Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study  Vibrio cholerae, the cause of a potentially fatal diarrheal illness, it has not previously been possible to identify precisely when or where virulence determinants are produced in the intestine. We addressed this question by investigating the expression of virulence genes by individual V. cholerae during infection of the small intestine. Virulence genes were found to be powerfully expressed early in the infectious process by bacteria in close proximity to epithelial surfaces. Increased replication rates were also localized to epithelial surfaces. During later stages of the infection, the population of V. cholerae bifurcates into two fractions: one subpopulation continues to express virulence genes, whereas these genes are silenced in the other subpopulation. The genetic program controlling the continued production of virulence genes may mediate the persistence of a hyper-infectious subpopulation of bacteria in the stools of cholera patients.Most pathogenic microorganisms infect in a stepwise manner: colonization of host surfaces is followed by invasion and injury of host tissues and, late in the infectious process, dissemination to other hosts occurs. During its residence in the host, the pathogen produces essential virulence determinants and often replicates rapidly, leading to a vast expansion of its biomass. Although this scenario is well established also for  Vibrio cholerae infecting the small intestine.Clinical and pathological studies of diverse bacterial pathogens disclose a common theme: the infectious process evolves as a series of spatial and temporal patterns during migration of the pathogen through different tissues and cellular compartments of the host. At each stage different microenvironments are encountered; in response to these, adjustments of the microbial transcriptome and phenotype are thought to occur: virulence genes are induced or silenced and changes in replication rate take place. Spatial differences at the site of infection are magnified by differences in scale between bacteria (microns) and infected host tissues (millimeters or centimeters) leading to situations where bacteria in the same organ could experience dramatically different microenvironments. Spatial and temporal sources of microbial heterogeneity can be compounded by stochastic events that cause cell-to-cell transcriptional and phenotypic differences between genetically-identical individuals in the same microenvironment V. cholerae is the etiological agent of cholera, a purging diarrheal illness that occurs in rapidly spreading, seasonally-determined epidemics in south Asia. Ingestion of contaminated water or food containing V. cholerae leads to colonization of the small intestine where production of a powerful enterotoxin induces epithelial cells to secrete water and electrolytes into the bowel lumen V. cholerae into a ligated ileal loop, bacteria migrated from the bowel lumen through the mucus gel to epithelial cell surfaces; eight hours post inoculation large numbers of V. cholerae collect on the surfaces of microvillae and fluid accumulation in the ileal loop lumen is evident; then, between 8 and 12 hours post inoculation, near synchronous detachment of bacteria from the mucosal surface occurs heralding the mucosal escape response, a process that is associated with RpoS-dependent down regulation of virulence gene expression. In humans and in the murine and rabbit models of cholera, the infectious process is accompanied by prodigious intra-intestinal replication of the organism leading to a vast expansion of its biomass and to the possibility that the progeny of the infection, shed in feces, will spread to other susceptible hosts or re-enter natural aquatic reservoirs where the organism resides between outbreaks of human disease V. cholerae in natural water sources was the identification of sets of genes induced late in the infection of a murine model of cholera that appear to be involved with adaptation of the organism to aquatic environments V. cholerae mutants show that the clinical hallmarks of cholera require the combined production of two virulence determinants tcp operon and promotes small bowel colonization ctx operon and elicits a secretory response by small bowel epithelial cells. Expression of the tcp and ctx operons is coordinately regulated by the ToxR regulon Human volunteer studies of tcpA and ctxAB by most bacteria during the early stages of infection of a murine model V. cholerae in the fluid that accumulates in ligated loops of the rabbit ileum failed to identify significant expression of tcpA and ctxAB even though CT is required for fluid secretion in this model V. cholerae virulence genes are either expressed at a low level by most fecal V. cholerae or at a very high level by a small sub-population of the bacteria Studies of cholera animal models illustrate the difficulty of discerning where, when and by which subsets of the bacterial population key virulence determinants are produced. Earlier work based on a recombination based reporter system showed induction of V. cholerae also showed that freshly-passed cholera stools from naturally infected humans harbor bacteria that are hyper-infectious when tested in an infant mouse model V. cholerae through the infant mouse intestine also induces a hyper-infectious state that was shown to be TCP-dependent V. cholerae in the stools of cholera patients could promote the rapid dissemination of V. cholerae within households and communities during outbreaks.Studies of fecal In the study reported below we have used the rabbit ileal loop model of cholera and site specific expression profiling to identify virulence genes that are differentially expressed on epithelial surfaces. Further resolution was obtained through the use of single cell expression methods and confocal microscopy to precisely localize where and when in the intestine the gene encoding the principal repeating subunit of the TCP filament is expressed. Expression analysis of a ribosomal promoter, imaged by confocal microscopy at the single cell level of resolution, showed that TCP gene expression and growth are co-localized in the intestine. The capacity to capture the expression behavior of specific virulence genes by individual bacteria led to the discovery that genes encoding TCP and CT are controlled by an epigenetic bistable switch that bifurcates the population into TCP/CT-expressing and TCP/CT-non-expressing subpopulations. The study of mutants in the ToxR regulatory cascade showed that this switch requires autocatalytic regulation of the gene encoding the ToxT transcription factor and the CRP-cAMP complex. Together these observations provide a mechanistic explanation for the presence of a subpopulation of TCP-expressing, hyper-infectious bacteria in the stools of cholera patients.V. cholerae O1 El Tor (strain A1552) in the rabbit ileal loop model of cholera 4, 8 and 12 hours after inoculation have previously shown that V. cholerae resides in at least three anatomically distinct sites in the ileal loop at the same time point: the epithelial surface; the mucus gel overlying the epithelial surface; and, in fluid that collects in the lumen of the loop V. cholerae collected from two anatomically distinct sites of the same ileal loop at 4, 8 and 12 hours post inoculation. Because the epithelial surface and overlying mucus gel could not be separated, these contiguous sites were obtained as a single fraction. V. cholerae in luminal fluid were collected as a second fraction by isolating the liquid contents of incised loops.Confocal microscopy studies of GFP-labeled tcpA (VC0828), the gene encoding the principal repeating subunit of the TCP filament, was 30.0-, 17.0- and 11.5-fold up-regulated in this fraction 4, 8 and 12 hours post-inoculation, respectively, compared to its expression in mid log phase LB cultures. Also strongly induced in the mucus gel/epithelial cell fraction were the eight downstream genes which together with tcpA compose the operon VC0828 –VC0837 within Vibrio Pathogenicity Island I (VPI-1) (tcpF (VC0837), which encodes a soluble colonization factor tcpA, 4.9- to 3.2-fold lower than its expression in the mucus gel/epithelial cell fraction at the same time point. Even more striking was the localized expression of ctxA and ctxB which encode the A and B subunits of CT: both genes were strongly expressed in the mucus gel/epithelial cell fraction. By contrast, they were not significantly up-regulated in luminal fluid compared to the mid log phase reference  . One of eference . Taken ttcpA-F and ctxABtcpA-F operon, was expressed 2.8-, 2.5- and 1.4-fold greater in the mucus gel/epithelial surface fraction 4, 8 and 12 hours post inoculation, respectively, compared to its expression in the LB broth mid log phase reference  reporter at a separate site on the same chromosome. The GFP protein variant encoded by this reporter was modified by the addition of 11 amino acids to the carboxy terminus which targets it for destruction by the ClpXP protease system Escherichia coli), yielding a reporter capable of monitoring both increased and decreased activity of the promoter to which it is fused V. cholerae tcpA-gfp(ASV) reporter strain was phenotypically indistinguishable from the wild type parent with respect to growth and its capacity to colonize the ileal loop and elicit a secretory response. The strain emitted only weak background fluorescence during growth in LB liquid medium. However, significant induction of fluorescence from the tcpA-gfp(ASV) reporter was observed in AKI medium, which induces the expression of the ctxAB and tcpA-F operons toxR, the global regulator of ctxAB and tcpA-F expression, was deleted from the tcpA-gfp(ASV) reporter strain and the ΔtoxR reporter grown in AKI medium (data not shown). Taken together, these results demonstrate that the fluorescence from the tcpA-gfp(ASV) construct parallels the expression behavior of tcpA.The V. cholerae tcpA-gfp(ASV) was inoculated into rabbit ligated ileal loops and expression of the tcpA-gfp(ASV) fusion monitored by confocal microscopy as a function of time and site. In addition, all V. cholerae  were visualized in the same sample using an O1 antigen-specific antibody so that even bacteria not producing GFP could be identified and distinguished from GFP-producing bacteria. Since the tissue samples were washed before microscopy to remove bacteria present in the luminal fluid, only the bacteria attached to epithelial cell surfaces or residing in the mucus gel coating these surfaces were visualized. Four hours after inoculation of rabbit ligated ileal loops, confocal microscopy showed tcpA-gfp(ASV) expression especially by bacteria which had reached the epithelial surface (tcpA-gfp(ASV) expression was not evident at this time point for most non epithelial surface-associated bacteria that were located in the overlying mucus gel. This distinction was quantified by correlating GFP fluorescence intensity with distance from the nearest epithelial surface. Quantitative image analysis of the ratio between green fluorescence from GFP and red fluorescence from the V. cholerae specific antibody showed significantly stronger expression of tcpA-gfp(ASV) by bacteria ≤5 µm from an epithelial cell surface (tcpA-gfp(ASV) declined rapidly for bacteria at greater distances from the epithelium: bacteria further than 5–10 µm from an epithelial surface showed an average fluorescence intensity five-fold lower than bacteria 0–5 µm from an epithelial surface (tcpA-reporter strain shown in tcpA-gfp(ASV) reporter (tcpA-gfp(ASV) expression data from the 4 and 8 hour post-inoculation time points suggest that bacteria encounter tcpA-inducing signals as they approach or contact the epithelial cell surface. Whether these signals emanate from the epithelial cell as a kind of chemical gradient or require physical contact with the cell surface was not investigated. surface . By cont surface . The ave surface . Using treporter . Taken ttcpA 4 hours post induction, whereas the tcpA-gfp(ASV) reporter showed the strongest induction 8 hours post infection. Thus these two expression methods yielded somewhat different temporal profiles for tcpA expression. However it is not possible to directly compare results from these two expression methods because microarray experiments estimate the average gene expression magnitude of all bacteria in the population, whereas the tcpA-gfp(ASV) reporter captures gene expression magnitude at the single cell level.Microarray experiments showed the highest expression of tcpA expression from the mucus gel towards the epithelial surface was observed in some locations of the mucus gel where V. cholerae, located at significant distances from the nearest epithelial cell surface, were found to express tcpA-gfp(ASV). Systematic examination of these non cell-associated, tcpA-gfp(ASV)-expressing bacteria showed that they were mainly found in aggregates (denoted by arrows in tcpA-gfp(ASV). This aggregation-associated, tcpA-inducing phenomenon was evident at the 4, 8 and 12 hour time points and is consistent with the previously-reported TCP-mediated auto-aggregation phenotype An exception to the increasing gradient of tcpA-gfp(ASV) was markedly reduced at this time point compared to eight hours post-inoculation (tcpA-gfp (ASV) confirmed that RpoS is required for decreased tcpA expression during the mucosal escape response  fused to a growth rate-regulated promoter.Beginning with an inoculum of 10ur hours . Increasur hours . HoweverE. coli rrnB ribosomal operon was coupled to gfp(ASV) and the resulting rrnBP1-gfp(ASV) fusion inserted as a single copy between VC0487 and VC0488 on the large chromosome of the same wild type V. cholerae strain that had been used to study in situ expression of tcpA. We selected this promoter fusion because the fluorescence intensity of a Pseudomonas putida strain harboring an E. coli rrnP1-gfp(ASV) reporter accurately reflected both increased and decreased growth and thus could be used in conjunction with confocal microscopy to study bacterial growth rates in complex environments The ribosome synthesis rate and thus the concentration of ribosomes in a cell is directly correlated with growth rate for a wide range of bacteria V. cholerae rrnBP1-gfp(ASV) reporter strain in LB medium showed strong fluorescence during exponential growth as quantified by flow cytometry (600 = 0.9), a reduction in growth rate was correlated with a reduction in fluorescence intensity. Further progression into stationary phase was accompanied by a rapid decline in fluorescence to approximately 10% of the level observed during exponential growth. Assuming that expression of gfp(ASV) from the rrnP1 promoter ceased after entry into stationary phase, then the rate of the decline in GFP fluorescence corresponds to a maximal GFP(ASV) half life of 40 minutes in V. cholerae.Growth of the ytometry . During V. cholerae rrnBP1-gfp(ASV) was inoculated into rabbit ligated ileal loops and samples obtained four, eight and twelve hours post inoculation. The fluorescence intensity of individual bacteria was then monitored by confocal microscopy as a function of time and anatomical site. Four hours post inoculation, bacteria juxtaposed to the epithelial cell surface were found to express the highest levels of GFP, indicating that these bacteria were replicating at a higher rate or were more metabolically active than bacteria residing in the mucus (V. cholerae O1-specific antibody) was quantified in multiple confocal planes from different images of epithelial tissue four hours post inoculation. Bacteria within 5 µm of the nearest epithelial cell surface produced nearly twice the amount of rRNA-associated fluorescence when compared to bacteria residing at distances further away from the epithelial surface (he mucus . The rat surface . In cont surface . While m surface . Most of surface .tcpA-gfp(ASV) and rrnBP1-gfp(ASV) promoters suggest that the replication of V. cholerae and the production of TCP are co-localized: both promoters are most active on or near epithelial cell surfaces  in rabbit ileal loops showed apparent variation between adjacent bacteria in the expression of tcpA twelve hours post-inoculation (tcpA-gfp(ASV) expression by V. cholerae in a liquid culture where all bacteria are exposed to the same condition. For this purpose we first used AKI medium, growth in which induces tcpA and ctxAB expression after the bacteria are cultivated four hours in a stationary test tube followed by one hour of growth in a shaken (and thus aerated) flask tcpA expression. To address this issue, we studied the expression of tcpA-gfp(ASV) under a homogeneous condition of growth that did not allow formation of chemical gradients. Expression from the tcpA promoter was induced by adding bicarbonate to stirred HEPES-buffered LB broth containing early exponential phase cultures (OD600 = 0.2), a variation of previously described methods that used bicarbonate or carbon dioxide to induce the production of CT through the activation of the ToxT transcription factor tcpA-gfp(ASV) expression 30 minutes after addition of bicarbonate to a shaken exponential culture was shown by flow cytometry to be an almost linear function of the bicarbonate concentration -expressing and non-expressing bacteria as a function of time after the addition of bicarbonate to an exponentially-growing culture (tcpA-gfp(ASV) was significantly induced in all cells 30 minutes after the addition of bicarbonate to an early exponential phase culture (OD600 ∼0.2). Growth of the culture to an OD600 of approximately 0.9, ninety minutes after the addition of bicarbonate, was associated with bifurcation of tcpA-gfp(ASV) expression. This time point corresponds to transition of the culture into stationary phase (tcpA-gfp(ASV) by ∼50% of the population and declining tcpA-gfp(ASV) expression in the remaining 50%. Several hours after entry into stationary phase, all cells eventually down-regulated tcpA-gfp(ASV) expression (data not shown). The concentration of bicarbonate not only controlled the initial induction of tcpA-gfp(ASV) during exponential growth  reporter strain was created in V. cholerae N16961. This strain also exhibited significant heterogeneity in the expression of the tcpA-gfp(ASV) after addition of bicarbonate and progression into stationary phase (data not shown).Three hours after the addition of bicarbonate to a stirred exponentially growing culture, at which time the culture had transitioned into early stationary phase, fluorescence microscopy showed a significant bifurcation of GFP-fluorescence: approximately 50% of the population fluoresced intensely whereas the rest of the population showed only background levels of fluorescence . This fi culture . Expressry phase . Serial ry phase  as the cl growth , but it l growth . To testtcpA-gfp(ASV) expression into two populations during entry into stationary phase, V. cholerae tcpA-gfp(ASV) was induced with bicarbonate at OD600 = 0.2 and grown to early stationary phase until the bifurcation phenotype was observed. Then, the bacteria were fixed with paraformaldehyde and sorted by fluorescence intensity using a fluorescence activated cell sorter (FACS). Cells that continued to produce GFP(ASV) from the tcpA promoter and cells that were GFP(ASV)-negative were collected as two separate populations. RT-PCR was performed to measure the abundance of the tcpA transcript in each of the two populations. The RT-PCR assay employed primers and probes corresponding to regions of the native tcpA gene in VPI-1; these regions were not present in the tcpA-gfp(ASV) construct. In this way, the expression of wild type tcpA could be monitored (by RT-PCR) in parallel with the expression of tcpA-gfp(ASV) (by flow cytometry). To determine if the expression of the genes encoding CT, which is co-regulated with tcpA, also segregate into the same two populations, ctxA mRNA abundance was also monitored. In addition, the RT-PCR multiplex assay employed primers and probes corresponding to three genes (cheA1-3) that are not regulated by ToxT and are located at three different sites on the genome, and six components of the ToxR regulatory network that governs the expression of the tcp and ctx operons tcpA and ctxAB promoters tcpA-F and ctxABtcpA and ctxAB expression as a function of population density cheA1-3, hapR, aphA, aphB or toxR in the two sorted populations (toxT transcript was six-fold more abundant in the GFP-positive cells. The accumulation of toxT mRNA in cells expressing the tcpA-gfp(ASV) reporter was associated with an 80-fold greater abundance of the tcpA transcript and a 20-fold greater abundance of the ctxA transcript in the GFP-positive population (tcpA-gfp(ASV) reporter can be used as a valid measure of tcpA expression, thus confirming that tcpA expression bifurcates into two populations. These data show that ctxA, long recognized to be co-regulated with tcpAV. cholerae colonization (TCP) and virulence (CT) in humans exhibits the bifurcation phenotype. Finally, these data demonstrate bifurcation of toxT expression; thus toxT expression, like the ctxA and tcpA promoters it binds and activates, also segregates into two populations after induction with bicarbonate and progression into stationary phase. The difference in the expression of toxT between the two populations was lower than the difference seen for tcpA, which indicate that smaller changes in the expression of the transcriptional regulator may affect expression of tcpA and ctxAB significantly.To further investigate the segregation of ulations . The genpulation . Taken ttcpA expression was caused by the presence in the culture of two pre-existing expression variants, only one of which sustains tcpA-gfp(ASV) expression during entry into stationary phase, a culture was induced with bicarbonate during exponential growth and allowed to undergo bifurcation. The two subpopulations [sustained or transient expression of tcpA-gfp(ASV)] were then separated by FACS and inoculated onto separate agar plates. Single colonies grown from each of the two sorted populations were picked, grown to early exponential phase in separate shaken flasks and then treated with bicarbonate to induce tcpA-gfp(ASV) expression. Bacteria from each of the sorted populations were found to exhibit the same bifurcation phenotype as the non-sorted progenitor (To test whether the observed bifurcation in ogenitor . From thtcpA-gfp(ASV) bifurcation phenotype, bicarbonate was removed once the bicarbonate-treated culture had reached early stationary phase by washing and resuspending the bacteria in filter-sterilized conditioned media from cultures grown in parallel to the same OD without bicarbonate. Then, flow cytometry was used to compare the fraction of GFP-positive cells in the bicarbonate-depleted culture with the fraction in a bicarbonate-containing culture as a function of time. tcpA expression in the fraction of cells that continue to be GFP-positive after progression into stationary phase. Although the mechanism by which bicarbonate enhances the activity of ToxT is not yet known tcpA and toxT thus causing this sub-population to continue to express tcpA even after transfer of the culture to a bicarbonate-free medium. If so, then the duration of sustained tcpA expression would depend on how long cellular concentrations of ToxT remain above a critical threshold concentration. This kind of biochemical memory is characteristic of systems that exhibit hysteresis: the capacity to sustain an induced phenotype for a period of time after the responsible inducer has been removed or its concentration reduced below the level required to elicit the phenotype To determine if the continued presence of bicarbonate is required to maintain the tcpA bifurcation phenotype described above are consistent with a variety of genetic, DNA modifying and epigenetic mechanisms by which a clonal population can generate, at high frequency and in a homogeneous environment, two or more subpopulations tcpA bifurcation phenotype because it typically results in two distinctive states, is reversible and demonstrates hysteresis tcpA by ToxT is such a system: the tcpA promoter reads through to toxT thereby creating a positive feedback induction of toxT expression tcpA expression, but also ctxAB expression Many of the features of the tcpA through binding of the two toxbox domains upstream of the tcpA promoter toxT described above, the dimerization of ToxT also may be important in the generation of bistability since it could render activation of the tcpA-promoter hypersensitive to the concentration of ToxT. This is supported by experiments using virstatin which blocks ToxT dimerization tcpA to a few percent of normal levels, thus reinforcing other findings that ToxT is essential for activation of the tcpA promoter and must dimerize to exert its effect.Like other members of the AraC family of transcriptional regulators, ToxT has an N-terminal dimerization domain that is required for transcriptional activation of toxT transcripts in GFP(ASV)-positive compared to GFP(ASV)-negative sorted cells (toxT expression through the tcpA promoter (tcpA-gfp(ASV) reporter strain by deleting the indigenous tcpA-promoter in VPI-1 thus interrupting the positive feedback loop. The tcpA-gfp(ASV) reporter, which is located at an ectopic site on the same chromosome, was left intact (tcpA promoter deletion version of the tcpA-gfp(ASV) reporter strain was then monitored for fluorescence intensity during induction with bicarbonate. Since the tcpA-gfp(ASV) reporter is inserted at a different locus  reporter was still observed in the tcpA-promoter deletion mutant 30 minutes after addition of bicarbonate to a mid exponential culture. However, in contrast to the wild type reporter strain  reporter during entry into stationary phase three hours post induction (tcpA-promoter therefore completely prevented bifurcation of tcpA-gfp(ASV) expression. Thus, in the absence of the indigenous tcpA promoter, ToxT is still capable of responding to bicarbonate induction through its own promoter during exponential phase growth and to induce expression of tcpA-gfp(ASV) at an ectopic site, but the tcpA promoter mutant has lost the capacity to sustain expression of tcpA in a fraction of the cells during entry into stationary phase. Therefore, bifurcation of the tcpA-gfp(ASV)-expressing phenotype appears to depend on positive feedback induction of toxT through the tcpA promoter. Examination of the flow cytometry data in tcpA-gfp(ASV) expression in the bicarbonate-induced tcpA-promoter deletion mutant is 20% lower when compared to the average level of expression of the tcpA-gfp(ASV) reporter in the wild type background. This result suggests that autocatalytic control of toxT expression may be required to increase ToxT concentrations above a critical threshold necessary to sustain tcpA-gfp(ASV) expression by a fraction of bicarbonate-induced bacteria during entry into stationary phase.The ToxT autocatalytic regulatory circuit and the ∼6-fold greater abundance of ed cells , led us promoter . We modit intact . The tcpnt locus , it contr strain , all celnduction . DeletiotcpA in response to bicarbonate during exponential growth results in a unimodal population of induced bacteria; bifurcation of the induced population into the two tcpA-expressing populations, depicted in tcpA bistable phenotype not only comes from positive auto-regulation of ToxT production, but also from other factors that are able to repress tcpA expression in a fraction of induced cells during entry into stationary phase. We reasoned that one such factor might be cAMP and the catabolite regulatory protein CRP with which it interacts. The CRP-cAMP complex is part of a global regulatory network that controls gene expression in response to the availability of carbon and energy sources in the environment. The effects of cAMP on gene expression are caused by an allosteric modification of CRP that occurs when cAMP binds CRP and the CRP-cAMP complex interacts with upstream promoter motifs. Depending on the position of the CRP-cAMP motif relative to other sites on the promoter, transcription of downstream genes is either increased or decreased  reporter strain. Results from flow cytometry studies of the tcpA-gfp(ASV) Δcrp reporter before the addition of bicarbonate, and then 30 minutes and 3 hours after addition of bicarbonate to an exponential phase culture are depicted in tcpA-gfp(ASV) by the wild type parent and the crp mutant was similar 30 minutes after addition of bicarbonate to log phase cultures (tcpA-gfp(ASV) expression in the wild type parent (crp mutant remained strongly induced 4 hours after bicarbonate induction as the cells entered stationary phase (crp mutant (7×101 FL-1 FITC) was 350% greater than the average intensity of the induced fraction of the wild type population (2×101 FL1-H FITC). This shows that CRP plays an important role in repressing virulence gene expression and that it is required for generation of the tcpA bistable phenotype. These results also demonstrate that CRP mainly acts as a repressor of tcpA expression during entry into stationary phase, since the crp mutant showed near normal levels of tcpA expression during exponential phase growth when compared to the wild type parent. To further test the role of CRP-cAMP in the bistable phenotype, we studied a mutant deficient in the production of adenylate cyclase (cyaA), an enzyme responsible for the synthesis of cAMP. As predicted by the requirement of cAMP for activation of CRP, results from flow cytometry studies of tcpA-gfp(ASV) expression by the cyaA mutant were identical to the crp mutant (data not shown).To test the hypothesis that CRP-cAMP is required for the cultures : both she parent , all celry phase . Moreovecrp and cyaA mutants with levels of cAMP during exponential growth and stationary phase, cAMP concentrations in the cytosol were measured during growth in LB medium. The concentration of cAMP was found to increase ∼2.5 fold between mid exponential phase and early stationary phase (OD600 = 0.9) where the bifurcation phenotype is first observed  during progression into stationary phase . Thus, the role of CRP-cAMP on bistability is not principally mediated through its regulation of hapR, but rather likely comes from the effects of CRP-cAMP at other sites in the regulatory cascade. As discussed above, one such site, predicted by the presence of a CRP promoter recognition motif, is the tcpA promoter CRP-cAMP has been shown to increase the expression of ry phase . A similtcpA expression bifurcates into two populations in a growth phase dependent manner and that this phenotype is controlled by a bistable switch that is governed by ToxT and by the CRP-cAMP complex. To determine if a bistable switch also segregates tcpA expression into two populations in vivo, we used single cell expression profiling to study tcpA expression by bacteria in the fluid that collects in the lumen of V. cholerae-infected ligated ileal loops. These fluids were used to address the following questions: (1) does luminal fluid contain a population of tcpA-expressing and a population of tcpA-non-expressing bacteria; (2) is the adoption of these two phenotypes a random and reversible process; (3) do tcpA-expressing bacteria in luminal fluid continue to express tcpA after they are transferred from luminal fluid to a medium that does not contain an inducer of tcpA expression; and (4), does disruption of crp abolish the bistable phenotype, yielding a unimodal, tcpA-expressing population of bacteria in the loop lumen. In addition, we sought to determine if the tcpA-expressing population of bacteria in luminal fluid coalesce into aggregates compared to the tcpA-non-expressing population.The in vitro studies reported above demonstrate that V. cholerae tcpA-gfp(ASV) reporter strain were examined by fluorescence microscopy and the ratio of tcpA-expressing to tcpA-non-expressing bacteria determined. Striking heterogeneity in the expression of tcpA was observed as shown in tcpA-gfp(ASV) reporter. Similar heterogeneity was observed in 10–20 micrographs from several ileal loops from two individual rabbits. Thus, tcpA expression bifurcates into two populations in vivo in a manner resembling the bifurcation phenotype revealed by the in vitro experiments depicted in tcpA promoter and down regulation by CRP-cAMP. To determine if crp and the tcpA promoter affect the bistable phenotype in vivo as demonstrated by the in vitro studies reported above, we monitored tcpA-gfp(ASV) expression by the crp and tcpA promoter deletion mutants in ileal loop luminal fluid. As expected, none of the cells of the tcpA promoter deletion mutant were observed to express tcpA in the lumen 12 hours post inoculation (data not shown). By contrast, deletion of crp resulted in strong and homogeneous tcpA expression by all of the bacteria observed in the lumen  reporter strain was obtained 12 hours post inoculation, plated onto rifampicin containing LB media. Twenty colonies were picked and individually tested in vitro for their ability to respond to bicarbonate by the induction of tcpA expression. No bacteria from the 20 tested colonies showed tcpA-gfp(ASV) expression during exponential growth in bicarbonate-free LB. With the addition of bicarbonate to the medium, tcpA-gfp(ASV) expression by all bacteria occurred; bifurcation of tcpA expression ensued during entry into stationary phase (data not shown).This parallels the in vitro findings depicted in tcpA expression with bicarbonate. This observation is consistent with the idea that the bifurcation phenotype observed in vivo also is caused by a reversible epigenetic switch.To determine if the heterogeneous expression of V. cholerae in luminal fluid were found as individual planktonic cells, dense aggregates of bacteria were also observed 12 hours post inoculation. Planktonic populations of individual bacteria were found to contain both tcpA-gfp(ASV)-expressing and non-expressing bacteria. By contrast, most bacteria in aggregates were found to strongly express the tcpA reporter; one such aggregate is depicted in V. cholerae. By contrast, no aggregates were found that were composed of bacteria not expressing the tcpA-gfp(ASV) reporter. Thus, while the planktonic population contains both tcpA-expressing and non-expressing bacteria, aggregates appear to be composed mainly of tcpA-expressing bactera.While most tcpA-gfp(ASV) by some bacteria in luminal fluid was dependent on the continued presence of a chemical inducer, an aliquot of luminal fluid, obtained 12 hour post inoculation, was diluted 10-fold in artificial seawater. This sample together with undiluted luminal fluid was incubated at 30°C for up to four hours and the two samples then assessed by fluorescence microscopy and compared to images of a sample of the same fluid examined immediately after incision of the loop (tcpA-gfp(ASV) reporter after four hours of incubation. By contrast, aggregates of bacteria continued to show very strong expression of tcpA  were powerfully up-regulated by bacteria on the epithelial surface or in the overlying mucus compared to their expression in fluid secreted into the lumen of the same ileal loop. Further, their expression was greater early in the time course before the mucosal escape response reduced the average expression magnitude of these genes tcpA expression and growth in the intestine, single cell gene expression analysis was performed using confocal microscopy and two reporters: tcpA-gfp(ASV) to monitor tcpA expression; and, rrnBP1-gfp(ASV) as a measure of growth. The magnitude of tcpA expression and the rate of growth as determined by fluorescence from the rrnBP1 reporter varied directly as a function of a bacterium's proximity to the nearest epithelial surface /protease of V. cholerae encoded by hap; and V. cholerae cytotoxins. In addition to its function as a secretory enterotoxin, CT also triggers the release of mucin from goblet cells in villus and crypt epithelia hap, which encodes the mucin degrading enzyme (HA)/protease, was found in our study to be 2.3-fold more strongly expressed in the mucus/epithelial surface compartment eight hours post inoculation than by mid log phase bacteria (data not shown). Thus, based on these site specific gene expression results, it is possible that CT-mediated mucus release and the degradation of mucus by (HA)/protease both occur in the mucus/epithelial compartment and that the hydrolytic products of mucin degradation provide growth promoting nutrients for V. cholerae. Two V. cholerae cytotoxins were also expressed in the mucus/epithelial cell compartment. hlyA, which encodes haemolysin A, a pore forming toxin that causes cell lysis, was expressed 2.5-, 4.3- and 7.4-fold more strongly in the mucus/epithelial cell compartment than by the mid log phase reference 4, 8 and 12 hours post inoculation (data not shown). Similarly, hlx, which encodes a hemolysin, was found to be expressed 3.6-, 3.2- and 3.0-fold more strongly in the mucus/epithelial cell compartment than by the mid log phase reference 4, 8 and 12 hour post-inoculation (data not shown). These cytolytic proteins could release intracellular growth promoting nutrients, including iron, from host cells; this effect might explain why islands of growing V. cholerae are found near extruded epithelial cells  in ileal loops indicated that a deterministic gene expression model could not explain all the results of the single cell expression study: in some locations on mucus membranes individual adjacent bacteria appear to produce dramatically different amounts of the TcpA-GFP(ASV) reporter .Review of confocal images of reporter . Similarculation . By conttcpA expression we turned to in vitro studies. To exclude the possible effects of physical or chemical gradients we studied well-stirred homogenous cultures of V. cholerae tcpA-gfp(ASV) containing bicarbonate, an inducer of tcpA and ctxAB expression. When bicarbonate was added to early exponential phase LB broth cultures to a final concentration of 100 mM, all cells in the population were induced as a monomodal peak typical of a normally-distributed, cell-to-cell variance in gene expression (tcpA-gfp(ASV) expression persisted undiminished for at least 4 hours whereas in the other fraction the average level of tcpA-gfp(ASV) expression declined to pre-induction levels. Studies using bicarbonate-induced cells sorted by FACS showed that the bifurcation phenotype was entirely reversible -expressing and non-expressing cells from the same population. In addition to tcpA, ctxA was also found to exhibit the bifurcation phenotype. This result shows that genes encoding the main determinants of virulence in humans, CT and TCP, both exhibit the bifurcation phenotype. Moreover, significantly larger numbers of the toxT transcript are produced in the same population of sorted cells that also contains larger numbers of the tcpA and ctxA transcripts. This result mechanistically implicates ToxT as a key component of the tcpA/ctxA bifurcation phenotype.The toxin-coregulated pilus was so-named because its biosynthesis is governed by the same ToxR hierarchy of transcription factors that regulate the production of CT toxT is positively autoregulated: ToxT dimerizes to activate the promoter of the tcpA-F operon; read-through to the next transcriptional unit activates the toxT promoter thus resulting in a positive feedback loop (tcpA promoter in VPI-1 was deleted in the reporter strain that carries the promoter-containing tcpA-gfp(ASV) construct in an ectopic location. Exposure of this mutant to bicarbonate during early exponential growth induced tcpA-gfp(ASV) expression, but abolished the bifurcation phenotype (tcpA expression (and by inference ctxAB expression as well) is governed by an autocatalyic process that renders it hypersensitive to the concentration of ToxT dimers in the cell.Expression of ack loop  [56]. Tohenotype . InsteadtcpA promoter, depicted in toxT very likely magnifies the cell-to-cell random variation in ToxT concentrations according to the following model. In cells with low ToxT monomer concentrations, few ToxT dimers form and a non-linear increase in ToxT production via the positive feedback loop does not occur. By contrast, in cells containing higher concentrations of ToxT monomers, ToxT dimer concentrations are correspondingly high. This favors an autocatalytic, non-linear increase in ToxT production resulting in yet higher concentrations of ToxT dimers. Cells with a high concentration of ToxT dimers are capable of sustaining tcpA expression after progression into stationary phase : the half-life of sustained tcpA expression is a function of the half-life of transcriptionally active ToxT dimers. These scenarios were not directly tested in the work presented here by measurements of ToxT monomers and dimers in each of the two populations. However, they are strongly supported by the demonstration that toxT transcript abundance is 6-fold greater in FACS-sorted cells that sustain tcpA expression compared to those that do not  expression during entry into stationary phase  reporter on epithelial cell surfaces and in the overlying mucus layer  reporter by the tcpA promoter and crp mutants. Three reasons led to our use of luminal fluids and the 12 hour post-inoculum time point to study the bistable control of tcpA expression in vivo: (1) this condition corresponds in time to the previously described mucosal escape response tcpA and ctxAB expression decline; (2) the bacterial growth phase in luminal fluid at this time point, as determined by viable plate counts, correlates with entry into or early stationary phase tcpA-expressing and tcpA-non-expressing subpopulations; and 3), expression of the tcpA-gfp(ASV) reporter by bacteria in luminal fluid is more easily studied compared to bacteria embedded in mucus or attached to epithelial surfaces. The tcpA promoter mutant lacking positive feedback induction of toxT from the tcpA promoter showed homogeneous low-level fluorescence from the tcpA reporter fusion after 12 hours of infection. This result likely indicates low and homogeneous ToxT concentrations. As expected, the crp deletion mutant showed strong and homogeneous expression from the tcpA promoter in luminal fluid late in the infectious process  strong expression of tcpA, particularly by bacteria within aggregates, even after dilution of the luminal fluid into artificial seawater. This finding indicates that tcpA expression by bacteria removed from the ileal loop can be sustained by a fraction of the population for a period of time and thus is likely not dependent on the continued presence of a chemical inducer  and an antitoxin (hipB) in E. coli. This system generates slow or non-growing persister cells with increased antibiotic resistance Bacillus subtilis is regulated by a positive feedback loop partly controlled by quorum sensing B. subtilis competence genes to be hypersensitive to changes in the concentration of ComK. As a consequence, only a fraction of the cells becomes competent for natural transformation. Sporulation in B. subtilis is also controlled by a positive feedback loop acting on Spo0A, which results in a subpopulation of cells that sporulate during entry into stationary phase B. subtilis biofilm formation are subject to bistable expression through regulation by Spo0A tcpA and ctxAB in V. cholerae presented here is the first example of bistable regulation of a major virulence pathway in a pathogenic organism.Other bacterial systems that employ a bistable switch to generate heterogeneity in the bacterial population include a positive feedback loop that controls the expression of a toxin  GFP(ASV) reporter used here reports real time levels of promoter activity. Consequently, activation of the gfp(ASV) linked promoter early in the infectious process, followed by silencing of the promoter subsequently, will be scored negative in samples taken at later time points and evaluated by in situ confocal microscopy. Consequently, the recombinase reporter system would not have detected silencing of virulence gene expression by a fraction of the V. cholerae population during the mucosal escape response that occurs late in the infectious process et al. In the present study, the rabbit ileal loop model was chosen since it is a well established model of V. cholerae was found to exist in luminal fluid as individual planktonic bacteria and as bacterial aggregates , which has been accredited by the Association of Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All animals were housed in a centralized and AAALAC-accredited research animal facility that is fully staffed with trained husbandry, technical, and veterinary personnel.V. cholerae O1 stool isolate from a cholera patient was used for all experiments. The strain, supplied by the California State Department of Health, was acquired from discarded material and previously de-identified. The isolate is a smooth phase variant of strain A1552 . The construction and properties of the rpoS and hapR deletion mutants of the parental strain were previously described E. coli strain DH5 was used for standard DNA manipulation experiments, and the E. coli strain S17-1λ pir was used for conjugation with V. cholerae. Bacteria were grown in Luria Bertani (LB) broth with 0.5% NaCl at 37°C. When appropriate, 100 µg/ml ampicillin, 100 µg/ml rifampicin or 50 µg/ml gentamycin was added to the media. Induction of virulence gene expression and bistability of tcpA expression was studied during bacterial growth in AKI conditions 3.A Intracellular concentration of cAMP was determined using the Biotrak cAMP competitive enzyme-immunoassay system RPN225  using the non-acetylated method according to the manufacturers instructions. Concentration of cAMP was normalized to the total protein concentration in the sample as determined by a standard Bradford assay V. cholerae A1552 by biparental mating. Sucrose-based counter selection was done essentially as described Non-polar deletions were generated essentially as described rrnBP1-gfp(ASV) vector pSM1695 tcpA reporter, the 192 nucleotides preceding the start codon in the tcpA promoter were amplified by PCR and inserted between SacI and SphI, immediately upstream of the gfp(ASV) ORF. These transposons were introduced into V. cholerae A1552 by triparental mating using helper plasmid pUX-BF13 (carrying the transposase genes) followed by selection on TCBS/gentamycin. Transposition into the chromosome was confirmed by PCR.The BglI fragment from pBK-mini-Tn7-eyfp-a rrnB-GFP reporter strain, the cells were grown in a total volume of 20 ml in a 100 ml shake flask in LB broth with strong agitation (250 rpm) at 37°C. Multiple samples were taken from mid-exponential phase and every half hour during transition into stationary phase. The bacteria were fixed in 2% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 2 hours, washed twice in PBS buffer and analyzed by flow cytometry . A total of 100, 000 bacteria were analyzed for each sample.For fluorescence analysis of the tcpA-gfp(ASV) reporter strain, the cells were grown with strong agitation (250 rpm) at 37°C in a 100 ml shake flask containing 20 ml of LB broth supplemented with 50 mM HEPES buffer. Bicarbonate was used to induce tcpA expression by the tcpA-gfp(ASV) reporter strain. At an OD600 of 0.4, NaHCO3 from a freshly prepared 1.0 M NaHCO3 stock solution in LB was added to the medium to a final concentration of 100 mM. Samples were subsequently taken during exponential phase and during entry into stationary phase, at which time bistable expression of tcpA-gfp(ASV) was first evident. At each time point, bacteria were fixed in 2% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 2 hours. Then, the cells were washed in PBS buffer twice before flow cytometry . A total of 100,000 bacteria were analyzed for each sample.For fluorescence analysis of the V. cholerae in order to separate and collect cells that either produced or suppressed fluorescence emission from the tcpA-gfp(ASV) reporter. A mid log phase culture was induced with 100 mM bicarbonate and grown for two hours at which time the bistable phenotype had developed during entry into stationary phase; bacteria were collected and placed on ice. A fluorescence activated cell sorter  was used to sort bacteria depending on their GFP fluorescence level. Forward light scatter was used as a second parameter to create a positive identification of bacteria in the solution. A total of 1,000,000 bacteria showing either high or low GFP fluorescence were collected.Fluorescence activated cell sorting (FACS) was used to sort For RT-PCR expression analysis, bacteria were induced with 100 mM bicarbonate and grown for approximately two hours until bistability was observed. Then, the bacteria were gently fixed with 0.5% paraformaldehyde in 100 mM phosphate buffer, pH 7.4 for 15 minutes. The fixed cells were washed three times with PBS buffer and placed on ice. A total of 1,600,000 bacteria showing either high or low levels of GFP fluorescence were collected using FACS. The cells were harvested by centrifugation and frozen on dry ice until RNA extraction. To extract RNA for RT-PCR analysis, the frozen cells were thawed, lysed with RLT buffer  and treated with proteinase K  for 10 minutes at 55°C in order to degrade protein cross linked to the RNA. RNA was isolated from the solution using an RNeasy kit  combined with DNaseI degradation of DNA .RNA from FACS-sorted bacteria was recovered as described above, and an equivalent of 20 ng of total RNA was used in a real-time RT-PCR reaction as previously described All animal work was conducted according to national and international guidelines. The animal experimental protocol was reviewed and approved by the institutional animal care and use committee of Stanford University. Ileal loop preparation and inoculation was performed essentially as previously described 600 = 0.3. Bacteria were diluted tenfold to OD600 = 0.03 in PBS buffer and kept at room temperature until injected into ileal loops. After appropriate incubation, ileal loops used for scanning confocal microscopy were cut open and stretched gently onto cardboard discs. The tissue was allowed to adhere to the cardboard before it was gently submersed into 2% paraformaldehyde in 100 mM phosphate buffer pH 7.4 and allowed to fixate for two hours. The fixative was washed away in three subsequent washes with PBS buffer. Then, blocks of approximately 0.3 cm2 were excised and transferred to a 96 well microtiter plate. The tissue samples were then permeabilized and blocked in staining buffer (PBS with 1% saponin and 3% BSA) for one hour before overnight staining with a 2.5 µg/ml anti-Vibrio cholerae O1 IgG monoclonal mouse antibody  in staining buffer. Samples were washed gently three times in PBS buffer and subsequently stained for 5 hours with 20-fold diluted Alexa Fluor 660 phalloidin  and 10 µg/ml goat anti-mouse Alexa Fluor 594 antibody  in staining buffer. After staining, samples were gently washed twice in PBS buffer and mounted for microscopy in Slowfade Light Antifade Kit . Samples were imaged with a BioRad MRC1000 confocal microscope adjusted to identical settings for all images. The z-stacks were reconstructed onto z-projections using the Imaris-software  and figures were assembled with Photoshop CS . For quantification of GFP fluorescence as a function of distance to the nearest epithelial surface, the epithelial surfaces in stacks of images was first outlined in Photoshop CS. The Border function was used to select incremental layers of 5 µm distances from the epithelial surface. The fluorescence in the green (GFP) channel versus the red channel (V. cholerae fluorescent antibody) was quantified using the program Scion Image .GFP-labeled bacteria were grown overnight, diluted 100-fold in fresh LB broth and grown to an optical density of ODtcpA-GFP reporter strain. A sample of luminal fluid was immediately fixed in 2% paraformaldehyde in 100 mM phosphate buffer pH 7.4. Another part of the luminal fluid was incubated at 30°C while an aliquot was diluted 10-fold in defined artificial sea water  and incubated at 30°C for up to four hours. Samples were isolated every hour and fixed in paraformaldehyde. Samples from the artificial seawater were harvested by gentle centrifugation and resuspension in a smaller volume of PBS buffer. For staining of V. cholerae, 20uL samples were spread out and allowed to dry on poly-L-lysine coated microscope slides and washed in 96% ethanol. Subsequently, the slides were washed in staining buffer (PBS with 1% saponin and 3% BSA) for 10 minutes before staining with a 2.5 µg/ml anti-Vibrio cholerae O1 IgG monoclonal mouse antibody  in staining buffer for 1 hour. The slides were washed three times with PBS buffer and subsequently stained for 30 minutes with 10 µg/ml goat anti-mouse Alexa Fluor 594 antibody  in staining buffer. The slides were then washed in PBS buffer and analyzed using fluorescence microscopy.Luminal fluid was isolated 12 hours post inoculation of the rabbit ileal loop with the V. cholerae, grown to mid-exponential phase in vitro, were used as the source of “reference RNA” for the two-color hybridization microassay assay described below. For this purpose, bacteria were grown to OD600 = 0.3 in LB medium at 37°C; then, the bacteria were quickly centrifuged, the bacterial pellet resuspended in Trizol reagent (GIBCO/BRL) and the suspension frozen on dry ice. Transcriptional analysis of wild type V. cholerae isolated from the fluid that collects in rabbit ileal loops during infection was performed by quickly pelleting bacteria from the ileal loop fluid after eight and 12 hours of infection. The bacteria were then resuspended in Trizol reagent and frozen on dry ice. The mucus and cell-associated bacteria were isolated as a single fraction from ileal loops four, eight and 12 hours post inoculation by scraping the epithelial surface of the intestine with a disposable plastic cell scraper after the loops had been cut open and gently rinsed in PBS buffer to wash away remaining luminal fluid. The mucus gel/cell associated fraction was suspended in Trizol and frozen on dry ice. Total RNA, which includes RNA from the infecting bacteria and from the host (see below), was isolated from the thawed Trizol suspension, treated with DNaseI  and cleaned by using the RNeasy kit . To avoid microarray expression artifacts imparted by the presence of host RNA, RNA was prepared from a healthy rabbit and added to the V. cholerae reference RNA, isolated as described above from bacteria grown to mid exponential phase in LB broth. The amount of host RNA added to the V. cholerae reference RNA was determined as follows. RT-PCR analysis using Taq Man probe specific for V. cholerae 16S rRNA was used to estimate the amount of V. cholerae RNA in the total RNA fraction. This estimate was used to determine the amount of rabbit RNA that was added to the mid log phase reference V. cholerae RNA. The presence of contaminating host RNA in the mucus gel/epithelial RNA extract was further managed as follows. Primers specific for over 85% of the ORFs identified by the V. cholerae genome sequencing project were used to prime reverse transcriptase reactions in order to enrich for V. cholerae cDNAs prepared from the RNA extracted from the mucus gel/epithelial surface fraction. Labeling of cDNA and microarray hybridizations were performed as described V. cholerae was labeled with Cy3. RNA from an exponentially growing LB culture was chosen as a reference since virulence gene expression and accumulation of virulence factors is not observed under these conditions. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. To avoid fluctuations in intensity values from genes that are not expressed to a measurable level, we designated a minimum background level for each channel http://smd.stanford.edu/.http://cmr.tigr.org/tigr-scripts/CMR/GenomePage.cgi?database=gvc) accession numbers used in this paper are tcpA (VC0828), toxT (VC0838), tcpA-F (VC0828-37), ctxA (VC1457), ctxB (vc1456), crp (vc2614), toxR (vc0984), toxR (VC0984), rpoS (VC0534), aphA (VC1049), aphB (VC2647), tcpP (VC0826), hapR (VC0583), tcpH (VC0827), toxS (VC0983), cyaA (VC0122), hapA (VCA0865), hlyA (VCA00219), hlx (VCA0594), cheA1 (VC1397), cheA2 (VC02063), cheA3 (VCA1095) and rrnB (E. coli b3968).The TIGR-CMR genome database , which encodes a destabilized GFP derivative, and the tcpA-gfp(ASV) fusion was inserted in a single copy into the large chromosome of a V. cholerae rpoS mutant. GFP fluorescence from the resulting transcriptional fusion reports on the expression of the gene encoding the principal repeating subunit (TcpA) of TCP. Bacteria harboring the tcpA-gfp(ASV) transcriptional reporter were visualized using scanning laser confocal microscopy 4 hours (A), 8 hours (B) and 12 hours (C) post inoculation of ligated rabbit ileal loops. The actin-rich epithelial surfaces were stained with phalloidin and shown with blue color, all V. cholerae were visualized using an O1-specific antibody and shown with red color, while GFP-producing bacteria are shown with green color. Strong induction of tcpA-gfp(ASV) expression was observed at all time points primarily in bacteria in close proximity to epithelial surfaces. Main images are reconstructed Z-projections and show horizontal sections of the villi, while side panels show vertical sections at the positions indicated by white lines. Scale bars correspond to 50 µm.(5.35 MB TIF)Click here for additional data file.Figure S2V. cholerae in extruded epithelial cells. Scanning confocal fluorescence microscopy was used to visualize V. cholerae harboring the rrnBP1-gfp(ASV) transcriptional reporter fusion 12 hours post inoculation. Actively growing V. cholerae were mainly observed inside extruded epithelial cells (white arrows). The actin-rich epithelial surfaces were stained with phalloidin and are shown with blue color, all V. cholerae were visualized using a V. cholerae O1-specific antibody and shown with red color (left image), while rrnBP1-gfp(ASV)-expressing bacteria are shown with green color (left and right image). Scale bar corresponds to 25 µm.Growth of (3.91 MB TIF)Click here for additional data file.Figure S3tcpA bifurcation phenotype is reversible. To test if the bifurcation in expression of tcpA was caused by genetic changes, a culture was induced with 100 mM NaHCO3 during exponential growth. After the culture had reached stationary phase and the tcpA expression had bifurcated three hours after induction, fluorescence activated cell sorting was used to isolate two populations with either induced (blue curve) or repressed (green curve) expression of GFP (A). The resulting populations were plated on LB plates and three colonies from each were grown in LB media and re-induced with 100 mM bicarbonate . Both populations showed the same degree of bifurcation in GFP production after entry into stationary phase.The (0.66 MB TIF)Click here for additional data file.Figure S4V. cholerae as a function of growth phase. V. cholerae grown in either Luria Broth media (A) or M9 minimal media with glucose as the only carbon source (B) showed significant increase in intracellular cAMP concentration during entry into stationary phase.Intracellular concentration of cAMP in (0.16 MB TIF)Click here for additional data file.Figure S5toxT, and thus the expression of tcpA and ctxAB, is positively regulated by AphAB and TcpPH and negatively regulated by HapR, which represses aphAB expression hapR is, in turn, positively regulated by the stationary sigma factor RpoS as a function of growth deceleration and nutrient limitation rpoS and hapRtcpA promoter discussed in the text, CRP may act to down regulate the expression of the tcpA and ctxAB by increasing the expression of hapR and rpoS. Because CRP-cAMP positively regulates hapR, we tested if CRP-cAMP also affects bistability through its capacity to increase the expression of hapR. If so, then disruption of hapR should increase the proportion of cells that show sustained tcpA-gfp(ASV) expression after they have been induced with bicarbonate and proceed into stationary phase. hapR was deleted in the tcpA-gfp(ASV) reporter strain and the effect of this mutation on the bistable phenotype was assessed. Thirty minutes after addition of bicarbonate to a mid-exponential phase culture (OD600 = 0.2), the average induction of tcpA-gfp(ASV) in the hapR deletion mutant was stronger (2.8-fold > uninduced culture) when compared to the wild type tcpA-gfp(ASV) reporter strain (1.8-fold > uninduced culture). This result is entirely consistent with the well-documented role of HapR as a repressor of tcpA expression. During entry into stationary phase the hapR mutant showed a significant increase in the fraction of cells that remained induced (∼85%) when compared to the wild type parent  during progression into stationary phase. Thus, the role of CRP-cAMP on bistability is not mediated entirely through its regulation of hapR, but rather likely comes from the effects of CRP-cAMP at other sites in the regulatory cascade.Effect of HapR, a CRP-responsive regulator, on the bistable phenotype. Expression of nt ∼50%, , thus cohenotype , deletio(0.25 MB TIF)Click here for additional data file.Figure S6tcpA in a CRP deletion mutant in luminal fluid. Fluorescence microscopy was used to analyze fluorescence from the tcpA-gfp(ASV) transcriptional reporter in a crp deletion mutant in luminal fluid obtained from rabbit ileal loops 12 hours post inoculation. The bacteria were stained with a V. cholerae specific antibody and shown in red (C) while GFP fluorescence from the tcpA-gfp(ASV) reporter is shown in green (B). An overlay of both colors is also shown (A). The majority of the bacteria showed strong and homogeneous tcpA expression levels.Expression of (1.15 MB TIF)Click here for additional data file.Table S1Primers and probes used for genetic manipulations and RT-PCR. TaqMan RT-PCR Primer nomenclature: Outflanking amplification primers ; TaqMan Primers ; 5′FAM-3′BHQ Taqman probe (TMP).(0.08 MB DOC)Click here for additional data file.Table S2V. cholerae virulence genes in ligated rabbit ileal loops using quantitative RT-PCR. Quantitative RT-PCR was used to monitor the expression of virulence genes in two compartments of ligated rabbit ileal loops. Eight and 12 hours post inoculation, samples were obtained from fluid collected in ileal loops during the infectious process. Four, eight and 12 hours post inoculation, samples were also obtained as a single fraction from epithelial surfaces and the overlying mucus gel. Each experiment was repeated four times. The expression of key virulence genes in each sample was compared to their expression during mid exponential phase growth in LB broth (Ref) using quantitative RT-PCR. Average values are shown and standard deviations are indicated in parentheses.Compartment-specific expression profiling of the (0.03 MB DOC)Click here for additional data file.Table S3V. cholerae A1552 in the mucus / epithelial surface fraction 4 hours post inoculation when compared to an exponentially grown reference. The gene expression data were analyzed using SAM with a 0% false-positive discovery rate and a 2-fold transcript abundance difference between samples in order to define significantly regulated genes. The genes are listed in gene order (Column 1), with Log2(expression ratio) (Column 2), and SAM score (Column 3).Complete list of differentially regulated genes in (0.71 MB DOC)Click here for additional data file.Table S4V. cholerae A1552 in the mucus / epithelial surface fraction 8 hours post inoculation when compared to an exponentially grown reference. The gene expression data were analyzed using SAM with a 0% false-positive discovery rate and a 2-fold transcript abundance difference between samples in order to define significantly regulated genes. The genes are listed in gene order (Column 1), with Log2(expression ratio) (Column 2), and SAM score (Column 3).Complete list of differentially regulated genes in (0.77 MB DOC)Click here for additional data file.Table S5V. cholerae A1552 in the mucus / epithelial surface fraction 12 hours post inoculation when compared to an exponentially grown reference. The gene expression data were analyzed using SAM with a 0% false-positive discovery rate and a 2-fold transcript abundance difference between samples in order to define significantly regulated genes. The genes are listed in gene order (Column 1), with Log2(expression ratio) (Column 2), and SAM score (Column 3).Complete list of differentially regulated genes in (0.60 MB DOC)Click here for additional data file.Table S6V. cholerae A1552 in the luminal fluid fraction 8 hours post inoculation when compared to an exponentially grown reference. The gene expression data were analyzed using SAM with a 0% false-positive discovery rate and a 2-fold transcript abundance difference between samples in order to define significantly regulated genes. The genes are listed in gene order (Column 1), with Log2(expression ratio) (Column 2), and SAM score (Column 3).Complete list of differentially regulated genes in (0.49 MB DOC)Click here for additional data file.Table S7V. cholerae A1552 in the luminal fluid fraction 12 hours post inoculation when compared to an exponentially grown reference. The gene expression data were analyzed using SAM with a 0% false-positive discovery rate and a 2-fold transcript abundance difference between samples in order to define significantly regulated genes. The genes are listed in gene order (Column 1), with Log2(expression ratio) (Column 2), and SAM score (Column 3).Complete list of differentially regulated genes in (0.45 MB DOC)Click here for additional data file.


A simulation model that relies on satellite observations of vegetation cover from the Landsat 7 sensor and from the Moderate Resolution Imaging Spectroradiometer (MODIS) was used to estimate net primary productivity (NPP) of forest stands at the Bartlett Experiment Forest (BEF) in the White Mountains of New Hampshire.2 = 0.5. Stepwise addition of 30-meter resolution elevation data values explained no more than 20% of the residual variation in measured NPP patterns at BEF. Both the Landsat 7 and the 250-meter resolution MODIS derived mean annual NPP predictions for the BEF plot locations were within ± 2.5% of the mean of plot estimates for annual NPP.Net primary production (NPP) predicted from the NASA-CASA model using 30-meter resolution Landsat inputs showed variations related to both vegetation cover type and elevational effects on mean air temperatures. Overall, the highest predicted NPP from the NASA-CASA model was for deciduous forest cover at low to mid-elevation locations over the landscape. Comparison of the model-predicted annual NPP to the plot-estimated values showed a significant correlation of RAlthough MODIS imagery cannot capture the spatial details of NPP across the network of closely spaced plot locations as well as Landsat, the MODIS satellite data as inputs to the NASA-CASA model does accurately predict the average annual productivity of a site like the BEF. Forested areas that consistently add carbon by growth in ecosystem production are potentially important as current and future sinks for industrial CO2 emissions. Conversely, land areas that do not consistently sequester carbon over time may be adding to already rising atmospheric CO2 levels from fossil fuel burning sources, Temperate forests recovering from disturbances, such as harvest for wood products or regrowth on abandoned agricultural lands, may represent important sinks globally for CO2 [The capacity of forests to sequester carbon from the increasing pool of atmospheric CO for CO2 . Nonethe2), patterns in forest carbon cycles has proven to be a challenge. At a landscape level, the influence of macroclimate is often less important than other sources of spatial variation, including disturbances, hydrology, and soil nutrient supply [Although regional- to global-scale relationships between forest carbon uptake and major climatic gradients have been demonstrated in several types of vegetation productivity models ,3, captut supply -6. Nevert supply -9.Satellite remote sensing from instruments like Landsat has been applied to forest canopy studies for over a decade ,11. The 2 = 0.91). The EVI has been found useful in estimating absorbed PAR related to chlorophyll contents in vegetated canopies [Operational MODIS algorithms were the first to generate the Enhanced Vegetation Index (EVI)  as globacanopies , and hascanopies ,17.In this study, we present the results of the NASA-CASA (Carnegie-Ames-Stanford Approach) model to predict net primary productivity (NPP) fluxes using both Landsat 7 and MODIS imagery as a means to infer variability in temperate forests of the eastern United States. Our NASA-CASA model -20 has b2 BEF is located within the White Mountain National Forest, a heavily forested, mountainous region in north-central New Hampshire. The BEF is representative of the larger White Mountain region, having similar vegetative composition  was established in 1932 as a U.S. Forest Service long-term research site. The 25 kmAcer saccharum), American beech (Fagus grandifolia), white ash (Fraxinus americana), paper birch (Betula papyrifera), yellow birch , red maple (Acer rubrum), pin cherry (Prunus pennsylvanica), eastern hemlock (Tsuga canadensis), red spruce (Picea rubens), balsam fir  and eastern white pine (Pinus strobus). Most plots contained mixtures of two or more species.In 1932, a network of permanent forest inventory plots (approximately 0.1 ha each) were established on a regular grid, spaced 200 m by 100 m apart . Plot elOllinger and Smith  hypothesA comparison of different modeling methods is presented in the following section. Each of these methods use remote sensing data inputs to characterize forest cover attributes that can influence NPP. The principal aim of this analysis is to determine how well satellite image data from Landsat and MODIS can capture landscape-level variability in forest production.-2 yr-1) for BEF plots has been estimated as the sum of wood production plus foliar production, based on plot measurements of tree diameters, litterfall, and allometeric scaling. Using these ANPP estimates for validation, a complete forest NPP (above- and below-ground) data set was generated from the forest process model PnET-II [Above ground net primary productivity  for plant production. Over large areas, the Topt setting will vary by latitude and longitude, ranging from near 0°C in the Arctic to the middle thirties in low latitude deserts. The W stress scalar is estimated from monthly water deficits, based on a comparison of moisture supply (precipitation) to potential evapotranspiration (PET) demand using the method of Thornthwaite and Mather . The Mod-1 PAR, a value that is globally 42% higher than previously used in the model for AVHRR-driven NPP predictions from 1982–1998 [2 = 0.91 for this NPP recalibration to 2001 MODIS EVI inputs was statistically significant (p < 0.01).Whereas previous versions of the NASA-CASA model ,18 used 982–1998 . The regIn the following section, an evaluation procedure at the landscape scale is presented for the NASA-CASA model at BEF using growing season EVI computed from Landsat 7 Enhanced Thematic Mapper (ETM+) imagery at a 30-m gridded spatial resolution. We hypothesize that to match estimated NPP across BEF sample plots, the spatial detail provided by Landsat imagery collected during the summer period when leaf area is near maximum for the year will compensate for the lack of repeated EVI sampling during each month of the year . The rationale behind this hypothesis is that monthly climate inputs to the NASA-CASA model that uses a single Landsat image may be sufficient to capture the canopy phenology patterns throughout any given year that MODIS monthly satellite observations are used to capture. Furthermore, 30-m elevation and Landsat data inputs to NASA-CASA may serve as proxies for local weather variation effects on NPP.In all the NASA-CASA simulation results presented for the BEF in this study, 4-km resolution spatial grids from PRISM (Parameter-elevation Regressions on Independent Slopes Model)  were useThe Landsat 7 imagery used for this NASA-CASA modeling was collected on August 29, 2001. Predicted NPP from the NASA-CASA model for the BEF Figure  shows va2 = 0.5 (p < 0.05) following the removal of 90 outlier plot values that were determined to have suffered heavy damage to forest stands during winter ice storms of January, 1998. The AVIRIS imagery used as an input to BEP plot NPP estimation was collected in 1997 and much of the Forest Service field data reflects pre-1997 tree status as well. Because we have used a more recent  Landsat 7 image for this study, NASA-CASA predicted NPP should be a more updated representation of BEF plot productivity.The Landsat 30-m resolution grid coverage was used to stratify 3 × 3 pixel areas around each BEF plot location into comparable predictions of annual NPP from the CASA model. Comparison of the NASA-CASA model-predicted annual NPP to the BEF-estimated values Figure  showed aIn an attempt to improve upon these initial results, we subdivided the BEF plots into three different forest types: Deciduous, Evergreen, and Mixed, defined by the National Land Cover Dataset (NLCD) , 1999; (2 coefficient by 0.17. The addition of 30-meter estimates for aspect direction (0–360°) in the stepwise regression increased the R2 coefficient by another 0.02, leading to a final R2 = 0.69 for correlation of NASA-CASA predicted NPP plus topographic variables with the measured NPP patterns at BEF plots.Previously published studies also suggested that low elevation stands at BEF tend to have lower NPP than mid- to higher elevation stands , a trendBecause we can compute both NDVI and EVI from the 2001 Landsat 7 bands, it is possible to compare NASA-CASA NPP results to the BEF NPP estimates using either vegetation index from the satellite imagery. It is evident that NDVI is saturating at the high end of the greenness  range, whereas EVI is saturating to a lower degree in this manner and instead spreads out the greenness inputs to NASA-CASA more linearly over the entire range of NPP estimates at the BEF Figure . The resMODIS EVI composite images for all months of 2001 were used in place of the single Landsat 7 EVI imagery to predict annual NPP at the BEF. These continuous gridded observations of canopy greenness cover from the MODIS instrument have been collected to improve the tracking of canopy green-up and green-down over a growing season, particularly for Deciduous and Mixed forest stand types. MODIS time series profiles for EVI at three 250-m pixel locations within the BEF could be identified at locations of nearly uniform coverage (> 90%) of single forest classes from the NLCD map. These profiles showed that Deciduous and Mixed forest types reached maximum greenness cover levels by end of July, whereas Coniferous forest types increased in greenness cover levels until mid-August , regardless of whether the entire collection of plot estimates were considered together, or were separated into predominantly Deciduous, Coniferous, and Mixed forest classes (data not shown). This implies that the relatively coarse spatial resolution of the MODIS 250-m EVI imagery cannot capture landscape-level variability in tree production in the same way that 30-m Landsat image data can for this eastern hardwood forest location.Nevertheless, using fully gridded 250-m MODIS EVI inputs to the NASA-CASA model (with no sub-sampling bias), comparison of predicted annual NPP to the BEF-estimated plot NPP values showed no significant correlation result  of landscape scale variation in the controls (such as foliar N content and moisture stress effects) on annual NPP at BEF. Nonetheless, a major limitation of Landsat imagery as a routine input to forest productivity models is the relatively low number of cloud-free scenes that can be collected over the course of a growing season in most temperate and humid climate zones. With a return schedule of approximately 16 days, the probability of collecting one cloud-free Landsat image per month of the year is low.In comparison, MODIS imagery can be used in the NASA-CASA model to generate accurate predictions of the average forest NPP for a site the size of the BEF. Because MODIS provides global daily coverage of the entire Earth surface, these 250-meter resolution images can be composited over several weeks to provide an economical and rapid method to capture the regional-scale factors controlling seasonal productivity over most forested areas of the United States. NASA-CASA model predictions from MODIS 250-meter resolution data inputs are best suited to assessments of large forested tracts of land where stand ages are relatively uniform.A next logical step in this methodology of using satellite image data as inputs to models of forest production at the landscape scale is to combine Landsat and MODIS data sets and enhance the advantages of both sensor measurements. We are investigating techniques to interpolate between dates of 30-meter resolution Landsat EVI data values by an appropriate sampling of monthly MODIS EVI values at selected locations that can represent much of the spatial variability in forest stand types and canopy properties detected in the higher resolution Landsat imagery.The author(s) declare that they have no competing interests.All authors have made substantial contributions to the analysis and interpretation of data, have been involved in drafting the manuscript and revising it critically for important intellectual content, and have given final approval of the version to be published.


In extensive disease of small cell lung cancer a doubling of the one-year-survival rate was reported in August 2007 by prophylactic cranial irradiation applied to patients who experienced any response to initial chemotherapy. We discuss the treatment concept of extensive disease in the face of the latest results and older studies with additional thoracic irradiation in this subgroup. A randomized trial with prophylactic cranial irradiation published in 1999 demonstrated an improvement of 5-year-overall-survival for complete responders (at least at distant levels) receiving additional thoracic radiochemotherapy compared to chemotherapy alone (9.1% vs. 3.7%). But, these results were almost neglected and thoracic radiotherapy was not further investigated for good responders of extensive disease. However, in the light of current advances by prophylactic cranial irradiation these findings are noteworthy on all accounts. Considering both, a possible interpretation of these data could be a survival benefit of local control by simultaneous thoracic radiochemotherapy in the case of improved distant control due to chemotherapy and prophylactic cranial irradiation. Furthermore the question arises whether the tumor biology indicated by the response to chemotherapy should be integrated in the present classification. Extensive disease of small cell lung cancer (ED-SCLC) is still a therapeutical challenge. The median survival time without treatment reaches two to four months . StandarThe investigations of the last years mainly concentrated on more effective and less toxic drug regimens or on targeted therapies, but until now these attempts did not bear essential success. Astonishingly, a doubling of the one-year-survival rate was reported in August 2007 for a "conventional" technique: Prophylactic cranial irradiation (PCI) applied to patients who experienced any response to the initial chemotherapy [For the first time an improved survival was shown for PCI by the mentioned EORTC-study in this favorable subgroup . The bottom line was an increase of overall survival  based on improved local control with a highly significant reduction of symptomatic cerebral metastasis for patients treated with PCI (41 vs. 17%). With the shortest of the applied radiation regimens this meant a gain in overall-survival of six weeks  with a therapy of one week. The assessed quality of life was significantly better in the radiation group due to acceptable toxicities and reduced morbidity caused by cerebral metastasis. An additional advantage of PCI and related lower morbidity was the increased applicability of second-line chemotherapy. The study was designed with focus on easy feasibility and cost-effectiveness. So, patients with symptomatic brain metastases were excluded and cross sectional imaging was not routinely demanded but performed in case of defined key symptoms. However, treatment of occult brain metastasis by PCI cannot be finally ruled out. Despite of this potential limitation, an older meta-analysis evaluating complete responders of SCLC illustrates the main principle that PCI improves survival (benefit of 5.4% at 3 years) [Local recurrence plays an increasing role in ED-SCLC. After thoracic radiation treatment, in-field relapse occurred in 24% of the cases as the first site of relapse [2) or TRT with daily carboplatin/etoposide (each 50 mg/m2). Subsequently, PCI and two more courses chemotherapy were performed. Interestingly thoracic radiochemotherapy improved 5-year overall-survival compared to chemotherapy (9.1% vs. 3.7%).Already in 1999 a study performed by a Serbian group  evaluateFour older studies -9 investth cycle) and PCI for good responders achieving complete remission at least at distant levels; chemotherapy and PCI for patients having less than complete response; second line chemotherapy or best supportive care for stable or progressive disease (Figure Taken together the results of Slotman et al. and Jeremic et al. lead to the question whether the treatment for extensive disease SCLC should be reconsidered. There could be three different treatment strategies according to initial response to chemotherapy: Chemotherapy plus TRT (simultaneously with the 4e Figure . If thisThe authors declare that they have no competing interests.FE drafted the manuscript, drafted the figure. ACM conceived the manuscript. All authors read and approved the final manuscript.


Anopheles gambiae mosquitoes exhibit an endophilic, nocturnal blood feeding behavior. Despite the importance of light as a regulator of malaria transmission, our knowledge on the molecular interactions between environmental cues, the circadian oscillators and the host seeking and feeding systems of the Anopheles mosquitoes is limited.timeless, cryptochrome and three takeout homologues significantly up-regulated the mosquito's blood-feeding propensity. In contrast, gene silencing of light pulse regulated olfactory factors down-regulated the mosquito's propensity to feed on blood.In the present study, we show that the blood feeding behavior of mosquitoes is under circadian control and can be modulated by light pulses, both in a clock dependent and in an independent manner. Short light pulses (~2–5 min) in the dark phase can inhibit the blood-feeding propensity of mosquitoes momentarily in a clock independent manner, while longer durations of light stimulation (~1–2 h) can induce a phase advance in blood-feeding propensity in a clock dependent manner. The temporary feeding inhibition after short light pulses may reflect a masking effect of light, an unknown mechanism which is known to superimpose on the true circadian regulation. Nonetheless, the shorter light pulses resulted in the differential regulation of a variety of genes including those implicated in the circadian control, suggesting that light induced masking effects also involve clock components. Light pulses (both short and long) also regulated genes implicated in feeding as well as different physiological processes like metabolism, transport, immunity and protease digestions. RNAi-mediated gene silencing assays of the light pulse regulated circadian factors Our study show that the mosquito's feeding behavior is under circadian control. Long and short light pulses can induce inhibition of blood-feeding through circadian and unknown mechanisms, respectively, that involve the chemosensory system. Drosophila melanogaster, have revealed a striking degree of conservation of these oscillators and their underlying molecular mechanisms, many of which involve transcriptional feedback loops . C. Validation of microarray gene expression data by qRT-PCR. The log2 transformed values generated by microarrays and qRT-PCR were plotted and showed a significant co-relation between the two assays.  Click here for fileThe light pulse and blood feeding regulated A. gambiae transcriptome.  List of genes regulated by both light-pulse and blood feeding, along with their previous and recent transcript IDs. Note that the recent AGAP-R IDs for some transcripts are missing in ENSEMBL and so they are left blank in column 2. The list includes only those genes that showed a significant  level of regulation in at least one of the eight assays ("a" to "h"). The non-significant values (if any) of these genes in other assays are also shown. The gene list is sorted according to the functional groups . The eight different assay types ("a" to "h") are listed in the first row.Click here for fileCorrelation of the microarray expression data with the qRT-PCR expression data  The log 2 ratios  of the different assay sets ("a" to "h"), from both the microarray (column 5) and qRT-PCR analysis (column 4) are shown. The gene name is given in column 1, the transcript ID in column 2 and the array set to which the values were compared is listed in column 3. Click here for fileDetermination of RNAi knockdown efficiency of the 10 circadian/chemosensory genes in A. gambiae female mosquitoes after dsRNA injections.  RNA was extracted from different body parts of gene silenced mosquitoes and the GFP dsRNA treated control mosquitoes, and the relative mRNA levels were determined for each gene in both samples. The mRNA level in GFP mosquitoes was set to 1.0, and the corresponding % silencing was determined by qRT-PCR. The AgS7 gene was used for normalization. The standard error values are shown.Click here for fileBlood-feeding propensity of mosquitoes after RNAi gene silencing  Ten circadian/chemosensory genes were individually silenced in mosquitoes, and the mosquitoes were allowed to feed on blood at the light-on stage in the insectary. The percentages of mosquitoes that did not feed (non-fed) were scored and are shown along with their standard error values. The P- values from statistical analysis; T-test and Mann Whitney Test are also shown.  Click here for fileFold change in expression of 10 circadian genes selected for RNAi assays.  The -fold change in expression of 10 genes (selected for RNAi assays) in different microarray assays , as determined by qRT-PCR and their corresponding values (if any) from the microarray analyses .Click here for filePrimers used for making dsRNAs for RNAi gene silencing. The first 20 bases in bold correspond to the T7 polymerase promoter site. The transcript ID numbers (AGAP-RA from ENSEMBL) are also mentioned for each gene.  Click here for filePrimers used for verification of RNAi silencing. The sense primers were newly designed (VERI Sense) and the antisense primers used were the same as those used for making the corresponding dsRNAs. The transcript ID numbers (AGAP-RA from ENSEMBL) are also mentioned for each gene.  Click here for file


A 40 year-old female, with a history of cardiac surgery for congenital aortic valve stenosis and von Willebrand's disease (VWD) presented with increasing shortness of breath due to mixed aortic valve dysfunction. With a paucity of such cases in the literature, we describe the successful outcome of a patient with VWD who underwent elective redo-redo aortic root replacement with a mechanical valved conduit. She was given a three-month trial of warfarin pre-operatively to evaluate the extent of bleeding risk. Her post-operative course was uneventful and she was discharged home after six days. VWD is an autosomal dominant bleeding diathesis with an incidence of 2-3% in the general population. The disease is characterised by a partial quantitative decrease of qualitatively normal von Willebrand factor (VWF) and Factor VIII (FVIII) . RecentlA 40 year-old female with type-I VWD and factor XII deficiency - a combination of haemostatic defects known as 'San Diego variant'- presented with exertional shortness of breath, tiredness and dizzy spells. Her basal FVIII/VWF: ristocetin cofactor and VWF Ag levels were 0.46 IU/ml and 0.40 IU/ml respectively.Streptococcus Viridans causing vegetations and a paravalvular leak resulted in a re-do homograft AVR four months later. After the second operation she developed complete heart block and a permanent pacemaker was implanted. During these first two cardiac operations she received Haemate P concentrate  which is a plasma-derived FVIII concentrate rich in VWF, with a ratio of FVIII:C to VWF ristocetin cofactor of 1:2.2 and no major bleeding occurred. Since that time the patient had not suffered from major bleeding.At the age of 25 years, she had undergone a homograft aortic valve replacement (AVR) for congenital bicuspid aortic valve disease and severe aortic stenosis. Soon after surgery the implanted homograft became infected with Regular follow-up in 2008 revealed that the implanted aortic homograft was degenerating. A trans-thoracic echocardiogram demonstrated mixed aortic valve disease with severe transvalvular regurgitation and a peak gradient of 59 mmHg accompanied by LV dilatation at 6.4 cm. LV systolic function was preserved with no hypertrophy and a mobile linear structure in the outflow tract suggesting prolapse of the cusp. A contrast computed tomography (CT) of the chest confirmed dilatation of the ascending aorta to 5 cm and it was appropriate to consider ascending aorta root replacement with a mechanical valved conduit. To evaluate whether the administration of vitamin K antagonists might pose bleeding problems post-operatively, she was warfarinised pre-operatively for a period of three months with an International Normalised Ratio (INR) range 2.0-3.0. After the trial period of warfarin had demonstrated no significant bleeding episodes, cardiac surgery was planned.Following administration of 1000 IU FVIII/VWF concentrate prior to induction she was taken to the operating room where, under general anaesthesia, re-opening of sternotomy was performed. She was placed on cardiopulmonary bypass (CPB) via femoro-atrial cannula with full heparinisation at 300 IU/Kg and cooled to 32°C. The heart was densely adherent and required careful dissection. Aortic cross-clamp was applied distally beyond the dilated ascending aorta root and 500 ml of cold blood cardioplegia administered retrogradely into the coronary sinus to stun the heart. An aortotomy was performed and 1000 ml cardioplegia was delivered into the left and right coronary ostia. Most of the ascending aorta was dissected and removed including the sub-coronary inclusion homograft, which was heavily calcified. A size 25 mm Carbo-Seal Composite  was implanted and coronary arteries on Carell patches re-attached to the neo-aorta. Cross clamp and bypass time were recorded at 132 and 156 minutes respectively. Post-bypass another 1000 IU of FVIII/VWF concentrate was given to ensure adequate replacement of VWF. Heparin was reversed in the usual manner with protamine 300 mg. The patient came off bypass uneventfully; however the suture lines continued to bleed due to a coagulopathic state confirmed by thromboelastography (TEG) requiring Fresh Frozen Plasma (FFP) and Bio-Glue . She was transferred to the Intensive Treatment Unit haemodynamically stable. Coagulation parameters are given in table Two chest drains were removed safely on day one when the total blood loss was 2250 ml. In total, four units of packed red blood cells; three units of FFP and two units of platelets were given, in view of coagulopathy and anaemia. VWF levels were maintained above 100% throughout the operation and remained above 100% for over 5 days post-operatively without the need for exogenous factor administration beyond those already stated peri-operatively. Thromboprophylaxis using unfractionated heparin (25000 IU/2 ml) 5000 IU three times a day subcutaneously was commenced on post-operative day (POD) one and then she was warfarinised the same day aiming for an INR range of 2.0-3.0. Her post-operative course was uneventful and she was discharged home on POD 6 when her INR was within the therapeutic range. The patient was very well and asymptomatic six weeks later at a follow up visit with no bleeding or thrombotic events reported.Due to a previous satisfactory response to FVIII/VWF concentrate and contraindication of desmopressin in patients with cardiac insufficiency because of fluid retention , FVIII/VClinically bleeding severity correlates with a reduction of VWF ristocetin cofactor and FVIII:C. Generally it is preferable to avoid anticoagulation in patients with VWD due to increased risk of bleeding. However in view of the patient's young age and previous homograft root replacement it was felt unwise to consider further, potentially multiple, redo homograft root replacements, and a mechanical valve was the prosthesis of choice. Our pre-operative evaluation of the patient required a 3-month period of observation whilst on warfarin to ensure that anti-coagulation could be controlled without major problems.Our case demonstrates that complex cardiac surgery can be performed in patients with underlying congenital coagulopathy, and that a successful outcome requires close multidisciplinary cooperation in terms of planning and monitoring peri-operative factor replacement therapy, the dilemma regarding the type of prosthetic valve and the level of anticoagulation required post-operatively.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.KS participated as first assistant in the operation, carried out the study, and wrote the initial manuscript, UB was involved in anaesthetising the patient and collected the relevant literatures, SM anaesthetised the patient and reviewed the manuscript before submission, JA was involved in pre- and post-operative haematology care, revised and corrected the manuscript, ETB performed the operation, revised and corrected the manuscript. All authors read and approved the final manuscript.


Newborn mice were more susceptible than 5-week-old mice to the development of lymphosarcomas, lung adenomas and hepatomas, whereas newborn rats were more susceptible than their weaned counterparts to the development of renal anaplastic tumours. Other tumours occured with the same frequency in newborn and mature animals. Tumours of the forestomach in mice were more frequenty found in animals treated at 5 weeks than in those treated at birth. Since NMUrea persists for only a very short time and breaks down spontaneously it seems that the paucity of enzymes related to immaturity in newborns is not a major factor in determining the different susceptibility of newborn animals to NMUrea carcinogenicity.N-Nitrosomethylurea (NMUrea) was given as a single intraperitoneal injection either to newborn or to 5-week-old (C57BL * C3Hf)F