Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapDHi, encoded by gene HI0450). Both vapDHi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapDHi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapXHi for the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections.Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable vapDHi were attenuated for survival inside human respiratory epithelial cells (NCI-H292) and human brain microvascular endothelial cells (HBMEC), the in vitro models of mucosal infection and the blood-brain barrier, respectively. Transcomplementation with a vapDHi allele restored wild-type NTHi survival within both cell lines. A PCR survey of 59 H. influenzae strains isolated from various anatomical sites determined the presence of a vapDHiallele in 100% of strains. Two isoforms of the gene were identified in this population; one that was 91 residues in length, and another that was truncated to 45 amino acids due to an in-frame deletion. The truncated allele failed to transcomplement the NTHi vapDHi survival defect in HBMEC. Subunits of full-length VapDHi homodimerized, but subunits of the truncated protein did not. However, truncated protein subunits did interact with full-length subunits, and this interaction resulted in a dominant-negative phenotype. Although Escherichia coli does not contain a homologue of either vapDHi or vapXHi, overexpression of the VapDHi toxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapXHi antitoxin on a compatible plasmid.Isogenic mutants in vapDHi and vapXHi may constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells.We conclude that  Haemophilus influenzae are acquired in the nasopharynx shortly after birth, and are thought to persist throughout life. H. influenzae adheres to and penetrates into and between cultured human respiratory epithelial cells, a mechanism that may contribute to its persistence in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) patients [H. influenzae can be found in the respiratory tracts of these patients even after they have undergone antibiotic treatment [H. influenzae can still be isolated from the subepithelial matrix [in vivo study that H. influenzae can persist in a human bronchiolar xenograft for at least three weeks [Culturable patients ,2. H. inreatment . As welll matrix . Finallyee weeks . This suH. influenzae to survive antibiotic treatment and reappear when growth is favorable may be responsible for the reseeding of the middle ear observed in chronic otitis media. Often, middle ear fluid from children presenting with otitis media with effusion are sterile when cultured, but PCR analysis of the fluid has determined the presence of H. influenzae [The ability of fluenzae . Furtherfluenzae .in vitro using a NTHi strain that was susceptible to β-lactam antibiotics. This strain was allowed to invade a human respiratory epithelial cell monolayer for 24 hours, which was subsequently treated with a 4 hour incubation in 10 × MIC concentrations of the β-lactam antibiotics ampicillin, imipenem, cefuroxim, amoxycillin/clavulanic acid, or cephalothin. The antibiotics killed all the extracellular bacteria, but none of the intra- or paracellular bacteria, suggesting that the organism was not replicating inside or between the epithelial cells [Persistence was investigated al cells . Non-repDichelobacter nodosus, the causative agent of ovine footrot, Katz et al. [vapA-D, for virulence-associated proteins. Homologues of these genes appear on the chromosomes and plasmids of a number of pathogenic microorganisms, including Neisseria gonorrhoeae, Helicobacter pylori, Reimerella anatipestifer and Actinobacillus actinomycetemcomitans. The chromosome of H. influenzae strain Rd KW20 (hereinafter referred to as strain Rd) contains vapA, vapBC, and vapD homologues, with one pair, vapBC, in duplicate. The genome organization of the vap genes in H. influenzae differs from that of D. nodosus, in that vapAHi (HI1250) is preceded by a conserved hypothetical protein, HI1251, and both genes are likely transcribed as an operon. As well, vapDHi is flanked by a gene encoding another conserved hypothetical protein, HI0451 (which we have named vapXHi), again in an apparent operon configuration (8 nucleotides separate HI0450 and HI0451). To determine if the vap homologues played a role in the persistence of NTHi, we chose vapDHi (HI0450) for further study, since this protein was found in a proteomic survey to be expressed in the soluble fraction of strain Rd [Hi is 40% identical and 67% similar to the Dichelobacter VapD and belongs to the Cluster of Orthologous Groups (COG) 3309 and Pfam 04605, termed the "N-terminal conserved domain of VapD".During a study aimed at identifying genes associated with virulence in pathogenic strains of the Gram-negative, strict anaerobe z et al.  reportedtrain Rd . VapDHi vapDHi mutant strain was used to invade the in vitro model of the blood-brain barrier, human brain microvascular endothelial cells (HBMEC) in 12-well plates, the amount of gentamicin-resistant bacteria recovered from the monolayer after three hours declined to approximately 60% of wild type levels, an average of 2.2 × 103 CFU/ml for the wild-type strain Rd versus 1.3 × 103 CFU/ml for the Rd vapDHi mutant (n = 3 (number of independent assays performed in at least duplicate); P < 0.05, Student's t test). No significant difference was observed between the wild type and vapDHi mutant in adherence to the human cell monolayers: the average number of cell-associated bacteria (both adherent and invaded) recovered for strain Rd was 1.5 × 105 CFU/ml and 1.8 × 105 CFU/ml for the Rd vapDHi mutant .When the Rd vapDHi mutant was a general phenomenon and not restricted to strain Rd, another isogenic pair was constructed and analyzed using a different strain, R3001. R3001 is a bronchoalveolar lavage isolate from a pediatric cystic fibrosis patient, and is considered invasive since it came from a normally sterile site [5 CFU/ml for the parent strain R3001 versus 7.1 × 104 CFU/ml for the R3001 vapDHi mutant . Although the absolute numbers of bacteria recovered were higher with strain R3001 than with Rd (as is often observed with invasive isolates), the attenuation of survival inside the HBMEC monolayer of ≤ 60% observed in the strain with a vapDHi mutation was maintained. There was no significant difference between the wild-type R3001 and the R3001 vapDHi mutant in adherence to the monolayer: the average numbers of cell-associated bacteria recovered for strain R3001 were 1.7 × 107 CFU/ml versus 1.3 × 107 CFU/ml for R3001 vapDHi . Unlike Rd, strain R3001 carries the high molecular weight (HMW) adhesins, which may account for its more efficient adherence to the HBMEC monolayer [vapDHi mutants versus their cognate parent strains were observed, whether grown in bacteriological media (sBHI broth) or on HBMEC or NCI-H292 monolayers (data not shown).To determine if the phenotype of attenuated survival observed in the Rd ile site . The aveonolayer . No signvapDHi mutation would affect the ability of H. influenzae to survive inside human respiratory epithelial cells over a longer period of time, 18-hour invasion assays were performed using NCI-H292 cells. The number of gentamicin-resistant bacteria recovered from the NCI-H292 monolayer after 18 hours for the parent strain Rd was an average of 6.4 × 104 CFU/ml versus 3.2 × 104 CFU/ml for the Rd vapDHi mutant . This represents a 50% reduction in survival of the vapDHi mutant within epithelial cells as compared to the parent strain, more attenuation than was seen for the three hour assays.To determine if the vapDHi locus from strain R3001 was cloned into the mobilizable broad host range plasmid pDD515, creating pDD564, and conjugally transferred into the Rd vapDHi mutant  within HBMEC monolayers. The amount of gentamicin-resistant bacteria recovered from the endothelial cell monolayer after a three hour invasion assay was an average of 8.0 × 102 CFU/ml for Rd (pDD515), the vector control, and 7.6 × 102 CFU/ml for the mutant strain that carried the wild-type vapDHi allele in trans, Rd vapDHi (pDD564) , indicating that there was no significant difference in the survival inside HBMEC monolayers of the wild type strain versus the transcomplemented strain. These data confirm that the phenotype of attenuated survival was due to the interruption in vapDHi and not to polar effects.The fluenzae . The survapDHi mutant mirrored the survival defect seen with the Rd vapDHi mutant, at approximately 60% of wild-type R3001 levels. However, attempts to conjugate the mobilizable broad host range plasmid carrying the vapDHi locus, pDD564, into strain R3001 for transcomplementation studies failed repeatedly. This clinical isolate likely has a plasmid or an origin of replication of the same incompatibility group incorporated into its chromosome and therefore will not maintain the broad host range plasmid for transcomplementation [The R3001 entation .vapDHi locus was transcribed during contact with a human cell monolayer, total RNA was isolated from the wild-type strain Rd recovered after 18 hours on HBMEC or NCI-H292 monolayers and was used as the template for RT-PCR. Figure vapDHi-specific primers, with a molecular weight marker in lane A. The template for lane B is Rd RNA from HBMEC endothelial cell monolayers, lane C is its cognate negative control, with no reverse transcriptase added to the RNA prior to PCR amplification. Lane D shows the results using Rd RNA from NCI-H292 epithelial cell monolayers; lane E is its cognate negative control. The vapDHilocus is transcribed when strain Rd is in contact with either human epithelial or endothelial cell monolayers.To determine if the vapDHi among the highly heterologous NTHi strains, a PCR survey of 59 commensal and disease-associated strains was undertaken (Table vapHI primer set was used. In Rd, these primers amplify a 769 bp PCR product that includes the full-length vapDHi gene.In order to estimate the carriage rate of en Table . The vapH. influenzae (types a through f) from the American Type Culture Collection (ATCC) reference strains described in Table vapHI forward and reverse primers. The NTHi strains included nasopharyngeal, blood, CSF, middle ear, tracheal aspirate, and sputum isolates. A PCR product was amplified in 100% of the strains. All of the ATCC encapsulated reference strains and Rd displayed a full-length vapDHiallele. Ninety-three percent of the nasopharyngeal strains carried a full-length allele, as did 71% of the blood and CSF isolates, and 50% of the middle ear, tracheal aspirate, and sputum isolates. Overall, only ten strains of the 59 included in the study displayed a truncated gene.Purified chromosomal DNA preparations from 53 randomly-chosen NTHi strains and one each of the six capsular serotypes of vapDHi in more detail, five out of the ten alleles that represented the smaller isoform of vapDHi from the PCR survey were sequenced on both strands. It was found that, in all cases, the gene had undergone a deletion event that had left the protein in frame, but missing 46 amino acids from the interior of the protein, resulting in a 45 amino acid protein rather than the full-length 91 amino acid protein  of LexA, homodimerization of identical protein subunits can be quantitated [Hi from strain R3001 was ligated to the DBD of LexA in plasmid pDD559 and the clones were analyzed on MacConkey agar with lactose. If there was no homodimerization of the LexA::VapDHi fusion protein, the colonies appeared red on MacConkey agar, as the native level of beta-galactosidase expression in the reporter strain was not inhibited. If the subunits interacted, the colonies appeared pale on MacConkey, as the engineered LexA operator controlling the lacZ reporter gene had been repressed by a homodimer of the LexA fusion protein. This repression was quantitated by beta-galactosidase activity assays. Each measurement is the mean of at least three experiments performed in triplicate.Using an ntitated ,15. In tHi interacted strongly with itself. The beta-galactosidase activity of the reporter strain SU101 carrying the vector control (pSR658) was an average of 975 (± 29) Miller units, and the activity of SU101 with the LexA::VapDHi fusion (pDD559) was an average of 16 (± 1) Miller units, indicating strong interaction. Full-length VapDHi forms homodimers in vivo. This protein may also form higher-order multimers, since this would result in a number of dimeric forms being available to act as a repressor of lacZ transcription in the reporter strain.It was found that VapDHi did not interact efficiently. The vector control for the homodimerization assays SU101 (pSR658) yielded 1490 (± 31) Miller units, and SU101 carrying the LexA fusion to the truncated VapDHi protein from strain R2866 (pDD577) displayed 1357 (± 54) Miller units of beta-galactosidase activity, showing little repression in this system.Homodimerization assays with the small allele revealed that the subunits of the truncated VapDHi subunits homodimerized strongly but the truncated subunits did not, the truncated subunit and the full-length subunit were examined for interaction in heterodimerization assays. In the reporter strain SU202, a LexA operator with a mutated half-site was engineered upstream of the lacZ gene. This strain was then transformed with two compatible plasmids, one that carried a fusion of the truncated VapDHi with a wild-type LexA DBD, and one that carried the full-length VapDHi fusion to a mutated LexA DBD that only recognized the mutated LexA operator half-site in SU202 [lacZ. It was determined that the truncated and full-length subunits could interact with each other. The vector control for the heterodimerization assays (SU202 with pSR658 and pSR659) yielded 1855 ± 196 Miller units, and SU202 carrying the LexA fusion to the truncated VapDHi protein from strain R2866, pDD577, coupled with the mutated LexA DBD fusion to the full-length VapDHi protein from strain R3001, pDD561, resulted in 792 ± 19 Miller units of beta-galactosidase activity, showing that the truncated subunit did heterodimerize with the full-length VapDHi subunit.Since the wild-type VapDin SU202 . If a heHi protein could transcomplement a mutation in the full-length gene, the truncated locus from strain R2866 was cloned into the mobilizable broad host range vector pDD515, creating pDD594, and conjugally transferred into the Rd vapDHi mutant strain. Three-hour survival assays using HBMEC monolayers were performed, and the number of gentamicin-resistant bacteria recovered from the monolayer was an average of 5.6 × 102 CFU/ml for Rd (pDD515), the vector control, versus 1.9 × 102 CFU/ml for Rd vapDHi(pDD594) . Survival in HBMEC by the mutant strain did not increase to wild-type levels, as was the case with the Rd vapDHi mutant transcomplemented with a full-length vapDHi allele on pDD564.To investigate whether the truncated VapDHi and the truncated protein interacted, the plasmid pDD594 carrying the truncated allele was conjugally transferred into wild-type strain Rd to determine whether expression of the small protein would interfere with the function of the wild-type VapDHi protein. The strain was used in three-hour assays of HBMEC monolayers, and it was found that Rd (pDD594) was attenuated in human cell survival as compared to Rd (pDD515), the wild-type strain carrying the vector without an insert. The average number of gentamicin-resistant bacteria recovered from the monolayer for Rd (pDD515), the vector control, was 5.7 × 102 CFU/ml versus 1.8 × 102 CFU/ml for Rd (pDD594) . The in trans expression of a truncated vapDHi allele in the wild-type strain Rd resulted in a dominant-negative effect on survival within HBMEC monolayers.Since subunits of the full-length VapDHi constituted the toxin, and that VapXHi encoded the antitoxin portion of a TA locus, both proteins were expressed in an E. coli background. E. coli does not contain a homologue of either protein. Initially, the vapDHi gene, HI0450, was cloned into the pTrcHisA vector (Invitrogen, Carlsbad, CA). This resulted in vapDHi being under the control of the strong Ptrc promoter, which is repressed for the most part until induced by IPTG. This plasmid was designated pDD560. Both the vector control, DH5α (pTrcHisA), and DH5α (pDD560) were grown to mid-log phase in LB broth with 100 μg/ml ampicillin and aliquots were spread on LB agar plates with 100 μg/ml ampicillin and 0.1 mM IPTG. Strain DH5α (pTrcHisA) grew on the plates, but DH5α (pDD560) did not, indicating that induction and overexpression of vapDHi was toxic to E. coli. The putative antitoxin VapXHi, was then cloned into the pTrcHisA vector. No growth disruption occurred in DH5α with the overexpression of VapXHi alone. The vapXHi gene plus the lacIq gene were then subcloned into the broad host range mobilizable plasmid, pDD515, resulting in pDD672. This strategy allowed the DH5α test strain to carry two compatible plasmids, one which encoded the vapDHi gene (pDD560) and the other expressing the vapXHi antitoxin gene (pDD672). Both genes were under the control of a Ptrc promoter and were therefore both repressed until induced by IPTG. When both genes were induced with 0.1 mM IPTG on LB agar plates that contained 100 μg/ml ampicillin and 10 μg/ml chloramphenicol in strain DH5α (pDD560, pDD672), growth was restored. This indicated that the concurrent expression of the vapXHi antitoxin with the vapDHi toxin ameliorated the cell growth arrest observed with expression of vapDHi alone, and that vapXHi was necessary for this rescue.To test the hypothesis that VapDvapDHi allele in strains Rd and R3001 resulted in attenuation of survival within both HBMEC and NCI-H292 monolayers, suggesting that in H. influenzae, the presence of a functional VapDHi facilitates persistence within epithelial and endothelial cells. Mutants invaded and survived in human cells at ≤ 60% of wild-type levels. Although relatively modest, this level of attenuation has been observed during the mutational analysis of other Haemophilus virulence factors, such as opacity-associated protein A as well as the high molecular weight (HMW) proteins [H. influenzae survival within human cells is multifactorial, and our data indicate that VapDHi contributes to this process. However, strain Rd contains three other vap genes (vapAHi, vapBHi, and vapCHi), and it is possible that these Haemophilus vap genes act synergistically, such that multiple mutations may result in a more attenuated survival phenotype. Indeed, a recent study has determined that a chromosomally-located homologue of the VapBC locus acts as a toxin-antitoxin module in the spirochete Leptospira interrogans [Haemophilus strain with mutations in all the vap genes.Mutation of the proteins ,16. H. ierrogans . It woulvapDHi mutants displayed differences in adherence to the monolayers compared to the parent strain, so the defect occurred after binding and affected the organism's ability to persist inside or between cells. Interestingly, the vapDHi mutants were not attenuated in growth rate when compared to the parent strains, either in bacteriological media or on the surface of human cell monolayers. The observed survival attenuation of the mutants could be transcomplemented with a full-length allele from a clinical isolate, R3001, demonstrating that the phenotype was due to the mutation in vapDHi and not polar effects. A truncated allele from another clinical isolate, R2866, did not transcomplement the Rd vapDHi strain, indicating that the truncated protein was not functional in vivo. RT-PCR analysis confirmed that the full-length vapDHi locus in Rd was transcribed during contact with both epithelial and endothelial cells. VapDHi has also been identified in the soluble fraction of strain Rd grown in bacteriological media [Neither of the al media . It remavapDHi allele on their chromosomes, suggesting that maintenance of a functional vapDHi gene was beneficial to survival in this niche  carried a full-length he Table . Of the Hi protein in Rd is approximately 10 kilodaltons, and small bacterial proteins often form multimers. Full-length VapDHi subunits exhibited strong homodimerization in a LexA-based protein-protein interaction system, and this may indicate that the subunits form higher-order multimers such as homotrimers or homotetramers in vivo. However, the protein encoded by the truncated allele did not homodimerize in the same system, further evidence of its loss of function. Interestingly, the truncated subunit did interact with full-length subunits in heterodimerization assays. Further evidence of this heterodimerization in vivo was that the expression of the truncated subunit in the wild-type strain resulted in a dominant-negative effect on survival within HBMEC monolayers, the levels of which mimicked the attenuation observed with the vapDHi mutation. This was likely due to truncated subunits forming hybrid complexes with full-length subunits and interfering with their structure and/or function, resulting in the observed dominant-negative phenotype.The calculated molecular mass of the VapDrelBE) was determined to be cleavage of mRNA in the ribosomal A site [relBE homologues (HI0710 and HI0711) as well as a homologue of higA (host inhibition of growth antidote protein) from plasmid Rts1. The higBA TA locus is unusual in that the toxin gene (higB) exists upstream of the antidote protein (higA). Interestingly, VapAHi of strain Rd is 29% identical and 53% similar to HigA.The activities of only a few toxins encoded by TA loci have been elucidated thus far. Two specific targets of plasmid-encoded toxins have been identified: CcdB of the F plasmid and ParE of plasmid RK2 inhibit DNA gyrase, and Kid of plasmid R1 was previously thought to interact with DnaB helicase, but has recently been shown to cleave cellular mRNA -23. The l A site . Strain Hi and VapXHi form a toxin-antitoxin pair, it is unusual to find homologues of VapXHi only in H. influenzae and the gonococcal plasmid. H. influenzae strains R2846 and R2866 both have a truncated vapDHi toxin gene, and possess a vapXHi which is 100% identical to that gene in strain Rd. Their respective genomes can be searched at . Interestingly, there are complete genome sequences available for two isolates of H. pylori, N. meningitidis and Haemophilus somnus, but only one strain of each has a homologue of vapDHi. Thus, several features of the vapDHi/vapXHi gene pair are unusual for a toxin-antitoxin locus.While the data acquired in this study suggests that VapDH. influenzae. Further studies are required to fully characterize the mechanism of VapDHi function and to define its role in the modulation of NTHi persistence in human cells.Persistence of NTHi is important in the progression of disease caused by this organism. Many investigators have previously reported the discovery of a number of virulence factors associated with adherence, invasion and survival of NTHi inside human cells ,16,18,25H. influenzae strains used are listed in Table H. influenzae was grown on chocolate agar (36 g Difco GC medium, 10 g hemoglobin, 10 ml Difco Supplement B (Becton Dickinson, Sparks, MD), 5,000 Units bacitracin per liter) or supplemented BHI (sBHI) broth or agar (37 g brain heart infusion media ± 15 g Bacto agar per liter (Remel, Lenexa, KS) with 10 μg/ml β-NAD, 10 μg/ml heme-histidine, and 5 Units/ml bacitracin). Strains containing the TSTE cassette [Escherichia coli strains used were DH5α, to clone fragments of NTHi DNA; DD12, as the host strain in conjugations; and SU101 or SU202 as the reporter strains for the homodimerization and heterodimerization assays, respectively [All cassette  were groectively ,15. T4 bvapDHi allele, along with 269 bp upstream and 227 bp downstream, was PCR-amplified using primers vapHI forward 5'-TATGTCTAGACAGTCGCTTCATAAGC-3' and vapHI reverse 5'-CCATTCTAGATTTGAGGTTAAATATGG-3'. Both primers included a XbaI site (underlined) and amplified Rd genome coordinates 472803 to 473572. The product was sequenced and cloned both into the XbaI site of pBluescript SK+ (creating plasmid pDD562) and into the NheI site of pDD515, a mobilizable broad-host range vector that could be conjugally transferred into and stably maintained in H. influenzae [vapDHi. The same primers were used to PCR amplify the 135 bp truncated vapDHi allele from strain R2866, the product of which was cloned into the NheI site of pDD515, creating pDD594. The insert was sequenced, then the plasmid was conjugally transferred into both Rd vapDHi and wild-type Rd. Conjugations were carried out as previously described [vapDHi by an aminoglycoside phosphotransferase gene (aph (3')II). Specifically, the 2184 bp BamHI fragment from pTSTE [BsaBI site of vapDHi. For the homodimerization assays, the plasmids pDD559 and pDD577 were derived from pSR658 and carried the NTHi strain R3001 vapDHi allele or the NTHi strain R2866 vapDHiallele fused in-frame to the wild-type LexA DNA-binding domain, respectively [vapDHi allele fused in-frame to the mutated LexA DNA-binding domain.For transcomplementation, the 276 bp R3001 fluenzae , creatinescribed . For allvapDHi genes in Rd and strain R3001 were disrupted by allelic exchange. Briefly, strains Rd and R3001 were made competent using the M-IV media technique [XmnI was used to transform each strain. Transformants were selected on chocolate agar supplemented with 15 μg/ml ribostamycin sulfate . The insertion in vapDHi was confirmed by Southern blotting using a digoxygenin-labeled denatured PCR fragment of vapDHi as the probe. The orientation of the aminoglycoside phosphotransferase cassette was determined by PCR using a primer that originated inside the aph (3')II gene and another that flanked vapDHi. The resistance gene was found to be transcribed in the opposite orientation of vapDHi in both strains.The echnique  and pDD55 cells per well and used 48 to 72 hours after seeding.Human brain microvascular endothelial cells (HBMECs) were a gift from K. S. Kim . Cells w5 cells per well and used 72 to 96 hours after seeding.NCI-H292 human respiratory epithelial cells  were passaged in collagen-1 coated T-25 flasks and monolayers for invasion assays were grown to confluency in 12-well collagen-1 coated BioCoat plates (Becton Dickinson, Bedford, MA). NCI-H292 media consisted of 870 ml RPMI 1640 medium with 25 mM HEPES and 2 mM L-glutamine, 10 ml of 100 mM MEM sodium pyruvate solution, 10 ml of 7.5% w/v sodium bicarbonate solution (Gibco, Grand Island, NY), 10 ml of 450 mg/ml filter-sterilized glucose solution, and 100 ml heat-inactivated fetal calf serum per liter. As above, media was changed every two days and cells were passaged every 3–5 days. Monolayers were seeded at a density of ~2.5 × 106 CFU of H. influenzae in a volume of 1 ml per well of a 12-well plate (an MOI of ≤ 10:1). After a 3 or 18 hour incubation in an atmosphere of 5% CO2 at 37°C, each monolayer was extensively washed with Dulbecco's PBS and 1.5 ml of media containing 100 μg/ml gentamicin was added to each well. Following a subsequent one hour incubation in the antibiotic, the wells were again washed extensively, harvested with 1% saponin, diluted in PBS-G and plated on chocolate or sBHI agar for viable intracellular CFU/ml. To quantitate total cell-associated bacteria (both intracellular and adherent), wells were also harvested and plated after the first wash and prior to gentamicin addition.Gentamicin-resistance invasion and survival assays were performed on HBMEC and NCI-H292 monolayers as previously described . Brieflyt-test was used and P-values of <0.05 were considered to indicate statistically significant differences.Statistical analyses were performed using the statistical analysis functions of Microsoft Excel (Microsoft Office 1997). For most comparisons of data, the Student's vapDHi cDNA.Total RNA was isolated using the SV Total RNA isolation system (Promega, Madison, WI) from the wild type strain Rd recovered from the media of 18-hour invasions of either HBMEC  or NCI-H292  monolayers. Standard procedures were used, with the modification that two separate DNAseI incubations were performed instead of the single one recommended with the kit. RT-PCR using the MasterAmp RT-PCR kit was then performed as per the manufacturer's instructions (Epicentre Technologies, Madison, WI). Negative controls of no reverse transcriptase added to the RNA followed by traditional PCR using Biolase DNA polymerase (Bioline, Madison, WI) were used to ensure that both RNA preparations were free of contaminating DNA. The primers for RT-PCR, 450 RT for (5'-CAGGCTTATACAGACATTGG-3') and 450 RT rev (5'-TCGTACCGACTGAGAAATCC-3') amplified a 153 bp internal portion of the vapDHi alleles from strains R3001 (full-length) and R2866 (truncated) were amplified by PCR and fused in-frame to the LexA DNA-binding domain (DBD) in pSR658, resulting in pDD559 and pDD577, respectively, and used to transform the reporter strain SU101 for homodimerization assays [lacZ gene controlled by a wild-type LexA operator site [lacZ, resulting in diminished levels of beta-galactosidase. The vapDHi allele from strain R3001 was also fused in-frame to the mutated LexA DNA-binding domain in pSR659, creating pDD561. The compatible plasmids pDD561 (full length vapDHi) and pDD577 (truncated vapDHi) were both used to transform the reporter strain SU202 for heterodimerization assays. Strain SU202 [lacZ gene controlled by a LexA operator, but this operator site is engineered such that only a mutated LexA DBD subunit (coded on pSR659) can bind to one half-site, while a wild-type LexA DBD subunit (coded on pSR658) can bind to the other half-site. Consequently, only a heterodimer composed of one mutated LexA DBD fusion subunit and one wild-type LexA DBD fusion subunit could bind to this engineered site and decrease transcription of lacZ in SU202. Three independent beta-galactosidase assays were carried out in triplicate as previously described [The n assays . Brieflytor site . If a hoin SU202  also hasescribed .DAD conceived of the study, carried out the protein-protein interaction and molecular genetics work, and drafted the manuscript. JJ carried out the intracellular survival experiments. ALS supported the study and participated in its design and coordination. All authors read and approved the final manuscript.


The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown.Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart.The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus. The Coxsackie and adenovirus receptor (CAR) ,2, a train vitro [Interest in CAR stems from its function as the primary high affinity receptor for Ad serotype 5, the most commonly used adenoviral vector in gene therapy protocols. CAR expression is the main determinant in gene transfer to normal tissue as ectopic expression of CAR in transgenic mice leads to several magnitudes of increase in adenovirus transducibility of tissues that are otherwise refractory to Ad-mediated gene expression -17. As win vitro . Thus, tin vitro . In contin vitro . Despitein vitro . This suin vitro  as well in vitro  with altin vitro ,5 [Fig. mdx mouse, CAR is re-expressed [To immunolocalize CAR, frozen sections of normal human muscle biopsies were probed with polyclonal antibodies raised against the extracellular domain of CAR  ab 2240024. We lxpressed  as is NCxpressed .CAR has been suggested to serve as a structural component at the intercalated discs in murine cardiac muscle  and receBoth of the cytoplasmic variants contain a PDZ recognition motif at their distal end, implicating CAR as a putative member of multiprotein complexes. This is indeed the case in polarized epithelial cells in which CAR is expressed at the tight junction where it associates with the tight junction scaffolding protein ZO-1  and contIt is interesting that a correlation can be drawn between disease incidence and expression levels of the CAR receptor. Cases of viral myocarditis in the human population outnumber those of myositis. This may be attributable to the difference of endogenous CAR expression between the two tissues. Considering that induction of CAR accompanies myocarditis  and its CAR is a novel member of the neuromuscular junction. In cardiac muscle, both CAR isoforms are found at the intercalated discs. The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.Figure To raise the chicken anti-CAR antibody (ChCT), purified His-tagged fusion protein encoding the C-terminal portion of CAR that is common to both isoforms (amino acids 259–339) was emulsified in an equal volume of TiterMax Gold adjuvant (CytRx Corp., Norcross, GA) and injected intramuscularly into chickens. One month post-injection, IgY antibodies to CAR were obtained from the eggs of injected chickens and subjected to affinity purification.Immunolabelling was performed using standard techniques. Briefly, frozen sections (5 μm) of normal human skeletal and cardiac muscle biopsies, murine skeletal and cardiac muscle were fixed in 2% paraformaldehyde (pH 6.8) for 1–2 minutes, followed by overnight incubation at 4°C with the primary antibodies . Incubation with a mouse anti-rabbit biotin-conjugated secondary ab  was followed by Cy-3-conjugated streptavidin . Controls consisted of sections treated in the absence of primary antibody. Neuromuscular junctions were revealed with Alexa-488-conjugated-α-bungarotoxin [α BTX] (1:40) (Molecular Probes, Eugene, OR). Slides were viewed on a Leica microscope-based imaging system using OpenLab imaging software (Quorum Technologies, St Catharines, ON).Cardiac muscle tissue was homogenized in extraction buffer [1% Triton X-100; 0.1 mM EDTA; 0.1 mM EGTA; 50 mM Tris-HCl; pH 8.0; with protease inhibitors (Roche)] at 4°C. After a 30 second sonication, samples were centrifuged at 3000 × g for 30 seconds at 4°C. Protein samples (10 μg) were anayzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (w/v) polyacrylamide gels, followed by electrotransfer to nitrocellulose. The blots were blocked in 10% BLOTTO (skim milk powder) in Tris buffered saline – Tween 20 (TBS-T) for 45 minutes at room temperature. Anti-CAR antibody was added in 10% BLOTTO at a dilution of 1:2500. Following incubation with peroxidase-labeled goat-anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories), the signal was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Baie d'Urfe, QC).Immunoprecipitation was carried out on cardiac muscle homogenates that had been pre-cleared with Protein-A Agarose slurry (Sigma), followed by overnight incubation at 4°C with RP291 or RP194. The samples were further incubated with Protein-A Agarose for 2 hours, washed twice with extraction buffer and then eluted with 2 X Laemmli SDS sample buffer with 5% mercaptoethanol. Following SDS-PAGE and electrotransfer, nitrocellulose membranes were probed with a primary polyclonal chicken anti-CAR (ChCT) in blocking solution (diluted 1:500) overnight at 4°C. Signal was revealed following incubation with a peroxidase-conjugated donkey anti-chicken IgY (Jackson Immunoresearch Laboratories) at a dilution of 1:2500 for 40 minutes, and enhanced chemiluminescence.CAS performed the immunoprecipitation, the immunoblotting, participated in the immunofluorescent staining experiments and drafted the manuscript. PCH and JN conceived the study, participated in its design and interpretation as well as drafting the manuscript. CA participated in the immunofluorescent staining experiments. KS prepared and characterized the isoform-specific antibodies. MS and GK participated in the design and interpretation of the studies. All authors read and approved the final manuscript.


How do people subjected to the endless dark days of winter in the far northern latitudes maintain normal daily rhythms? Though many might feel like hibernating, a highly regulated internal system keeps such impractical yearnings in check. From fruit flies to humans, nearly every living organism depends on an internal clock to regulate basic biological cycles such as sleep patterns, metabolism, and body temperature. And that clock runs on similar molecular mechanisms.Specific clusters of neurons in the brain are known to control the biological clock. Scientists believed these brain “clock cells” function as independent units. But new research described in this issue shows that the neurons do not act in isolation; rather, they collaborate with other neurons in a cell-communication network to sustain the repeating circadian rhythm cycles.Drosophila), called ventral lateral neurons, do indeed support the fly's circadian rhythms during periods of constant darkness and that the molecular expression patterns associated with these rhythms continue to cycle as well within other clock cells. These sustained expression patterns, however, require intercellular communication between different groups of brain clock cells.Clock cells within the brain maintain an organism's circadian rhythms, even in the absence of cyclical environmental signals like light, in a state scientists call “free running.” Though it has long been clear that the circadian rhythms of an organism persist under such free-running conditions (for example, constant darkness), it was thought that the gene-expression patterns within the cells governing these biorhythms did not require any external, or extracellular, signals to continue ticking. In experiments described here, Michael Rosbash and his colleagues show that the key brain clock cells in fruit flies  secreted by the ventral lateral cells.The PDF neuropeptide protein was thought to connect the molecular expression pattern of the ventral lateral neurons with the manifestation of circadian rhythms, but the researchers found evidence of a larger influence. When mutant flies lacking a functional PDF gene were exposed to constant darkness, the molecular expression patterns gradually stopped. The scientists say this suggests that the ventral lateral neurons and the PDF protein it produces help coordinate the entire neural network that underlies circadian rhythms.


The HIV epidemic is continuing to grow, year by year. According to the Joint United Nations Programme on HIV/AIDS (UNAIDS), in 2004 there were more people living with the virus than ever before, and in the same year more people than ever before died of it. So, although in the developed world HIV/AIDS is a controllable disease, one with which a treated person might expect to have a near normal lifespan, in much of the rest of the world AIDS is still a death sentence. Despite the fact that the cost of AIDS medicine has come down to around $150 per year in the developing world—a much lower cost than previously—the drugs are still unavailable to the vast majority of patients. What is more, every infected person has the chance of infecting many others. Although huge sums of money have been poured into combating HIV/AIDS—around US$4.7 billion in 2003—UNAIDS estimates this amount is less than half of what is required by 2005, and only a quarter of what will be required by 2007, to mount a comprehensive response to AIDS in low-income and middle-income countries. One of the real dilemmas, therefore, of HIV/AIDS policy is deciding whether it is better to concentrate resources on prevention of infections or on treatment of infected individuals. Each approach has ramifications for the other, as shown by the experience in some developed countries, where an increase in availability of treatment has been accompanied by an increase in risk behavior.PLoS Medicine suggests that trying to concentrate on one or the other of these alternatives is a false dichotomy, and that not integrating the two approaches could have a catastrophic effect on the global toll of HIV/AIDS by 2020. In this theoretical paper the authors analyze the epidemic in sub-Saharan Africa (where three-fourths of deaths from AIDS occur). With no change in current levels of prevention and care, it is predicted that there will be 3.7 million new HIV infections and 2.6 million adults dying of AIDS in this region each year within the next two decades. The authors predicted that concentrating on prevention alone could decrease yearly infections by half, and that concentrating on treatment could decrease yearly infections by 6%. However, combining both approaches could yield substantially greater benefits than the sum of the two alone—lowering projected new infections by 74% and projected annual mortality by half. These percentages translate into 29 million new infections and 10 million deaths averted between 2004 and 2020.An analysis by Joshua Salomon and colleagues in this month's The challenge now is obviously how to put these policy suggestions into practice. The current World Health Organization treatment target of having three million people on antiretroviral therapy by the end of 2005 (the “3 by 5” objective) provides a yardstick for only one part of the equation. The authors comment that the mobilization of communities that will be needed to achieve the 3 by 5 objective should also be harnessed for prevention, and that those who teach prevention must also be allowed to get care for those infected. As the authors say, only by doing so “will we at last move from slogans to impact.”


Recent studies have shown that honeybees flying through short, narrow tunnels with visually textured walls perform waggle dances that indicate a much greater flight distance than that actually flown. These studies suggest that the bee's “odometer” is driven by the optic flow (image motion) that is experienced during flight. One might therefore expect that, when bees fly to a food source through a varying outdoor landscape, their waggle dances would depend upon the nature of the terrain experienced en route. We trained honeybees to visit feeders positioned along two routes, each 580 m long. One route was exclusively over land. The other was initially over land, then over water and, finally, again over land. Flight over water resulted in a significantly flatter slope of the waggle-duration versus distance regression, compared to flight over land. The mean visual contrast of the scenes was significantly greater over land than over water. The results reveal that, in outdoor flight, the honeybee's odometer does not run at a constant rate; rather, the rate depends upon the properties of the terrain. The bee's perception of distance flown is therefore not absolute, but scene-dependent. These findings raise important and interesting questions about how these animals navigate reliably. After outdoor flight, honeybees represent distances they have travelled to find food differently, depending on the visual properties of the landscape When a scout honeybee discovers an attractive patch of flowers, she returns to the hive and performs the famous “waggle dance” to advertise the location of the food source to her nestmates . The danmax−Imin)/(Imax+Imin),(I where maxI and minI are the intensities of the bright and the dark bars of the grating, respectively.) At contrasts below 20%, the strength of the odometric signal starts to decrease. Another recent study revealed that the visual odometer of the honeybee is “color-blind” and driven exclusively by the green receptor (In a recent study, this question was explored by training bees to fly into a short, narrow tunnel (as described above), and analyzing the waggle dances of the returning bees as the texture and the contrast of the patterns lining the walls and the floor were systematically varied . The patreceptor .Here we examine the robustness of the honeybee's odometer when it is performing in natural conditions, during flight through varying landscapes. Specifically, we compare the strength of the odometric signal during flight over land, where contrasts tend to be relatively high and textures are relatively rich, to that during flight over water, where contrast tends to be low and texture is sparse. The experiments described here involve training bees to fly over stretches of water or land, and comparing their waggle dances.These experiments also provide us with the opportunity to address two further questions that are somewhat controversial and not yet completely resolved. One question relates to whether bees can, and do, fly safely over large bodies of water, such as lakes. In this experiment the feeder route was initially over land (segment 1), then over water (segment 2), and finally again over land (segment 3) . As the The situation with unmarked honeybees recruited to the feeder by the marked scouts was rather different. A few recruits were observed at the feeder as long as the feeder was on land. Once the feeder was on the water, however, very few recruits were observed. Recruits reappeared when the boat reached the island. In fact, they were observed searching at the shore of the island shortly before the boat reached that point. Recruits were present at all feeder positions on the island.For each feeder position along the route, the dances of the returning bees were filmed and analyzed as described in p < 0.02, pairwise comparison, Student t-test). On the other hand, there is no significant difference between the slopes for the land (segment 1) and the island (segment 3) segments (p > 0.2, Student t-test). Within each segment, the data fit a straight line quite well; the rms error between all of the data points and the piecewise linear approximation is 14.7 msec. This value is considerably lower than the rms error produced by the single straight line in Is this a good fit to the data? Visual inspection of the data in In experiment 2 the feeder route was of the same total length as in experiment 1, but was entirely over land see . In thisp > 0.10, pairwise comparison, Student t-test). The rms error between the piecewise linear approximation and the data was 41.6 msec, a value similar to that obtained with the single, best-fitting straight line (37.9 msec, p > 0.05, Student t-test).The variation of the mean waggle duration with feeder position for this experiment is shown in It is worth noting, however, that, in experiment 2, the three data points in the middle of segment 3 A and 3B We may summarize the findings of experiments 1 and 2 by saying that the mean waggle duration increases more rapidly with distance flown when bees fly over land, than when they fly over water. The slope of the mean waggle duration versus distance curve is three to four times greater over land. In other words, land provides a stronger odometric signal than does water.t-test results, than the slope in segment 1 (p < 0.01) and the slope in segment 3 (p < 0.001).Similar results were obtained when experiment 1 was repeated 3 weeks later using a different colony of bees. The new colony was placed in the same shed and allowed 10 d to acclimatize itself to the new location and familiarize itself with the surrounding terrain before the experiment commenced. The feeder was moved along exactly the same route as the original experiment 1.  were sparse and usually farther away than the ground, except in certain rare circumstances (see below).Our results demonstrate, first of all, that bees can be trained to fly reliably and without accident over stretches of water that span a few hundred meters. This has also been reported in earlier studies , althougAs in earlier studies, we observed that individually marked bees trafficked regularly between the hive and the feeder, regardless of whether the feeder was on land or water. The short flight times (of a minute or two across the whole stretch of water, as established by radio communication) indicated that they had no difficulty in flying the direct route between origin and destination. We found, however, that the feeder attracted far fewer recruits when it was positioned over water. Although the scout bees that were trained to visit the feeder on land readily followed the feeder when it was moved over the water, recruits failed to appear once the water was reached. This was not because the trained scout bees ceased to dance once the feeder was over the water; they continued to dance with high vigor, and their waggles encoded a position over the water. Recruits reappeared when the feeder arrived at the island; in fact, they seemed to “anticipate” its arrival by patrolling the shore of the island even before the boat had landed there. Recruits were observed all along the route on the island, as well as along the entire stretch of the route used in experiment 2, which was exclusively over land.One possible explanation for the lack of recruitment when the feeder was over water could be the relatively low slope of the distance indication curve in this region, which would lead to a less precise indication of the position of the feeder, making it harder to pinpoint. This explanation seems unlikely, however, since the boat in which the feeder was carried was large and conspicuous, and was the only object on the water.Another possible reason for the lack of recruitment could have to do with the lack of assistance from experienced foragers. A recent study showed that experienced foragers can sometimes aid recruits in pinpointing a feeder by synchronizing their flights and performing buzzing flights around an unscented feeder . Howevertrained bees fly at about the same height over land as they do over water (see below).A third possibility is that, when inexperienced recruits fly over water, they fly at a higher or lower altitude than the trained scouts, and thus experience a different magnitude of optic flow. Consequently, they may search for the feeder at the wrong location. This possibility remains to be explored. What our observations do suggest, however, is that the A fourth possibility relates to the controversial hypothesis, advanced by The primary contribution of the present study, however, is the demonstration that the honeybee's odometer does not run at a constant rate in outdoor flight. The results shown in Figures There are a number of possible explanations for this finding. One reason could be that while flight over water is likely to stimulate only the ventral fields of view of the eyes with image motion, flight over land is likely to provide image motion signals in the lateral fields as well. In three earlier studies, honeybee odometry was investigated by training bees to fly through short, narrow tunnels lined with visual textures on the walls and/or or the floor. One of these studies  suggesteA second reason for the different sensitivities of the odometer to flight over land and water might be that bees increase their altitude when flying over water, thus reducing the extent of image motion that a given forward motion of the bee would elicit in the eye. Although we do not have precise information on flight heights in this study, visual observation of bees approaching the feeder over the water suggested that they flew at heights of between 1 m and 2 m above the water surface, similar to the heights at which they are reported to cruise over land . In factA third possible explanation has to do with the fact that the visual spatial texture of land could be considerably different from that of water. Investigation of this possibility would require a detailed analysis of the spatial frequency spectra of samples of land and water images, which we have not undertaken in the present study. However, a recent study examined the effects of varying visual texture on perceived distance flown in honeybees . In thatThe same study, however, also found that the odometric signal dropped substantially when the contrast of the pattern was reduced to a level below 20% . This fiRegardless of which of the above explanations is the valid one, our results indicate that the differences in the odometric signal between flight over land and over water are due to differences in the visual environment.p > 0.08). Thus, we interpret this as implying that the bees experienced very little optic flow during flight over this region. The final data point in section 3 shows an abrupt increase in waggle duration, compared to the previous three points. Interestingly, as we see from Of course, the visual properties of land terrain can also vary considerably, depending upon the nature of the vegetation and on the existence of manmade structures. We suggest that this is the reason for some of the local fluctuations in slope that are evident in the data over land. In particular, we noted in the Results section that, in experiment 2, the three data points in the middle of segment 3 A and 3B It is instructive to compare our results with the findings from an earlier study by von Frisch and Lindauer . In 1962p > 0.3, two-way ANOVA). The mean waggle duration increased much more rapidly on land than it does over water, just as Figures To investigate this issue more closely, we have measured four parameters of our dance data: mean circuit duration, mean waggle duration, mean return duration (the duration of the return phase of the circuit made during the dance), and mean dance tempo. The mean circuit duration is equal to the sum of the mean waggle duration and the mean return duration, and is proportional to the reciprocal of the dance tempo. The results of this analysis, for flights of the same distance over water and over land, are shown in Our present findings confirm the suggestions from earlier work  that theGiven that the honeybee dance does not convey accurate information on distance flown, how do scout bees returning from a new food source usually manage to recruit other bees to visit it so quickly and so effectively? One explanation might be that the waggle dance conveys information on the direction as well as the distance of the food source. Thus, potential recruits that are persuaded by the scout's dance to seek out the source would fly in the direction signaled by the scout, and therefore experience approximately the same visual environment as the scout. Consequently, any terrain-induced variations in the odometric signal would be the same for the recruits as well as the scout, so that such variations would not compromise accurate pinpointing of the destination. The recruit would find the goal simply by flying in the specified direction until her odometric signal matched that indicated by the scout's dance. Thus, even though the scout's dance does not indicate distance in absolute terms, the recruits end up close to or at the correct location because their odometric signals evolve in the same way as that of the scout during the flight toward the food. Once the recruits are in the vicinity of the food source, the experienced foragers can assist them by providing visual and/or olfactory cues in the vicinity of the feeder to guide recruits to it .The experiments were conducted on the shore of Lake Burley Griffin in Canberra. A two-frame observation hive was set up 190 m from the shore of the lake, with the entrance facing the shore see . The gro(Apis mellifera) from the same hive. In experiment 1, bees were trained to fly a route due southwest toward the island. This route comprised an initial stretch over land, followed by a stretch over water, and then again over land (across the island), as shown in Two experiments were carried out, both using honeybees In experiment 1, about 20 bees were individually marked and trained to visit a feeder containing 1.0 M nonscented sucrose solution, initially placed 5 m from the hive. When the marked bees started to visit the feeder regularly, the feeder was moved step by step toward the shore. At each position, the dances of marked bees returning from the feeder were recorded at the hive. The distance from the hive to the lake shore was 190 m. The feeder was then placed in a rowboat and taken across the water, again stopping at several locations along the way to record dances back at the hive. In order to accustom the bees visually to the boat, we introduced them to the boat on land well before the water was reached. From about 50 m before the water's edge, the feeder was placed inside the boat. From this point on, the feeder was always in the boat, regardless of whether the location was on land or water. The stretch across the water was 240 m long. Upon reaching the shore of the island, the feeder (still placed in the boat) was moved stepwise across the island to the opposite shore, again recording the dances of bees returning from each location. The stretch across the island was 150 m long.The boat was an inflatable dinghy capable of carrying four adults in addition to the feeder. It was colored bright yellow to facilitate visual detection from a distance by the bees. A view of the boat on the lake is given in The weather was sunny and calm through most of the study, except for a few days (see below). The wind speed rarely exceeded 20 km/h. Most of the time, there were only a few ripples on the water surface. Temperature and weather conditions were recorded through the course of the experiment and were supplemented by records from the Canberra Meteorological Station.There were always at least two experimenters at the feeder (regardless of whether the feeder was on land or water), and two experimenters at the hive. The visit of each marked bee at the feeder, and its subsequent dance in the hive, were followed through radio communication between the feeder and the hive. At each feeder position, recording of a given bee's dances was commenced after it had made three visits to the feeder. This was done to allow adequate time for the dances to adjust to each new feeder position. Data were collected from between seven and 14 different individually marked bees at each feeder position. This procedure required a stay of about 30–60 min at each feeder position.In experiment 2, about 20 bees were trained by moving a feeder step by step from the hive, as in experiment 1, but along a route that was entirely over land, as described above. The total length of this route (580 m) was identical to that in experiment 1, thus enabling a direct comparison of the of the bees' dances along the two routes.The observation hive was housed in a specially constructed shed that afforded a weatherproof environment for observing and filming bee dances. Dances of marked bees were filmed at 25 frames/s, using a Sony (Tokyo, Japan) DCR-TRV310E video camera mounted on a tripod placed adjacent to one face of the observation hive, near the entrance. A mechanical gate at the entrance to the hive ensured that bees entered (and left) the hive only on the side facing the camera, thus facilitating the filming of dances.Bee dances were analyzed frame by frame to measure the mean duration of the waggle phase. The waggle duration was considered to be a measure of the bees' perception of the distance flown from the hive to the feeder: the longer the waggle duration, the greater the perceived distance . A totalThe dances were evaluated as follows. For each dance, the mean waggle duration was estimated by averaging the waggle durations over all loops. Then, the mean waggle duration for each bee was obtained by averaging the mean waggle durations over all of its dances at that feeder position. Finally, the mean waggle duration of all bees was calculated from the mean waggle durations for the individuals. The standard error of the mean was also calculated and displayed in the graphs , which produced color images of 1200 × 1600 pixel resolution. Sections of these images were analyzed to compare the mean visual contrast over land with that over water. The intensity of the image at each pixel was taken to be the average of the values of the three color subpixels at that location. These images were taken on a calm day with weather conditions similar to those on which the experiments were conducted. The surface of the lake was smooth, with relatively few ripples.The mean contrast in an image section was computed as the ratio of the standard deviation to the mean value of the intensities of all the pixels within that section, and was expressed as a percentage. It was recently demonstrated that the odometer is “color-blind” and is driven primarily by the green receptor channel , as are Figure S1(1.8 MB JPG).Click here for additional data file.Video S1(27.8 MB AVI).Click here for additional data file.


Integrin-linked kinase (ILK) promotes tumor growth and invasion. Increased ILK expression is correlated with progression of several tumor types, but the expression of ILK has not been investigated in patients with non-small cell lung cancers (NSCLCs).We investigated ILK expression in patients with NSCLC by means of immunohistochemistry.ILK expression was significantly associated with tumor grade, T status, lymph node metastasis and stage. . The 5-year survival rates for patients with strong and weak or no ILK expression levels were 20% and 59%, respectively: the difference was statistically significant (p < 0.0001). A multivariate analysis of survival revealed that ILK expression, T status, N status and vascular invasion were statistically significant prognostic factors .Our study demonstrates that increased expression of ILK is a poor prognostic factor in patients with NSCLC. Interaction of cells with the extracellular matrix regulates many physiological and pathological processes. These interactions are mediated by a large family of cell surface receptors known as integrins, which recognize several extracellular matrix proteins, including fibronectin, collagens, and vitronectin . IntegriIntegrin-linked kinase (ILK) interacts with cytoplasmic domain of both β1 and β3 integrins and is activated by cell- extracellular matrix interactions . OverexpA total of 134 patients  with NSCLC were studied (consecutive cases). All patients in this study had undergone curative tumor resection in our department between 1995 and 1998. Patients who died within 30 days after surgery and those who underwent exploratory thoracotomy were excluded from the study. Patients with a past history of another type of cancer were also excluded. With regard to histological type, 75 were adenocarcinomas, 54 were squamous cell carcinomas and 5 were large cell lung carcinomas. The lesions of these 134 patients were staged on both operative and pathologic findings according to the UICC TNM classification  (1997). Resected tissue specimens were fixed in formalin, embedded in paraffin, and cut into 3-μm serial sections. The sections were subjected to hematoxylin-eosin staining and immunohistochemical analysis with ILK. A mouse anti-ILK monoclonal antibody (Santa Cruz Biotechnology, CA, USA) was used, and immunohistochemical staining was based on the avidin-biotin-peroxidase complex method and was performed with a Vestastatin kit (Vector, Burlingame, CA). Negative control sections were treated using nonspecific IgG in the primary antibody.Immunoreactivity was graded as from - to +++, according to the number of cells stained. Grades was defined as follow as: -, no positive cells; ±, less than 5% of tumor cells showed immunoreactivity; +, 5–50% of tumor cells showed immunoreactivity; ++, 50–80% of tumor cells showed immunoreactivity; +++, over 80% of tumor cells showed immunoreactivity. Grades ++ and +++ were regarded as strong expression, and grades ± and + were regarded as weak expression. The results of immunohistochemical staining were evaluated independently by three observers with no prior knowledge of patients' clinical data. The evaluation was suitable for 96% of the samples. The other specimens (4%) were re-evaluated independently, then classified according to the classification given most frequently by the observers. In this study, we compared the group of tumors of strong ILK expression with the group of tumors of weak or no ILK expression.The association between ILK expression and clinical data was statistically evaluated by using Mann-Whitney U test or the chi-square test. Survival curves were calculated by the Kaplan-Meier method and were compared using the log-rank test. The correlation of variables with survival was analyzed by multivariate analysis using a Cox proportional hazards model. All statistical analyses were performed using the StatView software package (Abaracus Concepts, Berkeley CA). A P value of <0.05 was considered statistically significant.In non-neoplastic lung tissue, ILK was not detected in epithelial cells, while ILK expression was found in stromal cells including fibroblasts and infiltrating lymphocytes Figure . In the The 5-year survival rates for the groups with strong, and weak or no for ILK expression in their tumors were 20% and 59% respectively: the difference was statistically significant (p < 0.0001) Figure . The uniRecent studies have indicated that ILK facilitated the phosphorylation of AKT, which is required for AKT activation . ActivatIn the current study, we detected ILK expression using immunohistochemistry on tumors from patients with NSCLC. ILK was strongly expressed in 31% of tumor samples, whereas there was no ILK expression in noncancerous pulmonary tissue samples from the same patients, except for fibroblasts and infiltrating lymphocytes. These finding suggest that ILK expression may serve as a useful marker of NSCLC. ILK is very low in healthy cells. In cancer cells, however, ILK activity is often increased, possibly as a result of a malfunctioning of upstream components in the integrin and growth factor signaling pathways. We found a significant correlation between strong expression and advanced T status, N status and stage. No correlation was found age, gender or histology. These results suggest that ILK expression is correlated with tumor progression in NSCLC. Cases with strong ILK expression were reported to be significantly more frequent in advanced gastric carcinoma and advanOur study demonstrates that ILK expression is a poor prognostic factor in patients with NSCLC. Thus, the utility of the expression of ILK could open up a new window for the molecular marker and the treatment of NSCLC.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, α-, β-, and θ-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.Gallinacin 1–13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 β-defensin genes can be divided into two subgroups with Gallinacin 1–7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single β-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution.Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different β-defensins but with no other groups of defensins being discovered. These chicken β-defensin genes, designated as We conclude that the chicken genome encodes only β-defensin sequences and that all mammalian defensins are evolved from a common β-defensin-like ancestor. The α-defensins arose from β-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and θ-defensins have arisen from α-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity. HD5 are fully protected against the doses of Salmonella typhimurium that are otherwise lethal to the wide-type mice [S. typhimurium [Defensins constitute a large family of small, cysteine-rich, cationic peptides that are capable of killing a broad spectrum of pathogens, including various bacteria, fungi, and certain enveloped viruses -5. Theseype mice . Convershimurium .8–11-C-X3–5-C-X3-C-X9–12-C-X4–11-C-X1-C-X3-C; invertebrate defensin: C-X5–16-C-X3-C-X9–10-C-X4–7-C-X1-C; α-defensin: C-X1-C-X3–4-C-X9-C-X6–10-C-C; and β-defensin: C-X4–8-C-X3–5-C-X9–13-C-X4–7-C-C. The α- and β-defensins are unique to vertebrate animals with α-defensins only being found in rodents and primates, while β-defensins are present in all mammalian species investigated [Defensins have been identified in species ranging from plants, insects to animals and humans -5. CharaAnalysis of human and mouse genomes indicated that β-defensins form 4–5 distinct clusters on different chromosomes with each cluster consisting of multiple defensin genes . InteresExistence of a large number of expressed sequence tag (EST) sequences and recent completion of chicken genome sequencing at a 6.6× coverage  providedGal 1–3) [Gal 4–12, have been found in the EST database with at least two hits for each, and such sequences have also been confirmed in genomic sequences , EST, high throughput genomic sequence (HTGS), and whole-genome shortgun sequence (WGS) databases in the GenBank by using the TBLASTN program. All potGal 1–3) ,23, nine GENSCAN  and confGal genes constitute the entire repertoire of the β-defensin family encoded in the chicken genome. Although it is highly unlikely, we could not rule out the possibility that additional defensin-related genes with distant homology might be uncovered in the chicken by different computational search methods such as the use of Hidden Markov models [No other sequence containing β-defensin-like six-cysteine motif has been found in NR, EST or genomic databases, suggesting that 13 v models ,15. It iSimilar to Gal 1–3, 10 novel β-defensins, deduced from either EST or genomic sequences, vary from 63 to 104 amino acid residues in length. Alignment of these peptides revealed a conservation of the signal sequence at the N-terminus and the characteristic six-cysteine defensin motif at the C-terminus , while mean pN was 0.369 ± 0.040. In the mature defensin region, mean pS was 0.673 ± 0.027, while mean pN was 0.534 ± 0.051. Mean pN in the mature defensin was significantly greater than that in the propeptide , indicating lesser functional constraint on the amino acid sequence of the former. The high mean pS shows that chicken β-defensin genes have not duplicated recently, unlike β-defensin genes of the bovine [Gal6 and Gal7), mean pS in the mature defensin was 0.221 ± 0.082, while mean pN was 0.331 ± 0.076. While these values are not significantly different at the 5% level, the fact that pN was higher than pS suggested that positive selection may have acted to diversify the mature defensin region between these two genes.Comparison of the numbers of synonymous and nonsynonymous nucleotide substitutions provides a powerful test of the hypothesis that positive Darwinian selection has acted to favor changes at the amino acid level . This ape bovine . In the Gal genes except for Gal4 and Gal5. Later search of chicken WGS sequences released on February 29, 2004 confirmed the order of the genomic contig that we assembled and also revealed the locations of two remaining genes, Gal4 and Gal5, both of which reside on a WGS (accession no. AADN01058096) that overlaps with AC110874  using the TAM31-54I5 BAC DNA as probe  sequences, TAM31-54I5 (accession no. AC110874) and CH261-162O9 (accession no. AC146292), both of which were sequenced and deposited earlier by one of us (J.F. Chen). Alignment of these two sequences allowed to re-order three DNA fragments in AC110874 and to construct a continuous, gap-free genomic contig that includes 11 Gal gene. Unlike most mammalian β-defensin genes, which primarily consist of two exons and one intron, the Gal genes were found to be composed of four short exons separated by three introns with variable lengths ranging from 117 bp to 3,322 bp  and the majority of the last exon encodes 3'-UTR as well as a few C-terminal amino acids, two internal exons resemble mammalian β-defensin genes in that one exon encodes the signal and pro-sequence and the other encodes the mature sequence with six-cysteine motif [Gal genes have joined together during the evolution as a result of exon shuffling, which occurred in many other evolutionarily conserved gene families [Comparing the cDNA with genomic sequences also revealed the structure of each ne motif ,27-29. Afamilies , includiGal1 and Gal2 are expressed in bone marrow and lung, while Gal3 is more preferentially expressed in bone marrow, tongue, trachea, and bursa of Fabricius [Gal genes that we identified, RT-PCR was performed with a panel of 32 different chicken tissues. Similar to Gal 1–3, Gal 4–7 are highly restricted to bone marrow cells with Gal5 also expressed in tongue, trachea, lung, and brain at lower levels  are predominantly expressed in bone marrow and the respiratory tract, whereas the other six genes  are more restricted to liver and the urogenital tract. However, the functional significance and transcriptional regulatory mechanisms of these genes during inflammation and infection remain to be investigated.It has been shown that abricius . To studCTSB (Cathepsin B, accession no. NP_680093) and a human EST sequence (accession no. BE072524) immediately located centromeric to chicken defensins, were also found to be conserved in the defensin gene clusters on human chromosome 8p22 and mouse chromosome 14C3. Similarly, another gene, HARL2754 (accession no. XP_372011) that is 6-Kb telemetric to Gal4 is also conserved in another defensin cluster in human (8p23) or mouse (8A1.3) . In addition, a group of homologous β-defensin-like sequences, namely crotamine and myotoxins, have been found in several s snakes , which as snakes  is apparIn addition to the structural conservation between β-defensin-like sequences in the rattlesnake and mammals , a growiWe have showed that chicken genome encodes a total of 13 different β-defensin genes clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7, but with no α-defensin genes. These peptides exhibit homology to different subgroups of mammalian β-defensins-, consistent with the hypothesis that α-defensins and β-defensins arose by gene duplication after the divergence of birds and mammals. The θ-defensins are specific to primates; and thus appear to have arisen from α-defensins by gene duplication specific to the primate lineage. Apparently, the evolution of defensins is rapid and driven by duplication and positive diversifying selection. Collectively, this study represents the first large-scale detailed investigation of defensins in non-mammalian vertebrates. There is no doubt that further analysis of these defensin genes will lead to a better understanding of host defense mechanisms and evolution of innate immunity.To identify novel defensins in the chicken, all known cysteine-containing defensin-like peptide sequences discovered in plants, invertebrates, birds, and mammals were individually queried against the translated chicken NR, EST, HTGS, and WGS databases in the GenBank by using the TBLASTN program  with defS) and the proportion of nonsynonymous nucleotide differences per nonsynonymous site (pN) were estimated by the method of Nei and Gojobori [Multiple sequence alignment was constructed by using the ClustalW program (version 1.82) . A phyloGojobori . Again, To generate a continuous defensin gene cluster, the HTGS and WGS sequences containing the putative defensin genes were retrieved from the GenBank, aligned to generate a longer contig, which was confirmed later by searching through the assembled chicken genome released on February 29, 2004, by using the BLAT program  under thGal genes. Metaphase chromosome speads were prepared from mitogen-stimulated chicken splenocyte culture as we described [Fluorescence in situ hybridization (FISH) was used for chromosomal assignment of the chicken β-defensin gene cluster by using the BAC clone TAM31-54I4 as probe, which harbors 11 escribed ,42. The Total RNA was extracted with Trizol (Invitrogen) from a total of 32 different tissues from healthy, 2-month-old chickens (see Figure et al. reported independently discovery of seven novel chicken β-defensins in the chicken EST database by using homology search strategies [Gal 4–7 are primarily in bone marrow, while other genes are more restricted to liver and the genitourinary tract.Following submission of this manuscript, Lynn rategies . ConsistAbbreviations: Gal, Gallinacin; NR, nonredundant; EST, expressed sequence tag; HTGS, high throughput genomic sequence; WGS, whole-genome shortgun sequence; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; UTR, untranslated region; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.YX carried out the tissue collection, RT-PCR analysis of tissue expression patterns, and drafted the manuscript. ALH carried out the phylogenetic and molecular evolutionary analyses. JA and YM carried out the fluorescence in situ hybridization. JFC carried out the sequencing of two chicken defensin-containing BAC clones. DSN participated in tissue collection and preparation. GZ conceived of the study, carried out all computational analyses and annotation, drafted the manuscript, and participated in its design and coordination. All authors read and approved the final manuscript.