color coded to reflect the known regulatory action between two proteins. Red arrows between proteins indicates a negative regulatory effect, green arrows indicate a positive regulator effect, gray arrows indicate an unspecified regulatory effect.Click here for file2D on antioxidant and DNA protection at 6 hEffect of 1,25(OH). Figure showing the effect of 1,25(OH)2D  on transcripts controlling antioxidant and DNA repair systems in RWPE1 cells. Differentially expressed transcripts  were examined by time point for functional changes using GenMAPP and GSA. The GenMAPP local map for antioxidant responses to reactive oxygen (Hs_Oxidative_Stress) was identified as significantly up-regulated at 6 h. While not on this map, the up-regulation of G6PD is also relevant as this enzyme system contributes to glutathione production.Click here for file2D suppresses proinflammatory cytokine signaling1,25(OH). Figure representing regulation of transcripts controlling cytokine signaling in RWPE1 cells by 1,25(OH)2D treatment (100 nM). Differentially expressed transcripts  were examined for functional changes using GenMAPP and GSA. A large number of pathways related to the signaling through cytokine pathways were identified as down-regulated. Most of these pathways utilize a JAK-STAT intracellular signaling pathway. A selection of transcripts affected and their relationship to JAK-STAT signaling are shown.Click here for file2D suppresses STAT1, STAT3 and PU.1 networks at 48 h1,25(OH). Image representing suppression of transcripts regulated by STAT1, STAT3 and PU.1. Significantly differentially expressed transcripts at 48 h  were analyzed by using Metacore Network analysis (Transcription factor). Most of these transcripts are regulated by STAT1 or STAT3. Up--regulated transcripts are marked with red circles; down--regulated transcripts are identified by blue circles. Arrows are color coded to reflect the known regulatory action between two proteins. Red arrows between proteins indicates a negative regulatory effect, green arrows indicate a positive regulator effect, gray arrows indicate an unspecified regulatory effect.Click here for fileVDRE containing genes, comparison with Wang et al, 2005. Comparison of VD regulated transcripts in RWPE1 to those reported for EB1089 by Wang et al. in SCC25 cells.Click here for fileRT-PCR primers. List of RT-PCR primers used for validation of differential expression identified in 1,25(OH)2D-treated RWPE1 cells.Click here for fileGenes tested for functional VDRE. Table of genes tested for functional VDRE in promoter regions.Click here for file


S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the MSa phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-α, IFN-γ and Il-1β genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 µl/mouse, protected the animals  from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 µg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of  Staphylococcus aureus can cause minor infections as well as life-threatening diseases. Endocarditis, osteomyelitis, pneumonia, surgical wound and intravascular device infections caused by S. aureus represent a major public health concern S. aureus bloodstream infections and 40–60% of surgical wound infections are caused by methicillin-resistant strains of S. aureus (MRSA) S. aureus with reduced susceptibility to vancomycin are also emerging S. aureus infections are needed. This study describes the development of a vaccine active against S. aureus.S. aureus. In a mouse model of infection, this phage-resistant strain prevented infections from diverse and clinically relevant S. aureus strains.In the presence of an antibiotic or a bacteriophage (a bacteria-attacking virus), resistance is clearly beneficial to the bacteria. As might be expected, in such conditions, resistant bacteria are swift to emerge S. aureus strains A170, A177, A179 and A181 were isolated from clinical samples collected from patients with infected surgical wounds . Patients were hospitalized at the Medical School of the University of Naples Federico II. Clinical samples were streaked on trypticase soy agar (TSA)  and single colonies expanded in trypticase soy broth (TSB) (Oxoid). The strains listed above and their phage-resistant mutants  were all identified as S. aureus600: 1.5 to 1.8), centrifuged (8×103×g for 10 min) and washed with saline (0.15 M NaCl). Serial 10-fold dilutions in saline were then plated on TSA.6–108 bacteria (as detailed in each experiment) suspended in sterile saline (100 µl/mouse). In the case of aerosol infection, mice were placed in a nebulizing chamber and exposed to an aerosol solution (107 CFU/ml) for 10 min. When inspected on the day of infection, the lungs displayed about 1.6×105±5.5×103 CFU/g/mouse (average of 3 mice). Organs were dissected and weighed. Samples were homogenized in 1 ml saline and serially diluted in saline. Colony forming units (CFU) were evaluated by the plate count assay and expressed as CFU/g. Animal experiments were approved by the Animal Care Committee of the University of Naples (permit number 86/609/EEC).Experiments were carried out on female Balb/c mice (aged 8 to 10 weeks) at the animal facility of the University of Naples. Mice were infected via the intramuscular or aerosol routes. In the case of the intramuscular infection, mice received 5×10Sa was isolated from the S. aureus strain A170 following induction with mitomycin Sa1, MSa2 and MSa3 were isolated from S. aureus strains A177, A179 and A181, respectively; phage release was again induced with mitomycin S. aureus strains A172, A178, A180 and A182 were isolated by plating dilutions of overnight susceptible cultures on TSA containing increasing concentrations of the phage used for selection. Ten single colonies growing at the highest phage concentration were selected and subcultured twice on TSA agar in the absence of phage. To ensure stability of resistance, two colonies from each phage-resistant strain were further subcultured (20 times or more) in the absence of phage. Induction of the phage-resistant strains (including A172) with mitomycin excluded the presence of prophages in these strains.Phage M8 CFU/mouse) and two weeks later sacrificed and the blood pooled. The protein A gene is under-expressed in the A172 strain. Yet, to avoid interference with the protein A possibly present on the bacterial surface, the wells of a 96-well plate  were coated with the S. aureus protein A negative strain Wood 46 −2–10−4) in PBS , washed with PBS and incubated, in succession, with peroxidase-labelled rat anti mouse IgG  and peroxidase substrate .Mice were immunized with the A172 strain (10TA) and A172 (A172TA) isolates were prepared as described 3OH; 85°C, 24 h) and acetylation with acetic anhydride in pyridine , samples were analyzed by gas chromatography-combined mass spectrometry (GC-MS) and compared with standards. GC-MS analyses were carried out on a Hewlett-Packard 5890 instrument using SPB-5 capillary column . Ring size and attachment points were determined by methylation analysis as described 3I/NaOH in DMSO, hydrolysed with 2M trifluoroacetic acid , reduced with NaBD4, acetylated with acetic anhydride in pyridine and analyzed by GC-MS. The temperature programme was: 150°C for 5 min and then 5°C/min to 300°C over 10 min.Teichoic acids from the A170 (A1706×g for 10 min) and the supernatant precipitated with ethanol (four volumes) in the cold (−20C overnight). Supernatant and precipitate were separated by centrifugation (8×106×g for 10 min). The precipitate, insoluble in H2O and several organic solvents , was discarded. The supernatant was dialyzed  against water (3 days), lyophilised, weighed and tested for the capacity to protect mice. Mice vaccinated with the supernatant (25 µg/mouse) were fully protected against a lethal dose of A170 bacteria. This biologically active fraction (3.4 g) was hydrolysed with 0.1 M HCl at 100°C for 48 h. The mixture was neutralized with 2 M NaOH and reduced with sodium borohydride in distilled water (3 g/0.5 ml). The alditols were acetylated with pyridine (100 µl) and acetic anhydride (100 µl) at 85°C for 30 min. The monoses were identified as acetate alditols by gas liquid chromatography (GLC) The A172 strain (100 ml) was grown in TSB. When in the exponential growth phase, the culture was centrifuged (8×109 CFU/200 µl) were quantitatively spread over a phage lawn (1012 PFU/5 ml soft agar). Bacteria were thus surrounded by large numbers of virus particles. Plates were inspected for the presence of plaques of lysis during the next 4 days. The detection limit of the test was established by carrying out the test on the same A172 cultures spiked with 12, 6, 3 or 1 A170 MSa-sensitive bacteria. The dilutions with 12, 6, 3 or 1 A170 bacteria were assayed by plate count in triplicate.Individual wells of 96-well plates were filled with 200 µl TSB medium and inoculated with one colony of A172 bacteria isolated from mice vaccinated with the same strain and then grown in vitro for 15 generations under stress conditions (10 µg/ml novobiocin) 9 CFU/200 µl) were incubated for 1 h with the MSA phage (105 PFU). The cultures were then transferred quantitatively on a lawn of the A170 MSa-sensitive bacteria (105 CFU/4 ml soft agar) to determine whether the original phage titre increased. The detection limit of the test was established by testing in parallel the phage titre increase that occurred in the same A172 cultures (109 CFU/200 µl) spiked with 12, 6, 3 or 1 A170 MSa-sensitive bacteria. The dilutions with 12, 6, 3 or 1 A170 bacteria were assayed as described above.The presence of revertants was also investigated by an independent method, based on the increase of phage titre of a liquid culture upon addition of phage-sensitive bacteria 5 CFU/100 µl) were grown (4 h) in 2 ml TSB, in the presence or absence of: 5–20 mM N-acetyl-glucosamine (GlcNAc), 5–20 mM glucose , 0.1–4 nmol/tube teichoic acid from A170 (A170TA) or A172 (A172TA). The MSa phage (109 PFU7tube) was then added and bacterial growth (OD600) measured 30 min later.A170 or A172 bacteria (105 CFU/100 µl) were grown (4 h) in 2 ml TSB, in the presence or absence of N-acetyl-glucosaminidase from Canavalia ensiformis . The MSa phage was then added (109 PFU) and bacterial growth (OD600) measured 30 min later.A170 or A172 bacteria . Pulsed field gel electrophoresis (PFGE) of the restriction digests was performed by using the CHEF system  with 1% (wt/vol) agarose gels and 0.5×TBE as running buffer, at 10°C. Restriction fragments were resolved in a single run, at constant voltage of 6 V cm2 and an orientation angle of 120° between electric fields, by a single phase procedure for 24 h with a pulse ramping between 1 and 50s.The procedure adopted was that described in FITC) and A172 (A172FITC) bacteria were prepared as described S. aureus strains were determined by sequencing the hypervariable region of S. aureus protein A gene (spa) as described spa types were assigned using the Ridom spa-server (http://spaserver.ridom.de). Multilocus sequence typing (MLST) of S. aureus strains was carried out as described eta, etb, tst, lukS-PV–lukF-PV, lukE-lukD and lukM,  were detected by PCR se) genes se-a, se-b, se-c, se-d, and se-e were detected as described egc) type was carried out as described S. aureus strains A170 and A172 were calculated attributing to A170 and A172 the molecular weight of 6350 daltons  The transcription level of bacterial virulence genes and mouse cytokine genes was analyzed by real-time reverse transcription PCR (RT-PCR) as described spa-server (http://spaserver.ridom.de), t6668 represents a novel spa type. PFGE analysis displayed a close genetic relation between the parental phage MSa-sensitive strain A170 and the derivative phage-resistant strain A172, as proved by the identical PFGE pattern of the Sma I digests , the multilocus sequence type (ST 45) and spa-type (t6668) . According to the Ridom  digests . A172 wa11 bacteria . Plated with phage in excess (1012 PFU/5 ml soft agar), the 400 independent bacterial cultures did not yield any plaque of lysis. Control cultures (109 CFU A172/200 µl spiked with 3 A170 bacteria), assayed in triplicate in three independent experiments, displayed all at least one plaque and, on average, 2.2±0.7 plaques/culture (number of replicas: 9). The absence of A172 revertants was confirmed in a second independent experiment. When the MSa phage (105 PFU) was mixed with the A172 bacteria grown under stress conditions (109 CFU/200 µl), no measurable increase in the phage titre occurred. Control cultures (109 CFU A172/200 µl spiked with 3 A170 bacteria), compared to the non-spiked cultures, displayed an increase in phage titre from 1.4×105±24×103 to 1.2×106±0.5×103 . Conservatively, it is estimated that A172 revertants occur at a frequency <3×10−9.The strain was also highly stable in vivo. A172 bacteria were isolated from a vaccinated mouse, grown for 15 generations under stress conditions (10 µg/ml novobiocin) 600: 0.6) were assayed by plate count. In three independent experiments, A170 cultures contained about 3 fold as many CFU as the A172 cultures . A172 displayed reduced growth rate during the exponential phase  or from (A172TA) as inhibitor. Since the quantity of teichoic acid that is isolated from the A170 or A172 strains varies significantly , the experiment was conducted using an ample concentration range of the A170TA and A172TA reagents. Under these conditions, phage inactivation occurred with A170TA, but not with A172TA  and two weeks later infected with a lethal dose of A170. At the time of infection, in the serum of immunized animals the titre of the antibodies against A172-L ranged from 1∶5000 to 1∶12000. Control mice were also infected with a lethal dose of A170. The A172-L vaccine protected 90% of the mice (surviving mice: 18/20) from A170, while the control mice (10/10) died within 4 days , albeit considerable when the use of a large number of vaccine doses is prospected. The study therefore explored whether A172 remained protective once it was heat-killed (15 min at 100°C). Mice were vaccinated with heat-killed vaccine (A172-HK) and 2 weeks later infected with a lethal dose of A170. The vaccine was protective also in this form ; the vaccinated mice (37/40) were alive 14 days after challenge - was protective. No significant difference in survival between immunized and control mice was observed  (A174 and A175) and one vancomycin-intermediate S. aureus (VISA) (A176) isolates derived from patients with staphylococcal infections. Three groups of mice were immunized with A172-HK and 20 weeks later infected with a lethal dose of the A174, A175 or A176 isolates. The difference in survival between vaccinated and control mice was statistically significant in each case   were injected intramuscularly with normal mouse serum . Two more groups of mice were treated intramuscularly with serum from mice immunized with A172-HK . The titre of the antibodies against A172-HK was 1∶8000. The next day, the mice were all challenged with a lethal dose of S. aureus A170. Absorption of anti-A172 mouse serum completely eliminated the protective effect of the serum  and diluted to 100 µl with sterile saline. The next day, the mice were challenged with a lethal dose of 8 CFU/mouse). A172 modulated transcription of the genes coding for the pro-inflammatory cytokines TNF-α, IFN-γ and IL-1β, while inducing that of the genes coding for the anti-inflammatory cytokines Il-4 and Il-6, when these genes were analyzed by RT-PCR, 24 and 48 h after immunization  with the MSa1-resistant strain A178 (derived from the MSa1-susceptible isolate A177), MSa2-resistant strain A180 (derived from the MSa2-susceptible isolate A179) or MSa3-resistant strain A182 (derived from the MSa3-susceptible isolate A181), respectively. Two weeks later, immunized and control mice were challenged with A170 . The phage-resistant strains all provided protection in terms of survival and level of host colonization (S. aureus isolates use the same defence strategy against different phages.Three groups of mice were immunized (10nization . The expCapsular polysaccharide (CP) production is a characteristic common to the four protective strains  . In an aS. aureus are being investigated for their potential use as vaccines S aureus strain containing multiple antigens and therefore likely to elicit cross-protection against a spectrum of pathogenic strains. The attenuated strain (A172) proved effective as vaccine even when heat-killed. Thus, heat-killed A172 (A172-HK) possesses the advantages of a multiple component vaccine, without the problems of safety (reversion to phage sensitivity and the accompanying virulent phenotype), distribution and manufacturing of the live vaccines Several virulence factors of S. aureus. The approach had already been used successfully to produce a vaccine against Salmonella enterica serovar Paratyphi B Sa phage, yielded the phage-resistant strain A172. Given their common origin, the two strains share several properties, but display also crucial differences. They share the spa-type (t6668), the MLST type (ST 45), the egc type (egc-4) and part of the genome (S. aureusTA) inhibit lysis by the MSa phage, while glucose or the teichoic acids from A172 (A172TA) do not  occurs at a fitness cost e genome . At the e genome  that pha) do not . These rS aureus (VISA)  . Sometimstration .S. aureus A170. In two independent experiments, 10/10 of the animals passively immunized with an individual dose of 10 µl immune serum  survived, while 10/10 untreated control mice died  all display the characteristic of secreting CP  is limited by the difficulty of developing reliable procedures of bacterial attenuation Sa or the bacterial strain A172, but more general. Phage resistance, long considered a problem in the fight against bacteria In conclusion, given the multiple virulence factors expressed by


To the Editor: The first avian influenza (H5N1) outbreak in poultry in Laos occurred in 2003 and subsided in March 2004 after massive killing of poultry to contain the disease. Extensive surveillance from July 2005 through January 2006 did not detect any influenza virus subtypes in chicken, ducks, quails, and pigs in live bird markets in the Vientiane, Champasak, and Savannakhet Provinces (Avian influenza (H5N1) had not been reported in humans in Laos until February 27, 2007 (A diagnosis of infection with avian influenza (H5N1) was based on positive results obtained by reverse transcription–PCR (RT-PCR), viral isolation in MDCK cells inoculated with an endotracheal suction specimen collected on February 22, and a 4-fold increase in neutralizing antibody titers from 80 to 320 in paired blood specimens collected on February 25 and March 1, as assayed against autologous virus. This virus isolate was named A/Laos/Nong Khai 1/07(H5N1). Subsequent samples were collected on February 25 and March 7 (day of death). Results of RT-PCR were positive for the sample collected on February 25 only; virus isolation results were negative for both samples.www.who.int/csr/disease/avian_influenza/smaltree.pdf) showed that it belonged to clade 2.3.4 . One amino acid deletion and 1 amino acid change were found when compared with RERRRKKR, which is present in most avian influenza viruses (H5N1). There was no change in receptor binding site. This virus had glutamic acid at aa 627 in the PB2 protein, aspartic acid at aa position 92 in nonstructural protein 1, and 5 aa deletions at positions 80–84 in the nonstructural protein 1. Analysis of the neuraminidase gene showed a 20-aa deletion in the stalk protein; there was no mutation of histidine to tyrosine at aa position 274, a position shown to be the oseltamivir resistance marker in the neuraminidase 1 viral genome  have been reported in some East Asian countries. Genetic diversity in the hemagglutinin gene has classified those genotype V viruses into distinct clades. Viruses from avian species in South Korea in 2003 and Japan in 2004 (Highly pathogenic avian influenza viruses (H5N1) that caused outbreaks in Thailand since 2004 belong to genotype Z, clade 1. Introduction of genotype V clade 2.3.4 virus, A/chicken/Thailand/NP-172/2006, to Nakhon Phanom Province occurred in November 2006 , panel APhylogenetic analysis of avian influenza viruses (H5N1). A) hemagglutinin genes and B) polymerase A genes. Pseudosampling = 1,000. Known genotype V viruses are indicated in red, and genotype Z viruses are indicated in blue. Numbers on the right in braces indicate clades and subclades. Scale bars indicate genetic distances between sequences of different taxa. HK, Hong Kong.


Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits , and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higr virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production. Type A influenza viruses (IVAs) are classified into subtypes based on two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). IVAs infect a large variety of mammals and birds, occasionally producing devastating pandemics in humans . Epidemipandemic ,5. SinceThe IVA genomes consist of eight single-stranded RNA segments of negative polarity that encode up to 11 proteins ,8. TheseThe cellular response to growth factors, inflammatory cytokines, and other mitogens is often mediated by receptors that are either G protein-linked or intrinsic protein tyrosine kinases . The binRecently, we demonstrated that HA accumulation at the cell membrane and its tight association with lipid-raft domains trigger virus-induced ERK activation , showing50, viral growth, and the level of viral protein synthesized during infection. Logarithmic differences of viral infectivity titers were determined 3 days after infection via serial dilution. Infection with the H3N2 virus resulted in 2 log higher TCID50/ml than that seen with the H1N1 infection, which indicated higher production of infectious progeny virions of the H3N2 subtype. To determine the viral growth curve, we infected MDCK cells with either virus at m.o.i. = 2. New infectious progeny virions of H3N2 IVA were released within 4 h after infection, whereas almost no H1N1 virus could be detected within this time frame. Furthermore, a clear, at least 1 log increase in virus titers was observed in H3N2-infected cells between 6 to 12 h post infection (p.i.)  Fig. . Additioi.) Fig.  showing +. Around 40% more viral NP was found in H3N2-infected cells at 6 h p.i. and almost all of the cells were infected by H3N2 at 8 h p.i. This finding showed optimal replication of newly formed progeny virions of the H3N2 subtype. The amount of NP+ cells at 8 h after H1N1 infection was lower than that at 6 h after infection with H3N2. Overall, our results clearly showed that the studied H3N2 virus possesses better growth capacity and replicates more efficiently in tissue culture model than does the H1N1 subtype.To evaluate whether the amount of viral proteins synthesized during infection differed between these two strains, we measured NP production at different times in MDCK cells infected at m.o.i. = 1. Flow cytometry analysis revealed that the H3N2 IVA produced markedly more NP than did the H1N1 at 4, 6, and 8 h p.i. Fig. . Whole-cInduction of MAPK signaling is essential for influenza virus RNP export . As the To investigate the Raf/MEK/ERK signaling-dependent nuclear RNP export, we analyzed intracellular RNP localization in cells infected with either virus. In accordance with flow cytometry analysis showing a very low amount of viral NP produced by H1N1 virus at 4 h p.i., no H1N1-NP was detected at this time point by confocal laser scanning microscopy. RNPs were localized in the cytoplasm in nearly all H3N2-infected cells at 6 and 8 h p.i., whereas in H1N1-infected cells they were localized predominantly in the nucleus or at the nuclear membrane at those time points Fig. . These rThe Raf/MEK/ERK signal cascade can be activated by either protein kinase C alpha (PKCα)-dependent or Ras-dependent pathways . Upon thWe also assessed the effect of blocking ERK activity on H3N2-infected cells. The levels of ERK phosphorylation in H3N2-infected cells dramatically decreased Fig. . As a reP = 6.48 × 10-4 and 5.23 × 10-6)  Fig. . To provThe H3N2 virus replicated more efficiently in MDCK cells than did the H1N1 strain, and viral polymerase genes have been shown to contribute to virus growth and infectivity -28. TherTo test which viral protein in the RNP complexes affect viral polymerase activity the most, we exchanged each plasmid encoding PB1, PB2, PA, or NP of both viruses. Transfection without the PB1 plasmid was also assayed as an indication for background level of non-specific luciferase expression. The relative polymerase activity of the wild type H3N2 was higher than that of the wild type H1N1. The values obtained from the transfections comprising the wild type system of each virus are individually set as 100%. Replacing H1N1 PB1 or PB2 with those genes from the H3N2 virus significantly increased the viral polymerase activity of the H1N1 virus by about 35% Fig. . ConversThe PB1 and PB2 genes appeared to have the most influence on viral polymerase activity. Since PB1 plays a central role in the catalytic activities of the RNA-dependent RNA polymerases , we focuWe compared the viral replication efficiency of two strains of IVAs isolated from two different patients in Hong Kong in 2006. The isolated H3N2 subtype replicated more efficiently than the H1N1 in MDCK cells. Interestingly, growth capacity was related to the IVA's ability to activate the Raf/MEK/ERK (MAPK) signal cascade. The H3N2 virus upregulated MAPK signaling better than did the H1N1 virus. Accordingly, stimulation of MAPK signaling with TPA, a strong kinase activator, increased the H1N1 virus titers. In contrast, treatment of H3N2-infected cells with the specific MEK inhibitor U0126 abolished ERK activation and severely reduced the virus titers. These data show that replication of both viruses strongly depends on their ability to activate the MAPK signaling. Cell treatment with TPA or U0126 did not affect the synthesis of viral NPs at 6 and 8 h p.i. (data not shown). This finding showed that changes in virus titers, at least in part, are indeed influenced by nuclear export efficiency of the RNPs.Moreover, many studies have shown that the polymerase genes of more replication-efficient influenza viruses play a central role in virulence and virus replication ,31,27. TPreviously, we showed that a tight association of viral HA with lipid raft domains localized in the cell membrane is crucial for activating the virus-induced MAPK signal cascade . Three hNext, we tried to figure out the fundamental reasons why the H3N2 strain replicates more efficiently than the H1N1 subtype does. It is noteworthy that most of the currently circulating H5N1 strains with pandemic potential replicate very fast and exhibit high lethality in various hosts. The viral polymerase genes, particularly PB1 and PB2, contribute to the virulence of the human A/Vietnam/1203/04 (H5N1) influenza virus in mice and ferrets . SequencAll together, our findings led us to conclude that the viral polymerase complex contributes to the activation of HA-induced MAPK signaling. Influenza virus takes advantage of this event, in turn, to optimize viral growth. Our current data suggest that higher viral polymerase activity enhances the replication and transcription of viral RNA, which leads to greater expression of the viral HA protein and its accumulation on the cell surface late during virus replication. This in turn results in stronger ERK activation and thereby to more efficient nuclear RNP export and formation of infectious progeny virions. Understanding such a mechanism essential for influenza virus replication may also be a basis for the development of therapeutic implications, such as antiviral drug that reduces the polymerase activity leading to decreased HA-membrane accumulation and declined activation of the MAPK pathway.These results showed that HK/218449/06 (H3N2) influenza virus replicates more efficiently than HK/218847/06 (H1N1) subtype does. Infection with the H3N2 strain induced higher activation levels of the Raf/MEK/ERK (MAPK) signal cascade essential for virus replication. The previous study demonstrated the role of HA as an inducer of MAPK signaling causing enhanced nuclear RNP export at late time point of infectious cycle. Applying reverse genetic systems, we could show that the viral polymerase proteins (particularly PB1 and PB2) of the H3N2 influenza virus possess higher polymerase activity and that the PB1 protein of the H3N2 influenza virus contributes to the elevated HA-induced ERK activation, increased cytoplasmic RNP localization and higher virus titers.2. Human influenza viruses A/Hong Kong/218847/06 (H1N1) and A/Hong Kong/218849/06 (H3N2) were kindly provided by Dr. Malik Peiris (University of Hong Kong). We rescued the following viruses by reverse genetics (rg): rgH1N1, rgH3N2, and rgH1N1/H3N2-PB1. These five viruses were used to infect MDCK cells. Cells were washed with phosphate-buffered saline (PBS), infected at the indicated multiplicity of infection (m.o.i.), and further incubated as described previously [Human embryonic kidney cells (293T cells) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics. Madin-Darby canine kidney (MDCK) cells were kept in minimal essential medium supplemented with 10% FCS and antibiotics. All cells were cultivated at 37°C with 5% COeviously ,24.H1N1 and H3N2 IVAs were propagated in MDCK cells. RT-PCR using gene specific primers  was doneViral RNA was isolated directly from virus-containing supernatant by using an RNA isolation kit . The universal primer set for influenza A virus was used for RT-PCR . The Har50), we inoculated confluent monolayers of MDCK cells in a 96-well plate with 10-fold dilutions of influenza virus and incubated them at 37°C for 1 h. After inoculum removal, cells were washed with PBS and incubated for 72 h. A 50-μl sample of supernatant was drawn from each well, transferred to a new 96-well plate, and virus was titrated by HA test with a 0.5% suspension of chicken red blood cells. The TCID50 was calculated by the method of Reed and Muench [Confluent monolayers of MDCK cells in 35-mm dishes were inoculated with 10-fold dilutions of influenza virus (in DMEM with 3% BSA and antibiotics) and incubated at 37°C for 1 h. The inoculum was removed, and cells were washed with PBS and overlaid with MEM containing 1% agarose and 0.2% serum albumin. After 3 d at 37°C, cells were stained with 0.1% crystal violet in 10% formaldehyde solution, and plaque morphology was evaluated. Plaque size was measured using fine-scale magnifying comparator (6×). To determine the 50% tissue culture infecting dose (TCIDd Muench .O-tetradecanoyl-phorbol-13-acetate (TPA) (Sigma) at 4 h p.i.. U0126 (50 mM), a specific MEK inhibitor (Promega), was used to inhibit ERK activity as described previously [Activation of the Raf/MEK/ERK signal cascade was achieved by artificial stimulation of MDCK cells with 100 ng/ml 12-eviously .Cell lysate was cleared by centrifugation, and protein concentration was determined by Bradford assay before the protein was subjected to SDS-PAGE. Phosphorylated ERK (P-ERK) was detected with a specific monoclonal antibody (Santa Cruz Biotechnology). After stripping bound antibodies, we detected the total ERK2 using mAbs (Santa Cruz Biotechnology). Proteins recognized by mAbs were further analyzed with peroxidase-coupled, species-specific secondary antibodies and a standard enhanced chemiluminescence reaction (Amersham Biosciences). Quantification of specific bands was done with the PC-BAS software package (Fuji).MDCK cells grown on glass coverslips were infected and incubated as indicated below. The cells were washed with PBS at the indicated time points p.i. and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature (rt) for 30 min or at 4°C over night. Cells were permeabilized with 1% Triton X-100 (in PBS) at rt for 10 min. Then cells were incubated with a combination of the mouse anti-IVA NP mAb, clone AA5H (1:100) (Abcam) in PBS/3% bovine serum albumin (BSA) at rt for 1 h. The AlexaFluor488-coupled goat anti-mouse antibody (Invitrogen) was used as the secondary antibody. Cells were washed with PBS followed by double-distilled water and mounted with P-phenyldiamine (PPD) (Sigma) containing 500 nM TO-PRO-3 (Molecular Probes) for nuclear staining. Fluorescence was visualized with a multiphoton laser scanning microscope (Zeiss LSM 510 META). To analyze the expression of HA on the cell surface, cells were not permeabilized. The HA protein in infected cells was detected by anti-H1HA mAb  or by anti-H3HA (against HA of A/Mem/1/94) mAb  and AlexaFluor488-coupled goat anti-mouse antibody as secondary antibody.MDCK cells were infected with either HK/218847 (H1N1) or HK/218449 (H3N2) as indicated below. Cells were incubated for 4, 6, or 8 h. Then the cells were detached with trypsin, fixed in PBS/4% PFA, permeabilized with 1% Triton X-100, and stepwise incubated with FITC-conjugated mouse anti-NP mAb,  in PBS/3% BSA for 30 min on ice. Finally, the percentage of NP-expressing cells was determined by flow cytometry analysis using FACSCalibur (BD Biosciences). To analyze expression of HA on the cell surface, cells were not permeabilized. The HA protein in infected cells was detected by anti-H1HA mAb  or by anti-H3HA mAb  and AlexaFluor488-coupled goat anti-mouse antibody as secondary antibody.5 cells in 35-mm dishes) were transfected (Mirus Bio) with 2 μg luciferase reporter plasmid (EGFP open-reading frame in pHW72-EGFP substituted with luciferase gene [Subconfluent monolayers of 293T cells (7.5 × 10ase gene  and a miThe author(s) declare that they have no competing interests.HM performed most of the experiments and wrote the manuscript. HY and JF helped to generate the expression plasmids and designed the studies. HY, RW, SP and EH contributed to scientific ideas and analysis of the data.