Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed.We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of  Genetic elements, such as transposons and integrons, frequently carry antimicrobial resistance determinants and can be found widely disseminated among pathogenic bacteria. Their distribution pattern suggests dissemination through horizontal gene transfer. The role of natural transformation in horizontal transfer of genetic elements other than those that are self-replicative (plasmids) has remained largely unexplored. We have tested if natural transformation can facilitate transfer of transposons and class 1 integrons between bacterial species. We here provide experimental evidence showing that natural transformation can be a general mechanism for dissemination of genetic elements that by themselves do not encode interspecies transfer functions . We demonstrate that antibiotic resistance determinants present in such genetic elements can spread by natural transformation between species of clinical interest. We show by quantitative data that interspecies exchange of resistance gene cassettes is highly efficient among integron-containing strains and species. Our study also provides a plausible explanation for how sequence-conserved integrons can become distributed among bacterial species. The exposure of naturally competent A. baylyi cells to DNA from these sources led to the acquisition of novel resistance traits as well as entire integrons and transposons. Transposition-based integration occurred between unrelated hosts, whereas both transposition and homologous recombination facilitated acquisitions from related host species.Here we have investigated the potential for transposons, integrons and gene cassettes, supplied as fragmented DNA substrates, to move horizontally between bacterial species via natural transformation. Our model system relied on exposing the transposon- and integron-free and naturally-transformable bacterium A. baylyi were also used in subsequent transformation assays to confirm the nature of the initial interspecies gene transfer and determine further intraspecies transfer frequencies.Both the donor strains and the transformant isolates were characterized by antibiotic resistance profiling, targeted PCR, DNA sequencing by extensive primer walking, genome sequencing of two transformants, pulse-field gel-electrophoresis (PFGE) and Southern blot hybridization. A pairwise growth competition assay was undertaken to determine the impact of the acquired integrons on relative fitness. The integron-carrying transformants of A. baylyi BD413 (spontaneous rifampicin resistant mutant) A. baumannii 064, A. baumannii 65FFC, P. aeruginosa SM, all clinical isolates, S. enterica serovar Rissen 486 and S. enterica serovar Typhimurium 490, both isolated from pork processed food, and C. freundii C16R385, E. cloacae C2R371, E. coli C10R379, and E. fergusonii AS041A2 isolated from food-producing and wild animals  in the chromosome. The transformants yielded from exposure to DNA from these 3 latter strains were only characterized at the phenotypic level.The transformability of actamase . The NDMA. baylyi that were confirmed to have taken up the integron from the heterologous donor sources were also used as a source of donor DNA in subsequent transformation experiments  was also used as a recipient bacterium in subsequent transformation assays (see Some of the transformants of says see .A. baylyi BD413 strain employed as recipients (recBCD ΔsbcCD double mutant constructed as described by Harms and Wackernagel intI1::cat) and RAM (recA::cat) constructed as follows: an internal segment (724 bp) from the intI1 gene of A. baumannii 064 was PCR-amplified using the primers intI1-f (5′-AGCTTACGAA CCGAACAGGC-3′) and INCINTF (5′-TGATGCCTGC TTGTTCTACG-3′) and Phusion DNA polymerase , according to the manufacturer's instructions, and inserted into the SmaI site of pACYC177, resulting in pACYC177-int36. Next, a 1077 bp segment covering the cat (chloramphenicol resistance) gene from pACYC184 was amplified with primers cat-f (5′-CTCCGCTAGC GCTGATGTCC-3′) and cat-r (5′-GTAGCACCAG GCGTTTAAGG-3′) using Phusion polymerase and inserted into the singular PvuII site located in the intI1 segment of pACYC177-int36, resulting in pACYC177-int-cat. This plasmid was HincII-linearized and used to naturally transform A. baylyi SD2, giving strain KOI (verified by PCR). In parallel, an internal 938 bp segment of the recA gene of ADP1 was PCR-amplified with primers recA-f (5′-AGCAAGGCAT TACAAGCTGC-3′) and recA-r (5′-AATTCTGTAG AAATCTGAGG-3′) and Phusion and inserted into the HincII site of pUC19, giving pUC19-recA. The 1077 bp cat segment was cloned into the singular HincII site of pUC19-recA (located in the center of the recA fragment), resulting in pUC19-recA-cat which was XmnI-linearized and used to inactivate recA of A. baylyi SD2 by natural transformation to yield strain RAM (verified by PCR). The non-transformable strain KOC4 was constructed by transformation of BD413 by DNA from a strain carrying a ΔcomFECB::dhfr allele (trimethoprim-resistant) Other derivatives of the cipients  were SD9A. baylyi was cultivated in Luria-Bertani (LB) medium with rifampicin (R) 50 µg/ml; wild-type donor bacteria as well as transformants were grown, and selected for in LB supplemented with antibiotics according to their phenotype: ampicillin , cefotaxime , chloramphenicol , gentamicin , kanamycin , R (25 µg/ml) spectinomycin  and trimethoprim . A. baylyi cells and transformants were grown at 30°C, and the different donor bacteria at 37°C.Genomic DNA used in the transformation assays (10 µg) was isolated from bacterial cultures using anion exchange columns  columns according to the manufacturers protocol and resuspended in EB buffer, pH 8.5 . Plasmid DNA was isolated using a Plasmid Mini Kit . The DNA concentration was measured with a UV/VIS spectrophotometer  or a Nanodrop ND-1000 .For the preparation of supernatants (lysate) of the heat-treated bacterial cell suspensions, 5 ml overnight cultures of the bacteria were centrifuged at 20,000×g for 5 min and resuspended in water (50 µl), followed by heat treatment at 80°C for 15 min. The raw lysate was centrifuged, and the supernatant containing DNA was collected A. baylyi BD413 as recipient and DNA extracted from various wild-type integron-carrying species; ii) with A. baylyi BD413 as recipient and DNA extracted from integron-carrying A. baylyi transformants; iii) with integron-carrying A. baylyi transformant SD2 as recipient and DNA extracted from various wild-type integron-carrying species; iv) with an integrase deletion carrying A. baylyi transformant (KOI) as recipient and DNA extracted from various wild-type integron-carrying species; v) with an integron-carrying A. baylyi recA deletion (RAM) recipient and DNA extracted from various wild-type integron-carrying species; vi) with the double mutant strain A. baylyi SD9 (ΔrecBCD ΔsbcCD) as recipient; vii) and with competence mutant strain A. baylyi KOC4 (ΔcomFECB) as a recipient. Selection of the transformants was done with different antibiotics and concentrations . Both methods were performed according to the CLSI  guidelines IntI1The presence of class 1 integrons in the donor, recipient and transformant bacteria was assessed by PCR. PCR assays were set up in two different mixtures: 25 µl final volume of 22.5 µl PCR SuperMix , 0.75 µl of each primer 10 µM and 1 µl (approx. 10 ng DNA) of lysate DNA; or 50 µl final volume using 22.5 µl of the 2× PCR MasterMix Dynazyme II from Finnzymes , 0.75 µl of each primer 10 µM, 25 µl of sterile water and 1 µl (approx. 10 ng DNA) of lysate DNA. PCR amplification was performed with a T-personal , a MJ Mini  or a PTC-200  thermal cycler. Class 1 integrons were detected with a set of primers specific for the 5′-CS and the 3′-CS regions Rearrangements of gene cassettes during integron transfer and transformant cultivation were tested by PCR combining one primer for the 5′-CS region and one primer that binds in the distal gene cassette in the integron of the donor bacterium.aadB was determined by PCR with primers AADB1 (5′-ACGCAAGCACGATGATATTG-3′) and AADB2 (5′-CGCAAGACCTCAACCTTTTC-3′) for 5 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 55°C and 1.5 min at 72°C, followed by 10 min at 72°C.For some transformants, obtained with strains SD2, KOI and RAM as recipients, the presence of the gene cassette Aba1A. baylyi, in A. baumannii donors and in transformants with reduced susceptibility to ampicillin. For all PCR analyses, DNA extracted from the recipient and donor bacteria were used as negative and positive controls, respectively.Transformants with reduced susceptibility to some antibiotics but without a positive detection of the entire integron by PCR, were screened for the acquisition of different resistance determinants. PCRs were performed that targeted the gene cassettes present in the variable region of the integrons and the genes present in the 3′-CS of the integron of the corresponding donor bacteria, with specific primers for each cassette. The presence of the insertion sequence ISTransformants with reduced susceptibility to ampicillin, but that did not yield positive PCR products for the presence of integrons, were also tested for β-lactamase activity using a qualitative chromogenic method, with nitrocefin disks  according to the manufacturer's instructions. The randomly selected transformants showed reduced susceptibility to ampicillin; donors and recipient bacteria were used as positive and negative controls for β-lactamase production.A. baumannii 064 and S. enterica serovar Rissen 486, as well as the flanking genomic regions of donor and transformant bacteria were determined by direct sequencing of genomic DNA and primer walking using the BigDye 3.1 cycle sequencing terminator reactions (Applied Biosystems) and an ABI3130XL genetic analyzer, as previously described S. enterica serovar Rissen 486, VS1 (5′-CTGGCTGCGTAGTTGTTTCA-3′) and VS2 (5′-GGGCTGCGAGTTCAATAG-3′). The first primers used in integron flanking regions, CS3 (5′-TCTCTACGACGATGATTTACACG-3′) and CS2 (5′-CGAATGGACAGCGAGGAG-3′), were designed based on the conserved regions sequence of class 1 integrons (accession number M73819) and the flanking region sequences were obtained by primer walking, using the software Primer3 (http://fokker.wi.mit.edu/primer3/input.htm) and Oligoanalyzer . Sequences were edited and aligned in the Sequencher v.4.2.2 program  and identified using the BLASTN program (http://www.ncbi.nlm.nih.gov).The genetic composition of the integrons of two of the donor bacteria, http://clovr.org). Briefly, raw sequence data were filtered and trimmed for quality and adaptor removal, and assembled with Celera Assembler The genomes of transformants (St)3 and (AbII)3  were seqCeuI  that specifically recognize the rRNA operons CeuI fragments were separated in a 1% agarose by PFGE using a CHEF-DR III apparatus  at 15°C, 6 V/cm with pulse time ramped from 20 s to 120 s over 11 h, followed by ramping from 60 s to 100 s for 11 h  according to the manufacturer's instructions. Residual genomic DNA was removed by treatment with rDNase , followed by RNA ethanol precipitation. Reverse transcription was performed with 1 µg of RNA using the MonsterScript 1st-Strand cDNA synthesis kit with random 9-mer primers , and the resulting cDNA was used in PCR reactions. The primers HS464 and HS463a blaIMP-5 (transformant SD3); blaOXA-30+aadA1 ; dfrA12+aadA2 (transformant SD5); aacA4+PSEbla+aadA2 (transformant SD6); aadB . Selection was performed with CTX 20 µg/ml (transformant SD3), SC 10 µg/ml+AM 5 µg/ml , SC 20 µg/ml+W 250 µg/ml (transformant SD5), SC 20 µg/ml+AM 50 µg/ml (transformant SD6), or with K 25 µg/ml . Nineteen to 24 competition replicates were done for each transformant. The relative fitness (W) was calculated as the ratio of the Malthusian parameter of each competitor.The relative fitness (W) of integron-carrying transformants was estimated by pairwise competition experiments between transformants and the untransformed recipient strain in S2-minimal medium for 24 h, as previously described A. baylyi BD413 does not carry identifiable integrons A. baylyi, the composition and genomic context of all integrons in the donor genomes was assessed by integron-specific PCR, and DNA sequencing by primer walking. PCR with primer pairs specific for the amplification of integron gene cassettes yielded a single product, which in all cases, upon sequencing, contained at least one known antibiotic resistance gene. The direct sequencing of each PCR product indicated the presence of a single type of integron in each donor genome. In A. baumannii 064, the integron harbored a central region of 763 bp, including the aadB gene (CDS 597 bp), which is known to encode an aminoglycoside adenyltransferase, responsible for gentamicin, kanamycin and tobramycin resistance. The integron of S. enterica serovar Rissen 486 contained a central region of 1913 bp, with two gene cassettes: dfrA12 (CDS 498 bp), and aadA2, (CDS 792 bp). The dfrA12 gene encodes a dihydrofolate reductase that confers resistance to trimethoprim, and the aadA2 gene encodes an aminoglycoside adenyltransferase, responsible for streptomycin and spectinomycin resistance.The recipient strain intI1 and 16S rRNA probes was interpreted as indicating chromosomal location, whereas hybridization only with the intI1 probe was interpreted as indicating a plasmid location of the integron. A plasmid location of the integron was shown in the donors S. enterica serovar Typhimurium 490, E. cloacae C2R371 and C. freundii C16R385, while integrons in the A. baumannii 064, S. enterica serovar Rissen 486, E. coli C10R379, A. baumannii 65FFC P. aeruginosa SM A. baumannii 064 and S. enterica serovar Rissen 486 strains both contain a plasmid, as observed by agarose gel electrophoresis (data not shown). E. fergusonii AS041A2 also harbored a plasmid and repeated PFGE results were not conclusive for the determination of the location of the integron in this strain.The genomic location of the integrons in the donor genomes was determined by PFGE. Co-hybridization of the S. enterica serovar Typhimurium 490, the integron was inserted in an intact Tn21-like transposon A. baumannii 064 was inserted in a Tn1721-like transposon 26 on both sides, forming an IS26-composite transposon. The A. baumannii 65FFC integron was embedded in a defective Tn402-like transposon, flanked by a 439 bp Miniature Inverted-repeat Transposable Element (MITE) on both sides Sequencing of the flanking regions of the integrons revealed that their insertion sites varied in the donors, and that the integrons were often linked to transposable elements. In S. enterica serovar Rissen 486, the tnpA, tnpR and tnpM genes were detected next to the 5′-CS region of the integron. The chrA gene, which codes for a putative truncated chromate ion transporter, was found flanking the 3′-CS region. The closest homologue to the tnp genes was found in the transposon Tn1721, suggesting the integron in serovar Rissen 486 may be localized inside a transposon. However, for technical reasons, we were unable to obtain additional DNA sequence by primer walking.In P. aeruginosa SM, tnpR and tnpM genes were identified adjacent to the 5′-CS flanking region of the integron. The genes flanking the 3′-CS region could not be determined. In this case, the tnp genes were 100% identical to the genes found in the Tn5051-like transposon; also in this case indicating that the integron is located in a transposon.In C. freundii C16R385, E. cloacae C2R371, E. coli C10R379 and E. fergusonii AS041A2 were not determined.The flanking regions of the integrons of A. baylyi BD413 by integron-containing DNA from the related species A. baumannii occurred at frequencies up to 1.6×10−7 transformants per recipient over a 24 h period. Natural transformation was also seen after exposure to integron-containing DNA of strains from the unrelated bacterial species C. freundii, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, S. enterica serovar Rissen, and S. enterica serovar Typhimurium, at frequencies ranging from 4.4×10−9 to 2.1×10−7 (A. baylyi BD413 above the detection limit of <1 transformant per 109 bacteria were two Escherichia isolates (E. coli C10R379 and E. fergusonii AS041A2). In addition to purified DNA, the supernatant of a heat-killed bacterial cell suspension also gave rise to bacterial transformants 4 and (AbII)L1]; however, the molecular basis for the resistance was not determined. See footnote of table S1 for an explanation of the abbreviations of transformants.Transfer of the insertion sequence ISS. enterica serovar Typhimurium 490 , and another four transformants the entire integron from A. baumannii 064 , as confirmed by PCR (catA2). A. baylyi transformants.Among all the phenotypes with increased resistance observed after exposure of the BD413 strain to DNA extracted from various integron-carrying species, four transformants were shown to have acquired the complete integron from d by PCR  and DNA S. enterica serovar Typhimurium 490, the incorporated DNA region was found to consist a copy of transposon Tn21-like, which also contained the integron. In both cases, target site duplications of 4 and 5 bp, respectively, were identified providing evidence for that the (St)1 and SD1 transformants resulted from active transposition of Tn21-like (merR gene of the transposon. In transformant (St)1, the integron-containing transposon had inserted into the A. baylyi gene ACIAD1773, which encodes a putative transport protein permease (In the transformants (St)1 and SD1, obtained after exposure to DNA of n21-like . The trapermease . In tranpermease .A. baylyi transformant (St)3 revealed that the integron had transferred as part of a Tn21-like transposon. The incorporated DNA was found in the transformant genome in a region containing a 54 kb stretch of DNA not present in the published A. baylyi ADP1 strain. This DNA stretch has not been described before, and appears to be a prophage region, inserted in the ACIADtRNASer_34 gene. The transposon present in transformant (St)3 also showed rearrangement compared with its donor, S. enterica serovar Typhimurium 490. In (St)3, the tnpR, tnpA, ybjA, catA1 and IS1 genes were located at the 3′-CS region, flanking the mer operon, whereas in the previously described Tn21 sequences, they are located in the 5′-CS region 2 acquired the integron embedded in the TnA. baylyi transformant SD2, obtained after exposure to A. baumannii 064 DNA, also acquired considerable stretches of genomic donor DNA flanking the complete integron  recombination had facilitated the acquisition of the integron-containing DNA, a process recently investigated in detail for A. baumannii donor and A. baylyi recipient cells The integron . ExtensiA. baylyi transformant (AbII)3 revealed that it had acquired approximately 18,625 bps of DNA sequence from the A. baumannii 064 genome, which contained a IS26-composite transposon (tet (tetracycline resistance) gene and a tnpA transposase gene (74% similar to the tnpA gene of Tn1721), and further flanked on both sides by the IS26 elements. A target site duplication of 8 bp on both sides of the IS26 insertion suggests transfer of the entire 18,625 bp segment occurred by transposition facilitated by IS26 sequences, into the ACIAD0480 gene of A. baylyi (encoding a putative membrane protein). Analysis of additional transformants, exposed to DNA of A. baumannii 064 by primer walking allied with PCR confirmed that the integrons acquired by transformants (AbII)1 and (AbII)2 were also located within a Tn1721-like transposon, and flanked by a IS26 element. Although the exact locations in the A. baylyi genome were not determined for the latter, the obtained DNA sequence suggests a similar composition and mode of transfer as for transformant Ab(II)3.Whole genome sequencing of the ansposon ; the horS. enterica serovar Typhimurium 490 or A. baumannii 064. Although plasmids were clearly identified in both of the donor species by agarose gel-electrophoresis (data not shown) or PFGE, the same plasmids could not be detected in the transformants (intI1 or the 16S rRNA genes did not take place for DNA extracted from transformants (St)1 and (St)2. Chromosomal location is nevertheless assumed because the larger fragment, where the integron of these two transformants is located, is not expected to hybridize with the 16S rRNA probe even though it is chromosomal. This is due to the opposite orientation of the rRNA operons at nucleotides 1660700–1666022 and 2941950–2947363 (access. number CR543861), leading to a 1.28 MB I-CeuI fragment without the 16S rRNA gene. A. baylyi BD413 has seven rRNA operons, producing seven fragments after digestion with I-CeuI A. baylyiA. baylyi genome The possible transfer of whole plasmids was investigated in the transformants collected after exposure to DNA from the plasmid-harboring formants . The trarecBCD operon  protects transformable cells from transposon activity initiated by linear double-stranded DNA present in the cytoplasm.As several of the observed interspecies integron transfers appeared to have taken place through transposition, a transient existence of a linear double-stranded (ds) DNA intermediate in the cytoplasm was assumed. Such intermediates are thought to be substrates for the dsDNA-attacking exonuclease RecBCD A. baylyi BD413 by DNA extracted from A. baylyi transformants that previously acquired an integron  was performed. Intraspecies transformation was 10- to 1,000-fold more efficient than interspecies transfer of the same integrons 1, (SD1)2 and (SD1)3, obtained with the donor strain A. baylyi SD1, acquired at least 23,500 bp of the donor DNA. Transformant isolates (SD2)1, (SD2)2 and (SD2)3, obtained with the donor A. baylyi SD2, obtained at least 20,000 bp of the donor DNA. The exact size of the recombining regions for these isolates remains undetermined as the donor and recipient genomes have regions with identical DNA composition in the areas flanking the DNA insertion. Extensive DNA sequencing by primer walking of 6 transformants suggested that the composition of the donor DNA segments acquired in the initial interspecies transformation assay were maintained after subsequent intraspecies transformation  led to efficient replacement of gene cassettes rather than to the accumulation of additional integrons or gene cassettes in all tested transformants (76 out of 76)  lost the corresponding aminoglycoside resistance profiles upon transformation by genomic DNA containing different integrons  . Replacet of 76)  and the t of 76) . Recipientegrons . Gene lontegrons . Rearrananalyses , which mrecA mutant RAM were between 104- to 105-fold lower when compared to the recA-proficient recipient strain SD2  harboring a functional integrase gene , (St)3, (SD1)1 and SD4 (blaOXA-30+aadA1), SD5 (dfrA12+aadA2), SD6 (aacA4+PSEbla+aadA2), and SD2 and (SD2)1 (aadB). The mean relative fitness (w) of these integron-carrying strains ranged from 0.96 to 1.01 (n = 19 to 24 replicates); only transformant SD3 showed a clear negative fitness effect, with w = 0.91 . In general, no consistent major differences in fitness were observed, suggesting that the horizontal acquisitions of integrons  do not lead to immediate and severe growth inhibition of transformant cells.The biological cost of the acquired, integron-mediated, antibiotic resistance was determined for transformants SD3  is a key driver of bacterial adaptation and evolution Annealing of complementary DNA strands in the cytoplasm is considered necessary for recircularization and hence stable uptake of plasmids in competent bacterial species A. baylyi to DNA of an integron-containing Salmonella strain led to horizontal transfer of its integron due to transposition of a Tn21-like transposon that contained the integron. The Tn21-like transposon was found as single inserts at different chromosomal locations in 3 different examined transformant genomes. Exposure of A. baylyi to DNA of an integron containing A. baumannii strain also led to transformants with single chromosomal integrations of the integron. The integrations occurred by either DNA sequence similarity-based homologous recombination, or by genetic linkage to and movement of IS26 elements. Resistance profiling, PCR-based target amplification and extensive DNA sequencing confirmed the acquisition and chromosomal insertion of complete integrons and its flanking DNA by A. baylyi transformant cells. The general acquisition process involved is illustrated in Indeed, we provide experimental data demonstrating that natural transformation can facilitate interspecies transfer of integrons and transposons that is not limited by the genetic relatedness of the donor with the host. Exposure of A. baumannii 064 and S. enterica serovar Typhimurium 490 transformed A. baylyi cells at frequencies of 10−8 and 10−7 over a 24 h time period. In all examined cases, the composition and order of gene cassettes in the donor bacteria was maintained in the transformant cells. Also DNA lysates could transform A. baylyi cells suggesting variability in DNA purity is of minor importance for HGT, as observed previously Purified DNA substrates of A. baylyi BD413 chromosome. The genomic locations of the integrons were also maintained in subsequent transformants (n = 6) obtained after exposure to DNA isolated from the initial transformants. Sequencing of the flanking regions of the transferred integrons also revealed that not only the integron was acquired from the donor bacteria, but also additional DNA flanking the integrons. The length of the acquired, continual DNA fragments could be up to 23,000 bps long. The sequencing of the acquired DNA in 6 transformants obtained after exposure to DNA of various species, as well as Southern hybridization of a total of 8 transformants, revealed that integration had occurred in different regions of the A. baylyi genome.DNA sequencing of the flanking regions of the integrons in 6 initial transformants obtained after exposure to species-divergent donor DNA verified their incorporation into the A. baylyi chromosome had occurred by several mechanisms, depending of the flanking regions of the integrons. Transposition-based insertions were observed for integrons acquired from unrelated species. Specifically, DNA sequencing revealed that exposure to DNA substrates of S. enterica serovar Typhimurium 490 resulted in chromosomal integration of the acquired DNA due to the activity of transposase genes flanking the integrons; such as transposon Tn21-like. Integrons are often embedded in transposon Tn2121-like transposons A. baumannii DNA donor), homologous recombination-based insertions were observed, as expected 26-associated transposition was observed.Most interestingly, stable integration of the integron containing DNA in the 21-like, IS26 elements are often associated with antibiotic resistance genes and presumed active in Acinetobacter26-associated integrons 26-composite transposons has been suggested 26 could also allow the capture of additional resistance genes by homologous recombination-mediated DNA integration, if a homologous IS26 region is present in the donor DNA fragments. Such recombination scenario could explain, for example, the dissemination of the chromosomally-located integron-IS26 in A. baumannii in South Korea Similarly to TnA. baylyi ADP1 strain harbors two 2 prophages, a number of transposases, and 6 copies of IS1236, with two of them flanking Tn5613The biological properties of natural transformation as a mechanism that can facilitate DNA translocation over bacterial membranes and site-specific recombination (e.g. active transposition events of transposons) in bacterial genomes could be further utilized. For instance, both model and applied systems can be developed using small DNA fragments with site-specific recombination functions as donor DNAs. Moreover, possible interactions between recombinases encoded by the incoming DNA and host enzymes should be investigated. For example, the A. baylyi cells to the various DNA substrates resulted in surprisingly high transformation frequencies, as determined by phenotypic screening on antibiotic-containing growth media. However, subsequent genotypic screening (PCR) showed that only some of the transformants with changed antibiotic resistance profiles had acquired entire integrons. The observation of variable changes in susceptibility patterns, after exposure to various sources of bacterial DNA, suggest the broad potential of and the presence of a yet not described range of transferable resistance determinants in bacteria, of unknown mechanistic and genetic nature. Our study suggests that exposure of competent bacteria to heterologous sources of DNA may produce complex changes in resistance profiles, not necessarily predictable from the known resistance genes present in a given donor isolate. In two examined cases, changes in resistance profile were due to the acquisition of β-lactamases as determined by the nitrocefin test. The broader effects of the composition of the A. baylyi genome after the heterologous recombination with DNA from the related species A. baumannii will be determined in a separate study. A recent study by Mell et al.Haemophilus influenza genomes after natural transformation.The exposure of A. baylyi SD2 recipient with DNA isolated from different species of donor bacteria gave comparable transformation frequencies to those obtained with DNA of the same species. Thus, interspecies acquisitions of integrons that take place at low initial frequencies can be followed by high-frequency interspecies dissemination. Interestingly, all tested transformants acquired the gene cassette composition of the integron of the donor bacterium, rather than acquiring additional new gene cassettes in the existing integron. This result suggests that homologous recombination replaces gene cassettes through gene replacement caused by crossover junctions forming at the conserved end segments of the integron  that resulted four orders of magnitude reduction in transformation frequencies, confirm the role of homologous recombination Pseudomonas aeruginosa to have occurred by homologous recombination. Here we provide experimental evidence that homologous recombination can efficiently replace gene cassette arrays. Mobile genetic elements thus provide sufficient DNA similarity for homologous recombination to occur between otherwise unrelated bacterial species. The presence of DNA similarity can therefore likely contribute to the formation and spread of chromosomally-located complex mosaic regions, often formed by several resistance genes and mobile elements Transformation experiments performed with recipient cells containing an integron with the integrase gene inactivated (strain KOI), and also with recipient cells containing an integron, but with the lexA alleles, SOS induction can lead to increased integrase activity resulting in increased frequencies of cassette rearrangements V. cholerae. In contrast, and unlike many Enterobacteriaceae, the SOS response in A. baylyi is not regulated by a LexA orthologue Recent publications demonstrate that many integrase genes in integrons carry LexA binding sites in the vicinity of their promoter regions and can be controlled by the host LexA protein  in V. cholerae). The relative low frequency of integrase-facilitated cassettes recruitment and rearrangements, compared with the frequency of homologous recombination, can also help explain why some gene cassette arrays are common and shared between different bacterial species The absence of detected rearrangements of gene cassettes in our study might be explained by the absence (or low) expression of the integrase in the recipient Intervention policies aimed at combating antibiotic resistance often rely on the assumption that resistance genes are costly and will be lost from pathogenic bacterial populations when antibiotic pressure is removed E. coli, A. baumannii and Pseudomonas stutzeri, regarded as non-competent have later been shown to be naturally transformable under certain circumstances P. aeruginosaPseudomonas species Vibrio species, which are naturally transformable Natural competence is described in at least 60 species of bacteria, including several species present in clinical environments Our results indicate that natural transformation of DNA fragments is not necessarily limited by requirements of high DNA sequence similarity for stable integration to occur; and that genetically unrelated bacteria can exchange genetic material (such as integron-containing transposons) through natural transformation provided the transferred DNA fragments encodes functions for site-specific recombination. As observed in our studies, the genetic signature of events of natural transformation can be limited to the site-specific insertions of transposons. However, the observation of transposons or insertion sequences in a given bacterial genome is usually not causally attributed to events of natural transformation. Thus, retrospective DNA sequence analysis would not have considered natural transformation as the causal mechanism until now.This study suggests a significant potential for the interspecies spread of mobile genetic elements such as transposons, and insertion sequences through natural transformation. Hence, presenting a new pathway for horizontal dissemination of antimicrobial resistance determinants present in integrons embedded in transposons.aadA2 486S (5741162), ampC (2880712), dfrA12 (5741160), intI1 (11934322) recA (2879476), recB (2879477), recC (2879478), recD (2879479), lrp (2878604), sbcC (2879936), sbcD (2879937), 16S rRNA (2880271) GeneID and blaIMP-5 (AF290912), blaOXA-30+aadA1 (AY534545), aacA4+blaPSE+aadA2 (DQ219465), blaVIM-4+arr-7+aacA4+blaPSE-1 (FN397623), ISAba1 (AY758396), MITE (JF810083), Tn21-like (AM991977) and Tn5051-like (AJ867812) accession number from the NCBI GenBank database. The IS26-composite transposon was submitted to GenBank with the accession number JX041889.The reference numbers for genes mentioned in the text include: ACIAD0480 (2881081), ACIAD1773 (2878997), ACIAD3230 (2879415), ACIADtRNASer_34 (2877868) Figure S1Agarose gel electrophoresis of class 1 integron PCR-products. A) Lane 1 – A. baumannii 064 (donor strain); 2–5 – transformants from A. baumannii 064, 4.10.1, 4.31.1, 4.34.1 and 4.46.1, respectively; 6 – S. enterica serovar Typhimurium 490 (donor strain); 7–10 – transformants from S. enterica serovar Typhimurium 490, 2.1.1, 2.3.6, 2.24.5 and 2.26.1, respectively; 11 – A. baylyi BD413 (recipient strain); 12- 1 Kb Plus DNA ladder (Invitrogen). B) Lane 1 – 1 Kb Plus DNA ladder (Invitrogen); 2 – A. baumannii 65FFC (donor strain); 3–5 – transformants from A. baumannii 65FFC, SD3, [SD2](AbI)1 and [SD2](AbI)2, respectively; 6 – C. freundii C16R385; 7–9 – transformants from C. freundii C16R385, [SD2](Cf)1, [SD2](Cf)2, [SD2](Cf)3, respectively; 10 – E. cloacae C2R371; 11–13 – transformants from E. cloacae C2R371, [SD2](Ecl)1, [SD2](Ecl)2, [SD2](Ecl)3, respectively; 14 – E. coli C10R379; 15–17 – transformants from E. coli C10R379, [SD2](Ec)1, [SD2](Ec)2, [SD2](Ec)3, respectively; 18 – transformant SD2 (recipient bacterium); 19 – A. baylyi BD413 (negative control); 20 – 1 Kb Plus DNA ladder (Invitrogen); 21 – E. fergusonii AS041A2; 22–24 – transformants from E. fergusonii AS041A2, [SD2](Ef)1, [SD2](Ef)2, [SD2](Ef)3, respectively; 25 – P. aeruginosa SM (donor strain); 26–28 – transformants from P. aeruginosa SM, SD6, [SD2](Ps)1, [SD2](Ps)2, respectively; 29 – S. enterica serovar Rissen 486 (donor strain); 30–32 – transformants from S. enterica serovar Rissen 486, SD5, [SD2](Sr)1, [SD2](Sr)2, respectively; 33 – S. enterica serovar Typhimurium 490 (donor strain); 34–36 – transformants from S. enterica serovar Typhimurium 490, SD4, [SD2](St)1, [SD2](St)2, respectively; 37 – transformant SD2 (recipient bacterium); 38 – A. baylyi BD413 (negative control). C) Lane 1 – transformant SD1 (donor strain); 2–4 – transformants from SD1, (SD1)1, (SD1)2, (SD1)3, respectively; 5 – transformant SD2 (donor strain); 6–8 – transformants from SD2, (SD2)1, (SD2)2, (SD2)3, respectively; 9 – transformant SD3; 10–12 – transformants from SD3, (SD3)1, (SD3)2, (SD3)3, respectively; 13 – transformant SD4; 14–16 - transformants from SD4, (SD4)1, (SD4)2, (SD4)3, respectively; 17 – transformant SD5; 18–20 – transformants from SD5, (SD5)1, (SD5)2, (SD5)3, respectively; 21 – transformant SD6; 22–24 – transformants from SD6, (SD6)1, (SD6)2, (SD6)3, respectively; 25 – A. baylyi BD413 (recipient strain); 26 – 1 Kb Plus DNA ladder (Invitrogen). D) Lane 1 – 1 Kb Plus DNA ladder (Invitrogen); 2 – A. baumannii 65FFC (donor strain); 3–5 – transformants from A. baumannii 65FFC, [KOI](AbI)1, [KOI](AbI)2, [KOI](AbI)3, respectively; 6 – P. aeruginosa SM (donor strain); 7–9 – transformants from P. aeruginosa SM, [KOI](Ps)1, [KOI](Ps)2, [KOI](Ps)3, respectively; 10 – S. enterica serovar Rissen 486 (donor strain); 11–13 – transformants from S. enterica serovar Rissen 486, [KOI](Sr)1, [KOI](Sr)2, [KOI](Sr)3, respectively; 14 – S. enterica serovar Typhimurium 490 (donor strain); 15–17 – transformants from S. enterica serovar Typhimurium 490, [KOI](St)1, [KOI](St)2, [KOI](St)3, respectively; 18 – transformant KOI (recipient bacterium); 19 – A. baylyi BD413 (negative control).(TIF)Click here for additional data file.Figure S2Schematic presentation of the horizontal acquisitions. A) Acquisition of a transposon by transposition with duplication of sequences around insertion site; B) Acquisition or substitution of gene cassettes by homologous recombination occurring between conserved regions of a class 1 integron.(TIF)Click here for additional data file.Figure S3PFGE and Southern blot hybridization. A) Pulse-field gelelectrophoresis (PFGE) of I-CeuI-digested total DNA of recipient, donor and transformant bacteria; B) corresponding Southern hybridization with 16S rRNA probe; C) corresponding Southern hybridization with intI1 probe. Lane 1 – lambda PFG marker (New England Biolabs); 2 – A. baylyi BD413 (recipient); 3 – S. enterica serovar Typhimurium 490 (donor); 4–7 – transformants from exposure to DNA of S. enterica serovar Typhimurium 490, (St)1, SD1, (St)2, (St)3, respectively; 8 – A. baumannii 064 (donor); 9–12 – transformants from exposure to DNA of A. baumannii 064, SD2, (AbII)1, (AbII)2 and (AbII)3, respectively; 13 – S. enterica serovar Rissen 486 (donor); 14 – E. cloacae C2R371 (donor); 15 – E. coli C10R379 (donor); 16 – C. freundii C16R385 (donor); 17 – E. fergusonii AS041A2 (donor); 18 – lambda PFG marker (New England Biolabs).(TIF)Click here for additional data file.Figure S4RT-PCR of the class 1 integrase RNA in transformant A. baylyi SD2. Lane 1 (left) – SmartLadder (Eurogentec); 2–3 –PCR targeting the intI1 gene, 2 – cDNA; 3 – RNA; 4–5 – PCR targeting the 16S rRNA gene, 4 – cDNA; 5 – RNA. For primers description, see Material and Methods.(TIF)Click here for additional data file.Table S1Antimicrobial susceptibility of A. baylyi transformants determined by the E-test method.(DOC)Click here for additional data file.


Adaptive evolution is characterized by positive and parallel, or repeated selection of mutations. Mouse adaptation of influenza A virus (IAV) produces virulent mutants that demonstrate positive and parallel evolution of mutations in the hemagglutinin (HA) receptor and non-structural protein 1 (NS1) interferon antagonist genes. We now present a genomic analysis of all 11 genes of 39 mouse adapted IAV variants from 10 replicate adaptation experiments. Mutations were mapped on the primary and structural maps of each protein and specific mutations were validated with respect to virulence, replication, and RNA polymerase activity. Mouse adapted (MA) variants obtained after 12 or 20–21 serial infections acquired on average 5.8 and 7.9 nonsynonymous mutations per genome of 11 genes, respectively. Among a total of 115 nonsynonymous mutations, 51 demonstrated properties of natural selection including 27 parallel mutations. The greatest degree of parallel evolution occurred in the HA receptor and ribonucleocapsid components, polymerase subunits  and NP. Mutations occurred in host nuclear trafficking factor binding sites as well as sites of virus-virus protein subunit interaction for NP, NS1, HA and NA proteins. Adaptive regions included cap binding and endonuclease domains in the PB2 and PA polymerase subunits. Four mutations in NS1 resulted in loss of binding to the host cleavage and polyadenylation specificity factor (CPSF30) suggesting that a reduction in inhibition of host gene expression was being selected. The most prevalent mutations in PB2 and NP were shown to increase virulence but differed in their ability to enhance replication and demonstrated epistatic effects. Several positively selected RNA polymerase mutations demonstrated increased virulence associated with >300% enhanced polymerase activity. Adaptive mutations that control host range and virulence were identified by their repeated selection to comprise a defined model for studying IAV evolution to increased virulence in the mouse. The mutational basis for the control of host switching (host-specific infection) and virulence (disease severity) in influenza A viruses (IAV) or their interrelationship is poorly understood IAV are enveloped with genomes composed of 8 negative sense RNA segments encoding 11 proteins . IAV rep50 values of ≤103.5 pfu, Mammalian IAV species originate from the migratory aquatic bird reservoir of avian influenza viruses through processes that include reassortment of genome segments and adaptation of constituent genes Adaptive evolutionary theory states that phenotypic variation and speciation is explained by the selection of biological variants that function to increase replicative fitness 5 infectious viruses because populations of this size possess all single nucleotide polymorphisms . In addition parallel evolution of drug resistance occurs in the M2 ion channel and neuraminidase (NA) where S31N and H274Y mutations are diagnostic of adamantane Although phylogenic studies of humans and canine IAV show abundant variation, evidence of positive selection is generally lacking with nonsynonymous to synonymous mutation ratios (dN/dS) of <1 demonstrating stochastic variation Although IAV are host restricted such that high dose intranasal infection of mice with human IAV does not typically result in disease, virulent MA variants that cause primary viral pneumonia at low dosage can be selected by serial mouse-lung passage reviewed . The mou5 fold (reduction in LD50 from >107.7 to 102.7 plaque forming units (pfu)) −8 and P≤6×10−7, respectively, see methods). Sequence analysis of the NS1 gene of 42 MA variants of A/HK/1/68 identified 11 mutations with parallel evolution detected at position 106 (M106I and M106V) Mouse adaptation of A/HK/1/68 (H3N2) (HK-wt) by 20 serial passages in mouse lungs increased virulence by >10In this paper we extend our previous studies of HK-wt mouse adaptation by performing full genome sequencing of all 10 parental and 39 MA variants derived from 10 independent mouse-adaptation experiments in order to derive genetic and protein structural maps of adaptation to high virulence in the mouse. We identified adaptive regions within individual viral genes that included multiple instances of positive selection and parallel evolution.50 values of 105.4 to 103.6 pfu from 7.7 pfu for HK-wt, . After r HK-wt,  and 3 wi50 values for 7 of the 27 MA21 variants ranged from 101.1 to 106.5 pfu  possessing mutations; all were single mutations except one double mutation . Paralle++ ions in The PA protein was highly adaptive with 24 of 27 MA21 clones (89%) possessing 1 or 2 mutations . ConsideThe NP protein was mutated in 100% of MA clones with parallel evolution seen for D34N and D290N/E mutations in 8 of 9 HKMA21 populations and 85% of the variants (23 of 27),  and S5. 1, 2101, 2181, and HA2 1542) with 6 more showing positive selection . The mutations were clustered in 2 regions of the primary and 3D maps. One region in HA1  MA21 clones  and 2. PM1 and M2 are overlapping genes encoded in different reading frames. Both were highly conserved among MA21 clones; with 3 and 4 variants, respectively  and 2. MNS1 and NEP are also overlapping genes encoded in different reading frames but were more variable on mouse adaption than the M1 and M2 proteins. Four NEP mutants were observed among the MA21 clones and one in MA20B  and S8 tThe NS1 proteins possessed 8 mutations among 11 MA21 variants occurring as single or double mutations  that hav50 of HK-wt is >107.7 pfu 50 of each mutant virus was >5×106 pfu (Recombinant HK-wt (rHK-wt) and mutant viruses that differed from HK-wt due to each of the parallel PB2 mutations, K482R, D701N, D740N, as well as D701N+D740N were generated using reverse genetics. We assessed the ability of each of these combinations of PB2 mutations to cause disease in groups of mice that had been infected with each virus and monitored for weight loss and lethality. Because the LD×106 pfu . The D70×106 pfu .50>5×106 pfu except for D34N (LD50 = 2.7×106 pfu) that also caused a significantly reduced time to death relative to HK-wt (≤0.05 by t-test). All of these mutants enhanced disease severity as monitored by weight loss  . All mutants had an LDmutants) . Lethali5 pfu of each virus showed that the PA Q556R mutation resulted in significantly increased body weight loss (P≤0.001 by paired t-test) on the HK-wt backbone and a decreased time to death (P≤0.05 by t-test) in the MA20 virus relative to rMA20 + HK PA . NP mutations D34N, D209E, and PA Q556R did not significantly increase yields relative to rHK-wt at this time point  and NA proteins. In this study, we provide evidence for sites of natural selection in all of these genes . We obseD740N)X11KRIR-755; PB1 187RKR(R190K)VRDNMTKKMVTQRTIGKRKQR211; NP 1MAS(Q4K)GTKRSYxxM13; and NEP S23P in NES (11DILLRMSKMQLE(S23P)) (12)KRIR-755) to result in unfolding of the PB2 NLS structure, )  and 6  to ructure, . The PB2Both the PB2 and PA proteins demonstrated adaptive mutations in their respective cap binding domains, suggesting that increased ability to access host mRNA cap complexes for priming viral transcription is important for overcoming restricted viral replication in a new host. The N-terminal PA cap-binding domain also possesses RNA endonuclease, transcription, replication and protein stability functions that may be affected by mutations in this domain Influenza viruses replicate through the action of 11 genes that interact extensively with each other and with host proteins 1W/E and α2,3 sialic acid The NS1 protein is a multifunctional protein that binds RNA and protein factors to antagonize IFN functions and modulate infection 1S, Q2101R, G2181W) have been shown to increase the pH of fusion (except T1562N) and all of these were associated with increased mouse cell infection and virulence as a function of increased mouse-lung tropism and replication 2W mutation increases α2–3 sialic acid (SA) binding, consistent with the fact that α2–3 SA linkages are the only form of SA present in the mouse respiratory tract 1 and 1542 glycosylation sites (that were both lost due to adaptive mutations) suggests that differences in host mediated glycosylation may also be contributing to HA adaptive evolution  as outlined in the Care and Use of Experimental Animals, Vol.1, 2nd Edn. (1993), which are recognized as “best-practices” by the International Council for Laboratory Animal Science (ICLAS). The protocol was approved by the University of Ottawa Animal Care Committee (Protocol Number: BMI-85). Animal studies were also performed under the supervision of a veterinarian (DVM) and trained personnel. All efforts were made to minimize suffering and mice were euthanized at humane end-points, if infection resulted in greater than 25% body weight loss plus respiratory distress.Madin-Darby canine kidney cells (MDCK)  were maintained in autoclavable minimum essential medium (MEM) with Earle's salts, and both 293T human embryonic kidney cells  and mouse B82 fibroblasts  were maintained in Dulbecco's MEM . Media were supplemented with L-glutamine (2 mM), Penicillin (100 U/ml), Streptomycin (100 ug/ml)  and fetal bovine serum (FBS) (10%) .5 pfu HK-wt stock in 50 µL PBS was inoculated intranasally into each of 3, 20 g CD-1 strain mice under halothane anesthesia , that were housed for 3 days and euthanized by CO2 narcosis and surgical removal of lungs. Lungs were pooled in 3 ml PBS and a virus extract prepared by sonication for 2 minutes on ice, before sedimentation of debris by centrifugation at 250 G for 5 minutes. Virus extracts were diluted 10 fold in PBS before inoculating another group of CD-1 mice with 50 µL each with repeated passage for a total of 20 serial passages  with serial passage of undiluted lung extract into individual mice for a total of 10 passages before 5 serial undiluted passages of 2 days each in groups of 2 mice, followed by 6 serial undiluted passages of 3 days duration in groups of 2 mice. In each of these passages, infected lungs were suspended in 1 ml PBS each to make extracts that were sterilized using 0.22 µM Millipore Millex-GV PVDF  filtration before serial passage that involved infection with 50 µL of lung extract. All MA variants derived from the 9 independent mouse-adapted passage 21 populations clones were designated as HK(clone #) MA (mouse passage #)-(clone #).The prototype clinical isolate A/Hong Kong/1/68 (H3N2) (HK-wt) was obtained from the Laboratory Center for Disease Control, Health Canada, Ottawa that was originally obtained from H.G. Pereira . The passage history of HK-wt was: (2 passages in rhesus monkey kidney cells) +  before 2 plaque purifications (plaque-to-plaque) on MDCK cells before seed and stock preparation in chicken allantoic cavity. The generation of mouse-adapted clones was described previously Virus stocks were subjected to serial 10 fold dilution in PBS before application to PBS washed MDCK cells monolayers in 6 well plates as described previously 50) of IAV variants were determined in groups of CD-1 mice as described previously 2) with undiluted stock virus and serial 10 fold dilutions made in PBS. Mice were monitored for survival and weight loss over a 2 week period or until body weight increased. Mice that lost >25% body weight and were in respiratory distress where considered to have reached “humane endpoint” as required by our animal care protocol and were euthanized by CO2 narcosis. The median lethal dose (LD50) was determined using the Karber–Spearman method, using the formula (negative log10 of LD50)  =  (negative log10 of highest dose) –  – 0.5)) ×log10 of dilution steps)50 values calculated from n = 3 independent assays using groups of 5 CD-1 mice, has been determined to be 100.3 pfu Median lethal dosage  ×13,629 ntds)  = 0.2 mutations per genome . Virus populations of 2.0×105 infectious viruses are expected to possess all possible single amino acid substitution mutations , therefn  has P =   ≥2 or P≤2×10−5 again leading to rejection of the null hypothesis to support positive selection. Thus the selection of ≥2 mutants with the same mutation in the same or different populations is strong evidence of positive selection indicating that the mutation was positively selected and therefore adaptive.Positive selection results in the increased prevalence of mutation due to enhanced fitness versus random occurrence of the same mutations. In analysis of positive selection the null hypothesis is that the mutants occur at a frequency predicted by random probability or chance. Given that prototype A/HK/1/68 virus has a genome of 13,629 nucleotides and each position can be substituted with 3 alternative nucleotides, there are 3 times genome length  or 40,887 possible SNP variants. Thus any SNP mutant has a random probability of occurrence of 1/40,887 and thus the probability that multiple strains of influenza (n) will have the same mutation (SNP) is the product of the individual probabilities times the number of samples tested (N) to get HK-wt and mutants were produced using the 8 plasmid recombineering approach with pLLB plasmids Viral RNA was extracted from 140 µL of stock allantoic fluid from each virus using the QIAamp Viral RNA Mini Kit  and full length influenza genomic segments were amplified To compare the activities of viral RNP complexes in human and mouse cells, a Promega Dual-Glo Luciferase Assay System (Promega) was used The phPOL1-NP-LUC plasmid was constructed by insertion of the firefly luciferase gene and NP non-coding regions that were amplified by PCR using pGL3 Basic  as template and the following primers: Fw: 5′TATTCGTCTCAGGGAGCAAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAAATCATGGAAGACGCCAAAAACATA-3′ Bw: 5′ATATCGTCTCGTATTAGTAGAAACAAGGGTATTTTTCTTTACACGGCGATCTTTCCG-3′. The PCR product was digested by BsmBI and cloned into BsmBI digested pHH21, resulting in plasmid phPOL1-NP-LUC.ATGGAAGACGCCAAAAACAT-3′, Bw: 5′-TATTCGTCTCAAGGTAGTAGAAACAAGGGTATTTTTCTTTACACGGCGATCTTTCCGC-3′. The PCR product was digested by BsmBI and cloned into BsmBI digested pHL1261 The pmPOL1-NP-LUC plasmid (mouse Polymerase I promoter-Luc construct) was constructed by PCR amplification of the firefly luciferase gene flanked by NP non-coding region using the pGL3 Basic plasmid (Promega) as template and the following primers:Fw:5′ATATCGTCTCAGGGAGCAAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAAATC+ standard deviation of the ratios of firefly and renilla luciferase luminescence for three independent experiments of 3 replicates each.To perform the luciferase assays, 96 well plates of human 293T or mouse B82 cells were respectively transfected with 0.06 µg of the reporter plasmids, phPOLI-NP-LUC or pmPOLI-NP-LUC, in combination with 0.06 µg of each of the four pLLB-plasmids encoding the HK-wt or mutant forms of PB2, PB1, PA, NPNucleotide and amino acid sequences were aligned to identify mutations using BioEdit version 7.0.5.3 and Genedoc Multiple sequence alignment Editor and Shading Utility version 2.7.000 software.For each influenza protein, adaptive mutations were positioned according to their amino acid sequence location onto linear primary structural maps that indicated known site of function or interaction with other viral and host factors. Linear maps were made with CorelDRAW 10 v10.410 software. The linear maps of PB2, PB1, PA, and NP were derived from the previous maps of Boulo et al Structural maps were generated using the PDB ProteinWorkshop version 3.7 Samples were fractionated by SDS PAGE using 12.5% acrylamide gels as described previously E. coli using pET17b plasmids for 16 h at 21°C with 10 µM of IPTG except that the soluble fraction was employed for purification and was dialyzed against PBS. Purified NS1 protein was quantified using the Bio-Rad Protein Assay and standardized by comparative western blot. Plasmids were constructed by insertion of the NS1 genes of HK-wt and each mutant produced by PCR mutagenesis into pET17b after PCR amplification using pfu Turbo polymerase . CPSF30 or the CPSF30-F2F3 fragment was expressed in 1.5×107 293T cells transfected with 30 µg of pCAGGS-CPSF30-Flag or pCAGGS-CPSF30-F2F3-Flag plasmid  in 112 µl of Lipofectamine 2000 transfection reagent  for 24 hrs before lysis with 100 mM Tris, 250 mM NaCl, 0.5% NP-40, and 0.5% DOC, pH 8.5. Pull down experiments employed the lysate from 5×105 293 T cells, 1 ug of anti-FLAG M1 monoclonal antibody , defined amounts of NS1 protein, and 20 µl of protein G Dyna-beads  in a 0.25 ml volume with rotation for 2 hr at room temperature. Beads were washed three times in lysis buffer for 10 minutes before western blotting. Control pull-down assays used the lysate from 5×105 293 T cells transfected with empty vector that did not result in NS1 pulldown indicating a lack of nonspecific binding to anti-FLAG M1 monoclonal antibody. Similar results were obtained for both CPSF30 or the CPSF30-F2F3 fragment pull-downs and therefore only the CPSF-F2F3 fragment data were shown. Bound NS1 proteins were detected by western blotting with rabbit anti-NS1 antibody. Anti-FLAG M1 monoclonal antibody was monitored after pull-down by Coomassie Brilliant Blue staining of samples separated on 12.5% SDS-PAGE gels.Recombinant NS1 proteins with amino terminal 6xHis tags were synthesized as described previously Table S1Mutations found in previous studies of mouse adaptation of IAV.(DOC)Click here for additional data file.Table S2Number of mutations selected in each of 39 mouse adapted variants from 10 replicate mouse adaptation experiments.(DOC)Click here for additional data file.Table S3Amino acid changes in the PB2 protein of parental HK clones and their corresponding mouse adapted clones derived after 21 serial passages in the mouse lung.(DOC)Click here for additional data file.Table S4Amino acid changes in the PB1 and PA proteins of HK-wt clones and their corresponding mouse adapted variants derived after 21 serial passages in the mouse lung.(DOC)Click here for additional data file.Table S5Amino acid changes in the NP protein of parental HK clones and their corresponding mouse adapted clones derived after 21 serial passages in the mouse lung.(DOC)Click here for additional data file.Table S6Amino acid changes in the HA protein of parental HK clones and their corresponding mouse adapted clones derived after 21 serial passages in the mouse lung.(DOC)Click here for additional data file.Table S7Amino acid changes in the NA protein of parental HK clones and their corresponding mouse adapted clones derived after 21 serial passages in the mouse lung.(DOC)Click here for additional data file.Table S8Amino acid changes in the M1, M2, NS1, and NEP proteins of parental HK clones and mouse adapted clones derived after 21 serial passages in the mouse lung.(DOC)Click here for additional data file.Table S9List of Genbank accession numbers for nucleotide gene sequences of HK parental and mouse adapted variant clones for each genome segment with encoded proteins and nucleotide sequence length indicated.(DOC)Click here for additional data file.Table S10List of PB2 gene Genbank accession numbers for human H5N1 and canine H3N8 isolates that possess PB2 D701N and/or PB2 D740N mutations.(DOC)Click here for additional data file.Figure S1RNA polymerase activity effects of PB1, PB2, PA and NP mutations in mouse and human cells. Polymerase activity is shown for B82 mouse cells (A) and human 293T cells (B). Mouse adaptive mutations at the indicated positions are masked in gray and HK-wt is shown without mask in the table aligned with bars of activity. Influenza luciferase assays employed luciferase minigenomes expressed via a human or mouse POL1 polymerase in mouse B82 and human 293T cells respectively. Samples (i) has the HKMA12A and 12D combination of mutations; (j) has HKMA12E; and (m) has the HKMA20B, C, and D combination. Values were standardized relative to HK wt luciferase activity as 100% and are shown as averages for n = 3 experiments ± SD. Asterisks indicates significant difference from HK-wt polymerase activity for each cell type (* and ** indicate P≤0.05 and P≤0.01 by t-test respectively).(TIF)Click here for additional data file.Figure S2Blast alignment of mouse CPSF30 with human CPSF30. Amino acid sequence of mouse CPSF30 (query) is aligned above human CPSF30 sequence (Sbjct) with the consensus sequence indicated between each sequence. The F2F3 binding fragment is indicated in yellow mask showing identical amino acid sequence between human and mouse.(PDF)Click here for additional data file.


The incorporation of histone variants into nucleosomes is one of the main strategies that the cell uses to regulate the structure and function of chromatin. Histone H2A.Z is an evolutionarily conserved histone H2A variant that is preferentially localized within nucleosomes at the transcriptional start site (TSS). H2A.Z reorganizes the local chromatin structure and recruits the transcriptional machinery for gene activation. High expression of H2A.Z has been reported in several types of cancers and is causally linked to genomic instability and tumorigenesis. However, it is not entirely clear how H2A.Z overexpression in cancer cells establishes aberrant chromatin states and promotes gene expression.Through integration of genome-wide H2A.Z ChIP-seq data with microarray data, we demonstrate that H2A.Z is enriched around the TSS of cell cycle regulatory genes in bladder cancer cells, and this enrichment is correlated with the elevated expression of cancer-promoting genes. RNAi-mediated knockdown of H2A.Z in the cancer cells causes transcriptional suppression of multiple cell cycle regulatory genes with a distinct decrease in cell proliferation. H2A.Z nucleosomes around the TSS have higher levels of H3K4me2/me3, which coincides with the recruitment of two chromatin factors, WDR5 and BPTF. The observed recruitment is functional, as the active states of H2A.Z target genes are largely erased by suppressing the expression of WDR5 or BPTF, effects resembling H2A.Z knockdown.We conclude that H2A.Z is overexpressed in bladder cancer cells and contributes to cancer-related transcription pathways. We also provide evidence in support of the engagement of H3K4me2/me3 and WDR5/BPTF in H2A.Z-induced cancer pathogenesis. Further studies are warranted to understand how H2A.Z overexpression contributes to the recruitment of the full repertoire of transcription machinery to target genes in bladder cancer cells. Gene expression in eukaryotes takes place in the context of chromatin, which essentially acts as a physical barrier to the binding of transcription factors to certain genomic regions. The basic unit of chromatin is the nucleosome, which consists of approximately 147 bp of DNA wrapped around an octamer of four core histones  . HistoneH2A.Z is an essential variant of the histone H2A and has distinct functions in regulating chromatin dynamics. At the amino acid sequence level, H2A.Z shares about 60% identity with canonical H2A in mammalian cells -11. The In this work, we exploited microarray and chromatin immunoprecipitation sequencing (ChIP-seq) to examine the impact of H2A.Z overexpression on transcriptional states of bladder cancer cells, and identified groups of genes, the dysregulation of which is linked to H2A.Z enrichment around the TSS. We also found that H2A.Z nucleosomes are enriched for the active histone modification H3K4me2/me3 that is likely responsible for recruiting WDR5 and BPTF to H2A.Z target genes.As H2A.Z expression is often misregulated in cancer cells, we first examined H2A.Z levels in human bladder cell lines by Western blot. H2A.Z was expressed at much higher levels in the six bladder cancer cell lines  compared to the normal bladder cell line UROtsa -2, 5-diphenyltetrazolium (MTT) assays reproducibly showed that LD611 cancer cells expressing high levels of H2A.Z grow much faster compared to normal UROtsa cells , we found that 750 and 200 genes were enriched for H2A.Z in the cancer and normal cells, respectively. To identify the genes involved in bladder cancer development, we further classified the cancer-specific genes into the four distinct groups based on their FDR values and NLCS. A total of 480 out of 750 genes were classified into the groups 1, 2 and 3 with P <0.05, suggesting that these genes are specifically enriched for H2A.Z in the bladder cancer cells , we detected a robust signal for H2A.Z around the TSS , soluble chromatin was prepared from the transfected cells and digested with micrococcal nuclease to yield mainly mononucleosomes. Mononucleosomes containing ectopic histones and their bound proteins were purified by sequential immunoprecipitations with anti-Flag and anti-HA antibodies under stringent conditions (300 mM NaCl and 0.1% NP40). Coomassie brilliant blue (CBB) staining confirmed the stoichiometry of H2B, H3, H4 and ectopic H2A/H2A.Z in the purified nucleosomes , transcription start site (TSS) and coding region (CR). In agreement with the ChIP-seq data, we detected high H2A.Z signals around the TSS, but much reduced signals upstream and downstream of the TSS in CASP8 gene, which did not alter after individual depletion of H2A.Z, WDR5, and BPTF  cell line showed that H2A.Z is incorporated into the p21 promoter region in p53-dependent manner, and establishes a repressive barrier to p53-mediated p21 induction . HoweverAlthough the mechanism by which H2A.Z promotes chromatin transcription remains unclear, our interaction data demonstrated that the presence of H2A.Z in the nucleosome increases the levels of H3K4me2 and H3K4me3, marks that are associated with gene activation. These results are in good agreement with those of recent studies showing that H2A.Z colocalizes with active histone modifications in transcription regulatory regions ,10,30-32We show that H2A.Z is overexpressed in bladder cancer cells and establishes the active states of oncogenic transcription. H2A.Z enrichments at target genes increase the levels of H3K4me2 and H3K4me3, which in turn facilitate the recruitments of WDR5 and BPTF. These results unveil a specific contribution of H2A.Z to chromatin remodeling in bladder cancer cells and emphasize the importance of accurate levels of H2A.Z expression to prevent the onset of tumorigenesis.LD611 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). UROtsa cells were cultured in DMEM containing 2 mg/ml glucose and 5% FBS. For mammalian expression of H2A and H2A.Z, their cDNAs were amplified by PCR and ligated into the NheI and BamHI sites of pIRES expression vector containing 5′ Flag and HA coding sequences. Antibodies used in this study are as follows: H2A, H2A.Z, H3K4me1 and WDR5 antibodies from Abcam , Actin, HA and Flag antibodies from Sigma-Aldrich , BPTF antibody from Bethyl Laboratories , H3K4me3 and H3K9me3 antibodies from Active Motif , and H3K27me2 and H3K27me3 antibodies from Merck Millipore .The expression patterns of H2A.Z were determined in human bladder tissue microarrays, which include 16 cases of bladder malignant tissues and eight cases of normal bladder tissues . The tissues were deparaffinized, treated with 3% hydrogen peroxide to block endogenous peroxide, and subjected to antigen retrieval with citrate buffer . The tissue sections were blocked in 5% normal goat serum in Tris-buffered saline-Triton X-100  for 30 min at room temperature and incubated with H2A.Z antibody at 4°C overnight. Immunodetection was performed using Vectastain ABC kit  according to manufacturer’s recommendations. DAB (Vector Laboratories) and hematoxylin (Sigma-Aldrich) were used for color development and counterstaining. The staining intensity was evaluated by semiquantitative immunohistochemical assessment, and divided into four categories: negative staining (less than 20% of cells), weak staining (21 to 50% of cells), moderate staining (51 to 80% of cells) and strong staining (more than 80% of cells).8 cells) was carried out following a standard protocol as described previously [3). Crosslinking was reversed by overnight incubation at 65°C. After RNase A (200 μg/ml) and proteinase K (200 μg/ml) digestions, ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. The high-throughput DNA sequencing was performed using Illumina HiSeq 2000  at the University of Southern California (USC) Genomic Core.H2A.Z ChIP for UROtsa normal and LD611 bladder cancer cell lines (1 × 10eviously . Brieflyhttp://www.r-project.org).Approximately 17 and 13 million sequences were generated for the normal and cancer cells, respectively. MAQ was used to generate 14 and 11 million aligned reads to hg18 reference genome  . One of P <0.05 with fold change >1.5 were selected as suggested by the manufacturer (Illumina). Genes which are differentially associated with H2A.Z in LD611 cancer cells were functionally analyzed in the context of gene ontology and molecular networks by using Ingenuity pathway software . Differentially regulated genes were categorized into various functional groups (threshold P <0.05) and mapped to genetic networks.Total RNA was isolated from LD611 and UROtsa cells using the TRIzol reagent . Gene expression microarray experiments were conducted using a whole-genome expression array . This high-density oligonucleotide array chip targets more than 47,000 probe sequences derived from National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38  and other sources. Data were background-corrected using Genome Studio (Illumina) and were quantile-normalized. Genes showing detection For shRNA-based knockdown, DNA oligonucleotides encoding shRNAs specific for H2A.Z mRNA (5′-GCTTCAAAGAAGCTATTGATT-3′), WDR5 (5′-GAGAGTGGCTGGCAAGTTCAT-3′) and BPTF mRNA (5′-CGCCACTAACAGAGAAGGATT-3′) were annealed and ligated into the lentiviral expression vector pLKO.1 . Lentivirus particles were generated in 293T cells by co-transfecting plasmids encoding VSV-G, NL-BH and the shRNAs. For H2A.Z, WDR5 and BPTF knockdown, LD611 cells were infected with these viruses and selected with puromycin (2 μg/ml) for two weeks. For qRT-PCR, total RNA (2 μg) was reverse transcribed using the iScript cDNA Synthesis Kit  and the PerfeCta™ SYBR Green FastMix  as described . All samNuclei were prepared from LD611 cells expressing Flag and HA-tagged H2A or H2A.Z and were digested with micrococcal nuclease to produce predominantly mononucleosomes. Mononucleosomes containing ectopic H2A or H2A.Z were subjected to anti-Flag M2 agarose. After extensive washing with wash buffer , the eluates were further purified using anti-HA agarose affinity chromatography. The imunoprecipitated nucleosome samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described [4 and treated with the MTT labeling reagent (0.5 mg/ml) at 37°C for 1 h. The blue MTT formazan precipitate was dissolved with the MTT solvent (0.2 ml) and measured at a wavelength of 570 nm using a microplate reader (Bio-Rad). To measure the cell migration activity, Transwell migration assays were performed using 5 × 104 cells. Cells suspended in serum-free medium were plated on the Transwell filters , and DMEM supplemented with 10% FBS was used as chemoattractant. Cells were stained with 0.5% crystal violet and quantified after migration for 18 h.Cell proliferation was assessed by the 3–4, 5-(dimethyl-thyazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay as previously described . In brieThe authors declare that they have no competing interests.KK and WA conceived and designed the experiments. VP performed computational analyses. KK, JC, KH and JK carried out the experiments. PL provided reagents and technical assistance. KK, VP and WA wrote the manuscript. All authors read and approved the final manuscript.Gene dysregulation by histone variant H2A.Z in bladder cancer.Click here for fileList of H2A.Z enriched genes by ChIP-seq.Click here for fileDifferential gene expression in cancer vs. normal cells.Click here for fileGenes overlapping with ChIP-seq and gene expression array.Click here for fileH2A and H2A.Z nucleosomes interacting proteins.Click here for filePrimer sequences for RT-PCR and ChIP.Click here for file


Until 2001, H6N1 influenza viruses in the Hong Kong bird markets were represented by a single stable A/teal/Hong Kong/W312/97-like lineage. Beginning in 2001, despite a reduction in overall prevalence, an increase was observed in the number of H6 viruses isolated from chickens and other hosts. To assess any changes in H6 viruses, we characterized 18 H6 viruses isolated in the Hong Kong bird markets from 2001 to 2003. Experimental data showed that the 2003 H6 viruses had similar infectivity for chickens as did A/teal/HK/W312/97, and they were unable to transmit. Although all hemagglutinin genes were closely related to A/teal/HK/W312/97, 7 isolates were reassortant viruses containing similar gene segments of co-circulating H9N2 or H5N1 viruses. The receptor specificity was different from that of A/teal/Hong Kong/W312/97. Interestingly, similar observations have been documented in H9N2 viruses in Hong Kong. This evolution strongly suggests that some change in the ecology of influenza in the region selected for these changes. Taken together, these findings suggest that the H6 influenza viruses isolated in the Hong Kong markets are not well adapted to chickens and that the likely continued source of these viruses are other “minor” poultry species in which they are undergoing genetic and biologic evolution. The introduction and subsequent sustained global spread in the human population of influenza A viruses with a novel hemagglutinin (HA) subtype leads to an influenza pandemic. The novel influenza pandemics that occurred during the last century resulted in considerable mortality and morbidity. Genetic investigations revealed that these pandemic strains were partially or entirely derived from viruses of avian origin –4 and thThe avian H5N1 influenza virus that infected poultry and humans in Hong Kong in 1997 caused the death of 6 of 18 persons that were confirmed infected with this virus [H9N2 influenza viruses have become panzootic during the last decade and have been isolated from different types of terrestrial poultry worldwide . Two disDuring 1997, an H6N1 influenza A virus, A/teal/Hong Kong/W312/97 was isolated from a green-winged teal . SubsequThe viruses isolated in the Hong Kong region and the abbreviations used in this study are listed in Vibrio cholerae  to abolish interference by nonspecific serum inhibitors. The viruses, which came directly from infected allantoic fluid, were diluted to doses equivalent to four HA units for use in the tests.The H6 virus antigens were compared by using the hemagglutination inhibition (HI) test as previously described in . All serViral RNA was extracted from infected allantoic fluid by using RNeasy kits  according to the manufacturer's instructions. Reverse transcription and PCR were performed under standard conditions by using primers specific for the various genes of influenza viruses . PCR prohttp://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Nucleotide and amino acid sequences of other influenza viruses were obtained from the Influenza Sequence Database of the Los Alamos National Laboratory [DNA sequences were compiled and edited by using the Lasergene sequence analysis software package . Multiple sequence alignments were made by using CLUSTAL W , and phyboratory .α2-3- and α2-6-linked sialic acid whereas those from horse contain almost exclusively α2-3 linkages [The ability of the H6 viruses to hemagglutinate chicken, horse, and guinea pig erythrocytes was determined as previously described in . Brieflylinkages , thereby50 and CID50) of a selection of H6 viruses. Infectious allantoic fluid 10-fold serially diluted (1 : 10 to 1 : 108) was intranasally administered to three quails (0.2 ml inoculum) or chickens (0.5 ml inoculum) of each group. Birds were examined daily for disease signs, and cloacal and tracheal swabs were taken 3 and 5 days after inoculation. Tracheal swabs were placed in 0.5 ml of sample medium  that contained 1000 U/ml penicillin, 200 μg/ml streptomycin, 50 U/ml mycostatin, 100 U/ml polymyxin B, and 250 μg gentamicin); cloacal swabs were placed in 1 ml of sample medium. Each of two embryonated chicken eggs were inoculated with 100 μl of the sample medium containing tracheal or cloacal swabs and were incubated for 48 hours at 37°C. HA assays using chicken red blood cells were performed to verify virus growth in the harvested allantoic fluid [We used 5-to 6-week-old Japanese quail and white leghorn chickens to determine the 50% infectious doses (QIDic fluid . 5 pfu per bird) 1 day after inoculation. Trays between the cages were removed to allow efficient aerosol and fecal transmission. Animal work was performed under ABSL3+ biosafety conditions at St. Jude Children's Research Hospital. Transmission experiments were performed by following the method of Perez et al. . BrieflyIntensive surveillance systems in the Hong Kong live-bird markets have been in place since 1997. From 2001 through 2003, these systems showed that 253 isolates of 42495 tested specimens were H6 influenza viruses . We arbitrarily selected 18 H6 influenza viruses isolated from domestic poultry for further study .Since 1997, H5 viruses in Hong Kong have undergone antigenic and genetic change. To determine whether similar antigenic changes have occurred in H6 viruses in Hong Kong, we characterized the antigens of recent isolates by using HI tests. The H6 influenza viruses isolated in Hong Kong reacted with hyperimmune goat antisera raised against A/turkey/Massachusetts/1/65 (H6N2) (Ty/MA/1/65), hyperimmune rabbit antisera raised against A/shearwater/Australia/1/72 (H6N5) (Sh/Aus/1/72), and postinfection chicken antisera against A/teal/Hong Kong/W312/97 (H6N1) (Tl/HK/W312/97), A/quail/Hong Kong/YU1654/00 (H6N1) (Qa/HK/YU1654/00), A/quail/Hong Kong/YU39/01 (H6N1) (Qa/HK/YU39/01), and A/duck/Shantou/5540/01 (H6N2) (Dk/ST/5540/01) . The antThe full-length HA sequences of the 18 H6 influenza viruses were determined to analyze their phylogenetic relationships . The HA In addition to the HA genes, the NA genes of all 18 H6 isolates were sequenced. The N1NA genes from 14 virus isolates from domestic poultry, including the two isolates from chicken, belonged to the cluster represented by Tl/HK/W312/97. This cluster was distinguishable from that containing A/goose/Guandong/1/96-like (H5N1) N1NA genes. The remaining two N1NA genes, which were from two of the 2001 isolates (Qa/HK/FB611/01 and Qa/HK/FB801/01), were slightly different from those of the Tl/HK/W312/97-like N1NA cluster and instead were more similar to that of A/quail/Hong Kong/1721-20/99. Alignment analysis revealed a 19-amino acid deletion from the stalk region of the N1NAs of all contemporary terrestrial isolates. This deletion is characteristic of the H6N1 viruses isolated in the region after 1997 . UnexpecIn 1998 and 1999, the H6N1 viruses isolated from the Hong Kong markets were genetically stable and were of the same genetic lineage . To deteThe nucleotide sequences for 18 H6 strains presented in this paper have been submitted to DNA Data Bank of Japan (DDBJ) under the accession numbers AB586744 to AB586887.The receptor specificity of the HA is considered a likely determinant of host range for influenza viruses. Additionally, studies have shown that H9N2 viruses circulating in the Hong Kong live-bird markets have human virus-like receptor specificity . To expl7.5  to 108.0  EID50/0.1 ml. Tl/HK/W312/97 and the domestic poultry H6 isolates from 2001, 2002, and 2003 were able to infect quail and chickens  would substantially affect the isolation rates of H6 viruses . In 2001In addition to the reduction in isolation rates, the increase in the genetic diversity within the H6 viruses in the markets also corresponds with the removal of quail. Exactly what role quail may have had in maintaining a stable Tl/HK/W312/97-like lineage is uncertain. Reassortants were present as early as 2000, but Tl/HK/W312/97-like viruses were the dominant genotype. It could be speculated that the Tl/HK/W312/97-like viruses are more adapted for replication and transmission in quail than some of the reassortant viruses. Hence, while quail remained in the market Tl/HK/W312/97-like viruses predominated. The association of Qa/HK/G1/97 (H9N2)-like viruses, that share 6 gene segments in common with Tl/HK/W312/97, with quail would support this possibility. It is of course also possible that it is coincidence that links the removal of quail and the emergence of multiple genotypes. Studies on H9N2  and H5N1α2-3-linked sialic acids. Unlike the contemporary H6 viruses, Tl/HK/W312/97 has an avian virus-like binding pattern and retains the ability to bind to horse erythrocytes. Interestingly, the contemporary H6 viruses do not contain any of the amino acid changes in the HA that Matrosovich and colleagues associated with the human virus-like binding properties of the H9N2 viruses. Similarly, no correlation between predicted carbohydrate moieties and binding preferences of the H6 viruses was seen. It is interesting that both H9 and H6 viruses have evolved towards a human-like virus binding preference. The fact that it has occurred in distinct subtypes of influenza virus would strongly support a selective advantage accompaning the change. Exactly what environmental or host factor is driving the selective pressure is unclear.  Although we were unable to detect an increase in the chicken infectivity of recent H6 viruses, some biologic differences to Tl/HK/W312/97 were seen. Similar to what has been described for H9N2 viruses [The hypothesis that the host range of H6 viruses underwent expansion in the Hong Kong markets appears to be incorrect in view of our findings. H6 viruses do have the capacity to be maintained in chicken populations as demonstrated by the emergence of multiple genotypes of H6N2 virus in chicken in the United States –37. What


T2 values are related to the tissue water content, providing useful diagnostic information in cardiac diseases, especially in acute states such as myocardial infarction (MI). Visualization of edema, its regional distribution, and extent in acute and chronic heart disease may be useful as a diagnostic tool and help in guiding treatment in the patients with heart disease. Recently, edema detection (T2 elevation) using quantitative T2 maps has been shown more robust than qualitative clinical T2W imaging.Hypothesis: Whole heart coverage T2 mapping makes the global edema distribution available for quantitative myocardial edema assessment in the context of subacute MI.Under IACUC-approved protocol, MI was induced in swine (N = 4) by 120 min occlusion of the mid LAD. Imaging was carried out 2 to 9 days post MI using an Achieva 3T TX system . Normal animals (N=4) were imaged for reference T2 values. T2-mapping of the ventricles was carried using previously presented free-breathing 3D sequence. Post-contrast 3D Late Gadolinium enhancement with phase sensitive inversion recovery (PSIR) images were acquired ~30min post infusion of 0.2 mmol/kg of Magnevist. Both sequences were acquired in high-resolution  during free-breathing using independent respiratory navigator gating.3D T2 maps were calculated per voxel using linear regression of the log of the signal. Pixels with poor fits (corr. coeff. R2<0.9) were rejected. The left ventricle (LV) was manually segmented, excluding papillary muscle and epicardial/endocardial boundaries.MI with edema was observed in all animals; 3 animals displayed evidence of hemorrhage/clotting injuries as evidenced by microvascular obstruction and concomitantly reduced T2. Figs. The high resolution quantitative T2 maps enables monitoring of physiological changes over time, providing an objective assessment on the evolution of edema post MI.This work was funded in part by AHA-11SDG5280025.


Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK. Bladder cancer is the fifth most common cancer world-wide with an estimated 357.000 new cases and 145.000 deaths in 2010 2) into phosphatidylinsositol-3,4,5-trisphosphate (PIP3). This reaction can be reversed by the PI3K antagonist PTEN (phosphatase and tensin homologue deleted on chromosome 10). PIP3 recruits PDK1 (protein dependent kinase 1) to the cell membrane where it binds and phosphorylates Akt at amino acid residue T308. Akt is considered as the most critical signaling node in this pathway regulating various substrates affecting cellular processes involved in the control of cell growth and survival Normally, upon activation by tyrosine receptor kinases or RAS proteins PI3K converts phosphatidylinositol-4,5-bisphosphate  and RT112 (German Collection of Microorganisms and Cell Cultures) were maintained at 37°C in RPMI 1640 or DMEM supplemented with 10% FCS in a humidified atmosphere containing 5% or 7,5% CO5 cells were seeded on 6-well plates one day before infection and adenovirus particles were added at a multiplicity of infection of 30. Suppression of protein expression was analyzed by immunoblots 2–6 days after transfection or infection.siRNA oligonucleotides against 4E-BP1, S6K1 and control were designed by and purchased from Qiagen GmbH and Akt 1,−2,−3 stealth siRNA oligonucleotides from Invitrogen. Transient transfections were performed using X-treme gene transfection or lipofectamin reagent  according to the manufacturer's instructions, using 2 µg/siRNA/6-well. For triple transfection adenovirus mTOR shRNA and control shRNA was purchased from SIRION Biotech. 1.5×10Cells were lysed in RIPA buffer , phosphatase inhibitor Mix (SERVA) for 20 min on ice. Insoluble material was pelleted by centrifugation for 30 min at 4°C, 30,000 g. Protein concentration was quantified with the BCA™ Protein Assay (Pierce). Equal amounts of protein were subjected to SDS-PAGE on 7.5–12% polyacrylamide gels and transferred to PVDF membrane (Zefa). For immunoblotting, the antibodies used included: Akt (C67E7), Akt1, Akt2, Akt3, Phospho-Akt (Ser473), Phospho-Akt (Thr308), GSK3β, Phospho-GSK3β (Ser9), mTOR, Phospho-mTOR (Ser2448), 4E-BP1, Phospho-4E-BP1 (Thr37/46), S6K1, Phospho-S6K1(Thr389), S6RP, Phospho-S6RP, p44/42 MAPK, Phospho-p44/42 MAPK, c-Raf, Phospho-c-Raf (Ser338) . Secondary HRPO conjugated antibodies were purchased from Dianova. Immunoblotting was performed as described previously or modified according to the manufacturer's recommendation To evaluate cell growth, cells were seeded at 1000 cells/96-well and treated as described. After harvesting, cells were stained with trypane blue and living cells were counted in a Neubauer chamber.Cell proliferation was measured 24 h after treatment of cells with RAD001, NVP-BEZ235 and U0126 or 48–72 h after transfection/infection with siRNAs/shRNAs. Bromodeoxyuridine (BrdU) incorporation for detection of newly synthesized DNA and 7-amino-actinomycin D (7-AAD) staining for detection of double stranded DNA was performed using an APC BrdU Flow Kit (BD Biosciences) by pulsing cells for two hours according to the manufacturer's protocol followed by flow cytometry analysis.Apoptosis was detected by measuring activity of caspase 3/7 using the Caspase-Glo 3/7 Assay from Promega GmbH, according to the manufacturer's protocol.Cells were treated with RAD001, NVP-BEZ235 and U0126 in 96-well plates. At 36 h, cells were incubated for 3 h with XTT. The enzymatic reaction was analyzed at 450 and 650 nm in an ELISA reader.Student T-test was used to compare the means in different groups. Statistical calculations were done using Microsoft Office Excel.We analyzed expression and activation status of molecular key components in the PI3K pathway in a panel of 9 UC derived cell lines. Cells were harvested and lyzed for protein analysis in a subconfluent stage. Expression of Akt, mTOR, S6K1, S6RP, 4E-BP1 and PTEN was detected in all cell lines . PhosphoIn order to characterize the functional relevance of this pathway in UC we used the rapamycin derivatives RAD001 and CCI-779 and the PI3K-inhibitor NVP-BEZ235. Since effects of CCI-779 and RAD001 treatment were identical we only depict RAD001 results. First, dose response assays were conducted to assess the activity of RAD001 and NVP-BEZ235 1 h after cellular treatment. Activity of the compounds was characterized by the phosphorylation pattern of S6K1/S6RP/4E-BP1 for both substances and in addition for Akt when using NVP-BEZ235. Signal intensities were measured by a chemiluminescence imaging system and inhibitory concentrations of 50% (IC50) for phosphorylation level normalized to the protein level were calculated . RAD001 It has been described that S6K1 activation results in a negative feedback loop regulating not only PI3K activity which is probably responsible for the limited success of rapamycin derivatives in clinical use Whether S6K1 directly induced PI3K/Akt activity was addressed by silencing S6K1 expression using two specific siRNAs directed against S6K1. Two days after transfection, western blot analyses demonstrated a 90–96% reduction in S6K1 protein level . The dowThese results prompted us to characterize the signalling circuitry of PI3K, mTOR, Akt and 4E-BP1 in greater detail. In most suggested models, S6K1 and 4E-BP1 are downstream targets of Akt and mTORC1 and phosphorylation in most reports is simultaneously regulated In order to further examine this observation, we used an adenoviral shRNA approach to silence mTOR protein expression which should result in specific inactivation of both, mTORC1 and mTORC2 protein complexes. Two days after infection, western blot analyses confirmed an 85–96% knockdown in mTOR protein level that remained stable for 5 days . The knoLast, the influence of Akt on 4E-BP1 phosphorylation was characterized by using siRNAs directed against the three different isoforms of Akt. Three days after transfection, the combined knock down of all three isoforms resulted in >90% reduced protein expression of all three Akt isoforms . Only S6Given the importance of PI3K and MAPK pathway biology, we examined the interaction of the two pathways in bladder cancer. Cells were treated with RAD001, NVP-BEZ235 or with the MEK1/2 inhibitor U0126 and activation status of key proteins in both pathways 1–72 h after treatment were analyzed in western blots. Inhibition of mTORC1 by RAD001 induced activation of ERK1/2 phosphorylation 24–72 h after treatment . HoweverWe then analyzed the biochemical consequences when inhibiting both the PI3K/AKT/mTOR and the MAPK pathway, using RAD001, NVP-BEZ235 or U0126 in combination or U0126 alone. When cells were treated with U0126 alone, ERK1/2 phosphorylation was completely abolished whereas Akt phosphorylation on S473 and T308 was upregulated . InteresNext we determined how manipulation of PI3K and MAPK signaling influences cellular proliferation in UC. First, cells were treated with RAD001, NVP-BEZ235 and U0126 alone or in combination and cell proliferation was monitored over time. Three days after treatment, RAD001 inhibited cell proliferation by 62% in RT112 and 40% in T24 cells relative to the solvent control . TreatmeFinally, we addressed the question why NVP-BEZ235 affects cell growth more efficaciously than RAD001. We demonstrated that NVP-BEZ235 unlike RAD001 influences both, S6K1 and 4E-BP1 phosphorylation. Thus, we used either siRNA oligonucleotides directed against S6K1 or 4E-BP1 or combined RAD001 with 4E-BP1 siRNAs to mimic the presumed additional effect of NVP-BEZ235. Two days after transfection siRNAs against 4E-BP1 or S6K1 reduced protein expression by 80–96% , 2C. CelDespite a better understanding of bladder cancer biology within recent years, only minor improvements in the therapeutic management of this disease have been achieved during the last two decades. The PI3K/Akt/mTOR and MAPK signaling pathways are prone to mutations and aberrant activation in this tumor entity and might provide suitable targets for more effective therapies Both signaling pathways are constitutively activated in the majority of the examined UC derived cell lines supporting their importance in UC In tumor growth, 4E-BP1 is suggested to integrate signals from the PI3K and the MAPK signaling pathway and might be a potential target for therapy Akt has been described as a key molecule for the regulation of numerous processes including cell cycle progression, survival and apoptosis and is therefore considered a hopeful candidate for targeted therapies An important aspect of Akt activity in UC is its function on cell proliferation. As described, phosphorylated Akt induces a positive impulse towards cell proliferation but we observe the opposite  [13]. ThCrosstalk between the MAPK and PI3K signaling pathways has been established in recent years and we could also confirm that either pathway can partially substitute for the failure of the other in UC cell lines When interfering with MAPK activity using U0126, hyperactivation of Akt was observed but unlike postulated in the current model no or only weak responses on mTOR, S6K1 or 4E-BP1 phosphorylation status were evident . This miBased on our data, we suggest for urothelial carcinoma a molecular model depicted in