RNA amplification is required for incorporating laser-capture microdissection techniques into microarray assays. However, standard oligonucleotide microarrays contain sense-strand probes, so traditional T7 amplification schemes producing anti-sense RNA are not appropriate for hybridization when combined with conventional reverse transcription labeling methods. We wished to assess the accuracy of a new sense-strand RNA amplification method by comparing ratios between two samples using quantitative real-time PCR (qPCR), mimicking a two-color microarray assay.We performed our validation using qPCR. Three samples of rat brain RNA and three samples of rat liver RNA were amplified using several kits . Results were assessed by comparing the liver/brain ratio for 192 mRNAs before and after amplification. In general, all kits produced strong correlations with unamplified RNAs. The SenseAmp kit produced the highest correlation, and was also able to amplify a partially degraded sample accurately.We have validated an optimized sense-strand RNA amplification method for use in comparative studies such as two-color microarrays. One of the principal complications in microarray analysis of gene expression is the relatively large amount of input RNA required for each assay. On average, 1–20 μg of total RNA are required per study using glass microarrays . This is+ mRNA is reverse-transcribed and converted into double stranded cDNA using an oligo(dT) primer containing a promoter for T7 RNA polymerase. The second strand cDNA serves as a transcription template for amplified antisense RNA (aRNA) production. cDNA microarray studies using T7 amplified RNA have shown that the technique yields reproducible results that correlate with the results obtained from using total RNA  as described in the SenseAmp manual . For the degraded RNA samples, random 9 mers were added to the reverse transcription reaction at twice the mass of the input total RNA . Superscript II (Invitrogen) was used for all reverse transcriptions. The cDNA was purified using a MinElute PCR Purification Kit (Qiagen).For all versions of SenseAmp, total RNA was reverse transcribed using 100 ng of an anchored dT primer [d(T)E. coli DNA polymerase I (Invitrogen) at 16°C for 2 hours as described by the manufacturer. Double-stranded cDNA was purified using the MinElute PCR Purification Kit and used for in vitro transcription using the MegaScript kit (Ambion). Amplified sense RNA was purified using the RNeasy Kit (Qiagen) and the manufacturer's recommendation for RNA clean up.The purified cDNA was 3' tailed with dATP. A T7-promoter/oligo d(T) primer was used to initiate second strand cDNA synthesis using 10 overhang on the 3' end prehybridized in 6X ligation buffer . Excess unligated ds T7 promoter was removed using the MinElute PCR Purification Kit. The purified cDNA, which contained a double-stranded T7 promoter linked to a single stranded cDNA template [in vitro transcription using the MegaScript kit (Ambion). Amplified sense RNA was purified using the RNeasy Kit (Qiagen).The purified cDNA was poly d(T) tailed as described in the SenseAmp (Genisphere) product manual. Excess double-stranded T7-promoter was ligated to the 3' poly d(T) tail on the cDNA in 1X ligation buffer (Roche) at room temperature for 30 minutes. The double stranded (ds)T7 promoter consisted of equal molar amounts of a T7 promoter oligo hybridized to a complementary oligo have a 10 base d(A)template ,24, was in vitro transcription using the MegaScript kit (Ambion). Amplified sense RNA was purified using the RNeasy Kit (Qiagen) and the manufacturer's recommendation for RNA clean up.The complete process is described in the Gensiphere SenseAmp product manual. Briefly, the purified cDNA was poly d(T) tailed. A T7-promoter/oligo dA template strand with a 3' blocking group was hybridized to the poly d(T) tail of the purified cDNA. Double-stranded T7-promoter was synthesized at room temperature for 30 minutes using Klenow fragment of DNA Polymerase I (Invitrogen). The 3' blocker was used to prevent the synthesis of complete second strand cDNA during the T7 promoter "fill-in" reaction. Excess T7 promoter template was removed using the MinElute PCR Purification Kit. The purified cDNA, which contained a double-stranded T7 promoter linked to a single stranded cDNA template, was used for Five μg of each amplified RNA sample and unamplified control (with the exception of the NuGen Ovation amplification which yielded cDNA directly) was reverse transcribed into cDNA. RNA was added to 1 μl of 50 ng/μl random hexamer primer, 1 μl 10 mM dNTP mix , and RNase-free water to make 12 μl. The mixture was denatured at 65°C for 5 min and immediately chilled. Reaction buffer and SuperScript II (Invitrogen) was then added and the volume was adjusted to 20 μl. The mixture was then incubated at 25°C for 10 minutes, 42°C for 50 minutes, and finally 70°C for 15 minutes to stop the reaction.t comparable to that derived from the 1:10 dilution in the unamplified control. cDNAs for the NuGen, 2 rounds of SenseAmp version 1–2, and SenseAmp version 1–2 on degraded RNA were all diluted 100X. cDNAs from SenseAmp version 1 were diluted 120X. The remaining version 2, version 1–2, and messageAmp cDNA samples were diluted 200X. 2 μl of diluted cDNA was added to the primer pair mix and SYBR Green Master Mix (Applied Biosystems) in each well. qPCR was conducted on the Prism 7900HT Sequence Detection System (Applied Biosystems). Plates were run for 40 cycles and fluorescence intensity measured after every cycle. For each target sequence the average cycle number at which fluorescence was detected above background in the exponential phase of amplification was obtained for the brain and liver samples. This value, Ct, or cycle number at threshold, was used for calculations of relative abundance of mRNA molecules in the liver samples compared to the brain samples for each of the amplification methods.Each treatment assay was conducted across a total of four 384-well plates per assay. Each plate targeted 48 different genes for PCR amplification, each with 3 brain and 3 liver samples as well as 6 GAPDH wells for normalization across plates. A calibrator plate was used for each treatment to determine the concentration of cDNA required from each amplification technique to produce a GAPDH Cslope of the linear regression of the normalized log fluorescence values within the "window of linearity" for each well. As per the recommendations for this calculation, PCR primer pairs with strongly deviating PCR efficiencies or correlation coefficients below 0.999 were discarded [The efficiency of each of the 192 target gene PCR primer pairs was calculated using the LinRegPCR software . Normaliiscarded  Average t qPCR analysis method, an average cycle number was determined at which fluorescence crossed a threshold above background. The resulting Ct value was recorded for each tissue type and target gene. This Ct value was normalized across plates by subtraction of the Ct value from the housekeeping gene GAPDH. This value represents the ΔCt. The ΔCt from the reference brain samples was subtracted from the ΔCt obtained from the liver samples. This gave the change in ΔCt between the two tissues or ΔΔCt. With the addition of calculated primer pair efficiencies, the ratio of gene expression for each target mRNA sequence between the two tissues was determined using equation (1). Ratios of expression values for liver tissues relative to brain (RQ) for each amplification technique were plotted in Excel  and Pearson correlations to the unamplified control were determined using Analyze-It , an Excel Statistics Add-on.The following algorithm was applied to the results from each amplification method as well as the unamplified control. As per the ΔΔCLAG performed all qPCR assays, selected and designed primer pairs, developed databases holding all data, and prepared the first draft of the manuscript. JB, JS, KH and RCG participated in the design of the study, developed the SenseAmp reactions, and modified the reactions in response to qPCR validation results. RCG supervised SenseAmp development. RPH conceived the study, supervised the qPCR assays, and coordinated work. All authors read and approved the final manuscript.


These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies , we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac+ cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v) lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.“Adaptive mutation” denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase  Cells can respond to stress by apparently increasing their mutation rate. This study provides evidence that there is more than one pathway by which cells achieve such a response Escherichia coli . In most clones, Lac+ is conferred by a compensatory −1 frameshift mutation in the lac gene (Lac+ point mutants). Lac+ point mutation during stationary phase differs from mutation during rapid growth in that it requires the recombination proteins RecA, RecBC, RuvA, RuvB, and RuvC  do not readily yield Lac+ point mutants when resubjected to selection on lactose medium, led us to propose that amplification reflects a pathway of genetic change wholly or partly separate from point mutation . Lacome Lac+ . This inome Lac+ . In keepome Lac+ . These d+ point mutants is proposed to result from transient hypermutation as part of a stress response, one consequence of which is acceleration of genetic change  . We also report the first genetic requirement specific to the adaptive amplification pathway, Pol I.lac-amplified clones grown on rich medium with the dye 5-bromo-4-chloro-3-indoyl β-D-galactoside   e.g.,   e.g., . We use l) e.g., B and 1C.c region . Sectoric region C may refmutation A.lac amplification , unstable sectoring (lac-amplified), or both. A significant problem in interpreting data from such analyses is that the picked microcolony carries with it many Lac− background cells from the selection plate . Second, because some sectored colonies are not unstable , all sectored colonies found were retested by plating a sample on the same X-gal antibiotic medium to determine whether there was continued instability indicative of amplification (lac-amplified cells (colony-forming units [cfu]) it should contain   and, second, by imposing stringent methods to reduce contamination of microcolonies with neighboring cells and microcolonies from the selection plate . First, fication E. Third, contain , and bec sample) Table 1.−2–10−3 in almost all microcolonies . In general, we looked at 50 to 100 cells from these replated sectored colonies, but, for three of them, we screened 3,000–4,000 cells with the same result: we could find no unstable cfu within them. In contrast, cells from lac-amplified colonies, upon replating, again give rise to sectored colonies. Therefore, most of the sectored colonies that we found give no evidence of carrying amplification of the lac locus. Sectoring in these colonies may occur by chance juxtaposition of Lac+ point-mutant cells with non-mutated background cells (which are numerous) during the plating  Those unstable cfu might be clonally related to the rest of the microcolony, or might result from chance juxtaposition of two different clones carrying the same antibiotic resistance marker. To determine the frequency at which such chance juxtapositions are expected, we analyzed the cells carried on the agar plugs on which microcolonies were harvested. We did this by plating the microcolony suspensions on medium containing an antibiotic to which cells of the microcolony were sensitive, to ascertain the frequency of marked Lac+ cells that are unrelated to a microcolony but get harvested with it. The microcolony plugs contained from 900 to 7,000 antibiotic-resistant lac− background cells each. Of eleven such plugs analyzed, one contained three unrelated (different antibiotic resistance) point-mutant Lac+ cfu, one contained one unrelated unstable cfu, and three contained unrelated cfu of the very pale sectored phenotype described above. The presence on agar plugs of cells giving rise to sectored colonies that are unrelated to the microcolony, but occur at a frequency comparable to the frequency of unstables in a microcolony of the same antibiotic-resistance genotype . Another possibility is that they contain multiple F′ plasmids (lac-amplified colonies shows that they do not advance to a Lac+ phenotype efficiently (so are not obvious precursors to point mutation).The very pale sectored colonies that we found were examined further and were shown to be unstable upon retesting but contained no cells capable of forming colonies on lactose medium under the conditions of an adaptive mutation experiment. Presumably they contain too few copies of the plasmids . Their flac-amplified and point-mutant cells, as reported by + point mutants in the colony.Thus, colonies composed of a mixture of lac-amplified cells in point-mutant microcolonies, we devised a genetic selection capable of revealing single lac-amplified cells among numerous non-amplified cells. The selection depends on the quantitative relationship between the number of copies of the chloramphenicol acetyl transferase gene (cat) and the concentration of the antibiotic chloramphenicol that cells resist  could grow on rich Luria-Bertani-Herskowitz medium (LBH) containing 100 μg/ml chloramphenicol, whereas Lac+ point-mutant cells, and those amplified at lac but not at cat, did not form colonies at this concentration. We determined that 61% or more of all lac-amplified cells are detected by this selection  were found to contain a few chloramphenicol-resistant (CamR) cfu . As discussed above, this contrasts with the report of + microcolonies contain lac-amplified cells. Also, these data verify independently that the high levels of sectored, but not unstable, cfu found among microcolonies  cfu . All of n above;  shows thnies see  were notlac-amplified and showed CamR. The similarity of this proportion to that for lac-amplified cells in point-mutant microcolonies suggests that none of the mixtures detected represents an occurrence of lac-amplified cells that arose in the same cell clone as the point mutation. Thus, we conclude that few, if any, point-mutant microcolonies include lac-amplified cells.A negative control was performed by plating samples of cells from agar plugs that did not carry a microcolony visible on a dissecting microscope. Five of 70 such samples yielded one or a few cells that were + colonies as young as the two-cell stage and up to about 40 cells. The sequential amplification–mutagenesis model demands that all colonies as young as 2–40 cells must consist only of lac-amplified cells, because these are proposed not to generate Lac+ point mutations until the number of lac copies reaches 107 or more . This frequency of events is compatible with the observation of 30–60 Lac+ colonies per 108 cells per day, which we see routinely in adaptive mutation experiments  detected as CamR. The cell numbers ranged from one to 16 cells . Two amplification events gave Lac+ cells that were chloramphenicol sensitive, detected on lactose-minimal medium plates. Many more amplification events were detected by CamR selection than by growth on lactose medium , implying that the CamR test is more sensitive, and capable of detecting levels of amplification that are too low to allow colony formation on lactose medium.Twelve samples gave + cells (cfu), ranging in number from one to 20 cells from half the sample (implying colonies ranging in size from 2–40 cells). None of these eight contained amplified cells, as determined by CamR, or by rescoring the lactose plates after 7 d, by which time any pre-existing lac-amplified cells would have formed colonies . This model predicts that lac-amplified microcolonies of approximately 105 cells should precede point-mutant colonies. As shown in 5 cells, show a very distinct pattern that conflicts with this expectation. Only 5% of microcolonies isolated on day 3 consisted of lac-amplified cells (the rest were stable point mutant). The proportion of unstable microcolonies increases on later days. This is the pattern of arising of lac-amplified visible colonies, which are rare in the first few days of an experiment and increase in frequency later (lac-amplified cells 1 to 4 d later (lac-amplified microcolonies produce lac-amplified (not point-mutant) visible colonies.If point-mutant colonies arose from cy later . The pro d later , as expelac-amplified cells . We overcame this problem by scoring the same colonies as microcolonies and found that these small lac-amplified colonies do not grow to become point mutant  allele incubated at semi-permissive temperature, because polA null mutation causes inviability in this strain background (Harris 1997). In repeat experiments, we saw a 2- to 6-fold reduction in amplification rate. In the same experiments, adaptive mutation was unchanged or increased by up to 8-fold.If adaptive amplification were a prerequisite for point mutation, then all genetic requirements for amplification should also be required for point mutation. We report the first genetic requirement found for amplification that is not also required for point mutation: Pol I. Pol I, encoded by fication A, but adA mutant C. We use+ colonies are unable to grow to visibility in the time-span of an adaptive mutation experiment is ruled out by reconstruction experiments, in which three independent lac-amplified isolates carrying polA(Ts) or +polA formed visible colonies in 5.25 ± 0.17 d and 5.39 ± 0.24 d, respectively (mean of three strains ± standard error of the mean [SEM]). Another possibility that we considered is that the polA mutation, which causes an increase in SOS induction (lexA confers low viability in combination with polA(Ts) (data not shown). However we tested this possibility using dinB-defective cells. DinB/Pol IV accounts for most, if not all, of the requirement of adaptive mutation for SOS induction (dinB polA(Ts) show the reduction in point mutation (dinB (polA(Ts). This demonstrates that the decrease in lac-amplified colonies in polA(Ts) strains is not due to conversion/channeling of lac-amplified cells into point mutants, but rather is caused by reduction of the number of lac-amplified cells formed. We conclude that Pol I is required specifically for adaptive amplification and not point mutation. These data are incompatible with models in which amplification is a prerequisite for point mutation, and support models in which point mutation is an independent mechanism.The possibility that amplification occurs in the Pol I–deficient strain but that the Lacnduction , might cnduction . The resmutation C expecteon (dinB  and the on (dinB B reporte+ adaptive point mutants (sulA promoter (see sulA is one of about 40 SOS (DNA damage inducible) genes in E. coli  or without (glycerol medium) selection for lactose utilization. Control experiments showed that 3 h after exposure to 20 J/m2 of ultraviolet light, all cells were induced for SOS (data not shown). Thus, amplification per se does not induce an SOS response.A final aspect of the sequential model of  mutants , Hendric mutants  suggest  mutants  is a conoter see E and 1F. E. coli . Figure lac-amplified, and screened other cells from the microcolonies for GFP production. The data presented in lac-amplified microcolonies in the distribution of cells producing GFP (p = 0.8 by Student's t-test) and that neither shows persistent SOS induction. Thus, even under adaptive mutation experimental conditions, amplification is not sufficient to induce the SOS regulon. The results also show that SOS induction does not persist for many generations after stress has been released. The data are compatible with hypermutation models for adaptive mutation  X-gal rifampicin (rif) plates to determine which colonies were point mutant and which mutation , whereinlac amplification and point mutation are alternative end points, either of which allows growth on lactose. These data are reviewed conveniently by contrast with the sequential, amplification–mutagenesis model, as follows.The experiments reported here were designed to determine whether DNA amplification is an intermediate in the formation of adaptive point mutations in the Lac system, or whether the two are independent end points of genetic change under stress, each of which adapts cells to their environment. All of the data presented support the idea that E. coli Lac system  lac-amplified cells. This model derives most of its support from the observation of sectored-colony-forming cells in colonies of point-mutant cells (The experiments reported test the amplification–mutagenesis model for the mechanism of adaptive mutation in the c system . The modc system . In the nt cells .+ colonies are not mixtures of stable point-mutant cells with 10−2–10−3 unstable (amplification bearing) cells, but rather are pure  or the same one (a duplication), but either way it is required for error-prone repair leading to mutation, and its counter-selection would decrease point mutation, even though amplification is not an intermediate in the process.An additional result previously interpreted as support for sequential models was that when a gene counter-selectable at more than one copy was placed next to ecreased . Howeverlac amplification in E. coli  genetic change in response to stress, is compatible with Darwinism, which also encompasses changing rates of heritable variations  stable, alternative outcome  mutations in other genes efficiently—and by the same recombination-protein- and DinB-dependent mechanism as Lac+ adaptive point mutation—both in the F′ that carries lac (E. coli Lac assay (Unlike the hypermutable state model of ries lac  and the ries lac . A reporac assay .S)  . RpoS upS) , which mS) . HoweverS) . RpoS is, SMR3598 (FC40 zff3139::Tn10Kan), SMR533 , and PJH223, a spontaneous streptomycin-resistant mutant of SMR4562, are resistant to 10 μg/ml tetracycline, 30 μg/ml kanamycin, 25 μg/ml chloramphenicol, and 100 μg/ml streptomycin, respectively. SMR4481 was made by transposition of Tn10dTet from lambda NK1323  carrying zff3139::Tn10Kan, respectively. SMR4562 codA::cat (PJH220) was constructed by linear replacement into SMR4562[pKD46] (plasmid from cat gene from pKRP10 (fadAB3165::Tn10Kan (SMR3490) and FC40 polA12(Ts)fadAB3165::Tn10Kan (SMR3491) were constructed by P1 transduction of FC36 (fadAB3165::Tn10Kan from CAG18495 (fadAB3165::Tn10Kan from CAG18495 linked with polA12(Ts) (polA(Ts)fadAB3165::Tn10KandinB10 and the UV-resistant isogenic control strain PJH310 (FC40 fadAB3165::Tn10KandinB10). polA mutant strains were grown at 30 °C, and experiments were carried out at 37 °C (a semi-permissive temperature). SMR6039 is a derivative of SMR4562 carrying Δattλ::PsulAΩgfp-mut2: the gfp-mut2 reporter gene   with lyster gene  under thgfp-mut2 gene , as per  cells were mixed with 3% each of three or four different derivative testers that carried antibiotic-resistance markers see . MicrocoH plates  containiplug see . The celplug see A, indicaplug see B, indicad colony . The col− tester cells. The number of these that form colonies during the analysis is reduced by use of antibiotics (as described above) because most cells are not resistant, but the background of tester cells may still be equal in number to those in the microcolony  of the suspension was spread on LBH X-gal rif medium to determine cell number and which microcolonies were point mutant. Point-mutant microcolonies were then plated to select CamR on LBH plates containing X-gal, rif, and 100 μg/ml chloramphenicol. Plates were scored after 15 to 20 h at 37 °C.lac amplification (blue and white sectoring) directly on that plate. All CamR colonies were seen to be lac-amplified.To analyze background cells without microcolonies, plugs of agar were taken from areas of the plates where no microcolony was visible at 20× magnification and the cells suspended in 200 μl of buffer. Half of each suspension was plated on chloramphenicol-containing medium as above, and the other half was plated with scavenger cells onto lactose-minimal medium. Chloramphenicol plates were scored between 15 and 18 h, and lactose plates were scored at 2 d, and again at 7 d. Colonies appearing on lactose medium were scored for amplification by plating a sample of the cells on LBH X-gal rif medium. Colonies appearing on chloramphenicol-containing medium were scored for lac-amplified cells detectable by CamR selection was determined as follows. There are two reasons why lac amplification might fail to be detected with the CamR selection: first, because some lac amplifications have not co-amplified the neighboring codA::cat gene, and, second, because some cat-amplified microcolonies obtained from the lactose plates (grown selectively for Lac+ and not for CamR) might have too few cat copies for growth on 100 μg/ml chloramphenicol. We determined each of these separately. We found that 59 of 77 lac-amplified microcolonies were CamR (at least 77% had co-amplified cat), and that 84% of cells from CamR (cat-amplified) isolates grown on lactose without chloramphenicol were then able to form colonies on chloramphenicol medium. The cells that did not form colonies on chloramphenicol presumably had lost or reduced their amplification of cat and lac. This would indicate that 77% of 84% (65%) of all lac-amplified cells should be detected by the CamR selection. An independent determination of the number of cat-amplified cells that grow on chloramphenicol after growth on lactose was obtained in a reconstruction experiment in which a few CamR cells were plated with a 104-fold excess of Lac+ point-mutant cells, mimicking more closely the conditions of the experiments in which microcolonies are obtained. A total of 79% of the cat-amplified cells formed colonies on chloramphenicol medium, so 77% of 79% (61%) of all lac-amplified cells should be detected. The selection for CamR is actually a more sensitive assay for lac amplification than is selection for Lac+, as shown by the fact that only 60% of cells from the cultures that showed CamR were able to form colonies on lactose medium under the conditions of an adaptive mutation experiment, though all carriedlac amplification (indicating that 40% had too few lac copies for growth on lactose), whereas 79% were detected on chloramphenicol medium.The fraction of Microcolonies were touched with the point of a needle, and cells were transferred to LBH X-gal rif plates, where they were spread by streaking for scoring. Control experiments (not shown) showed that this procedure removed, on average, 5% of the microcolony from the plate. About 50 separate colonies from each microcolony were scored for sectoring. The remainder of the colony was left for 1 to 4 d to form a visible colony in situ. The visible colony was then sampled, plated, and scored for sectoring .+ point-mutant and lac-amplified cells were grown (separately) to saturation, then mixed in various proportions, and 0.5 μl of the mixed cell suspensions was put onto the surface of prescavenged lactose plates and grown into visible colonies. These “pseudocolonies” were suspended, and cells were spread on LBH X-gal rif plates at 100 to 200 cells per plate and scored for sectoring blue phenotypes after 2 d growth.M9 lactose liquid cultures of Lachttp://www.ebi.ac.uk/swissprot/) and Ecogene ( http://bmb.med.miami.edu/EcoGene/EcoWeb/) accession numbers for the genes and gene products discussed in this paper are cat (Swiss-Prot P62577), codA (Swiss-Prot P25524), DNA Pol I (Swiss-Prot P00582), DNA Pol IV/dinB (Swiss-Prot Q47155), fadAB (Swiss-Prot P21177), malB (Swiss-Prot P02943), gfp-mut2 (Swiss-Prot P42212), lac (Swiss-Prot P00722 and P03023), recA (Swiss-Prot P03017), recBC (Swiss-Prot P08394 and EcoGene EG10825), rpoS (Swiss-Prot P13445), ruvA (Ecogene EG10923), ruvB (Ecogene EG10924), ruvC (Ecogene EG10925), and sulA (Swiss-Prot P08846).The Swiss-Prot (


In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity Rabankyrin-5, which is activated by the small GTPase Rab5, coordinates two disparate methods that cells use to take in solids and fluids, playing a key role in both endosome fusion and macropinocytosis In mammalian cells multiple mechanisms of endocytosis operate within a single cell to perform nutrient uptake, cellular homeostasis, neurotransmission, signal transduction, antigen presentation, and defence against pathogens. Endocytosis comprises two major categories, phagocytosis and pinocytosis, depending on the uptake of particles or fluid, respectively .We have previously purified a large number of Rab5 effectors by an affinity chromatography approach based on glutathione S-transferase (GST)-Rab5-GTPγS . Among the numerous proteins purified, we focused on a prominent 130-kDa protein A. The prTo investigate the function of human Rabankyrin-5, we raised antibodies against the recombinant full-length protein. By Western blotting, these antibodies detected a predominant band of 130 kDa in both bovine brain and HeLa cytosol, in the corresponding GST-Rab5-GTPγS but not GST-Rab5-GDP affinity column eluates C. EvidenAfter EEA1  and RabeWe next investigated whether Rabankyrin-5 plays a role in homotypic early endosome fusion as well as fusion of CCVs with early endosomes, similar to EEA1  and RabeSurprisingly, we noted that some peripheral structures harbouring Rabankyrin-5 overlapped with Rab5 but either poorly or not at all with EEA1 in A431 cells A, arrowsMacropinosomes have been so far defined by a combination of criteria rather than specific molecular markers. To test whether the Rabankyrin-5 vesicles fulfil the previously described requirements of macropinosomes, we determined whether they (1) are stimulated by growth factors , (2) do In NIH3T3 fibroblasts, the staining pattern of endogenous Rabankyrin-5 was similar to that of EGF-stimulated A431 cells. Rabankyrin-5 was detected on enlarged structures, predominantly at the outermost periphery and in membrane protrusions of the cell A, arrowsIn NIH3T3 cells, Rabankyrin-5–enlarged structures were labelled by an 8-min uptake of fluorescein isothiocyanate (FITC)-dextran . Consistent with their macropinocytic nature, these structures were largely depleted of transferrin see C, arrowsTo confirm that the Rabankyrin-5 structures originated from the plasma membrane through an actin-driven process, we employed time-lapse video fluorescence microscopy on NIH3T3 cells transiently coexpressing yellow fluorescent protein (YFP)-Rabankyrin-5 and cyan fluorescent protein (CFP)-β-actin. In the sequence depicted in The GTP-dependent interaction with Rab5 suggests that Rabankyrin-5 may function as a downstream effector of Rab5 in the macropinocytic pathway. Accordingly, one would expect that Rab5 localises to macropinosomes and, together with Rabankyrin-5, is required for macropinocytosis. We found that Rab5 indeed colocalises with Rabankyrin-5 on macropinosomes see , (2) of Whereas the aforementioned results demonstrate a general requirement for Rab5 and Rabankyrin-5 in pinocytosis, they do not discriminate between clathrin-dependent and-independent endocytosis. To distinguish between the two pathways, we compared the effects of Rabankyrin-5 overexpression on transferrin and horseradish peroxidase (HRP) uptake in NIH3T3 cells, biochemically. A recombinant adenovirus was generated to overexpress Rabankyrin-5 in these cells. In comparison with mock-infected cells, Rabankyrin-5 overexpression increased the uptake of HRP, particularly after longer (10 min) incubation times A. In conTo determine whether Rabankyrin-5 is also required for macropinocytosis, we attempted to ablate the protein by RNA interference using endoribonuclease-prepared small interfering RNA (esiRNA) . Since wTo confirm the requirement of Rabankyrin-5 for fluid-phase endocytosis we used an independent method: quantification of the uptake of fluorescent dextran in NIH3T3 cells microinjected with affinity-purified anti–Rabankyrin-5 antibodies. Following microinjection, FITC-dextran uptake was internalised for 30 min to label macropinosomes. Microinjection of anti–Rabankyrin-5 antibodies inhibited FITC-dextran uptake by 53% ± 5% when compared with cells injected with preimmune serum , whereasApical pinocytosis in polarised epithelial cells and growth factor-induced macropinocytosis share mechanistic properties, like dependence on actin  and PI3-This was confirmed and extended by immunogold electron microscopy on ultrathin frozen sections of mouse proximal tubule. Rabankyrin-5 showed weak labelling of the apical microvilli-covered surface of proximal tubule cells but labelled large electron-lucent structures underlying the apical surface A and 9B.To functionally address the role of Rabankyrin-5 in pinocytosis, we used filter-grown Madin-Darby canine kidney (MDCK) cells as an established system to study polarised trafficking. Alexa 488 dextran was internalised for 13 min from either the apical or the basolateral chamber. Although endogenous Rabankyrin-5 exhibited partial colocalisation with fluid-phase marker internalised from the basolateral side (unpublished data), a striking degree of overlap between the internalised tracer and Rabankyrin-5 on often ring-shaped structures underlying the apical region was observed A. These We next measured fluid-phase uptake biochemically by quantifying the internalisation of HRP either from the apical or the basolateral side for various periods of time. Filter-grown MDCK cells were either infected with adenovirus encoding Rabankyrin-5 or control virus prior to HRP internalisation. While internalisation of HRP from the basolateral plasma membrane did not significantly vary D, uptakeSince the pioneering work of Here, we provide evidence that Rabankyrin-5 is a novel Rab5 effector which, in addition to being localised to early endosomes, is associated with macropinosomes and promotes their formation according to previously established criteria. First, Rabankyrin-5 localised to enlarged vesicles that were stimulated by growth factors . Second, these vesicles differed from early endosomes by the lack of constitutively endocytosed molecules, such as transferrin, and components of the transport machinery, such as EEA1. Third, the vacuoles originated from the plasma membrane and engulfed extracellular fluid. Fourth, their formation depended on PI3-K activity, and fifth, they followed actin rearrangements at the plasma membrane.Macropinosomes are formed by the closure of membrane protrusions generated upon actin-mediated membrane ruffling. Although membrane ruffling is required for macropinocytosis, it seems not to be sufficient for macropinosome formation . Our datHow does the function of Rab5 and Rabankyrin-5 in macropinocytosis relate to early endosome trafficking? We have previously proposed a model whereby Rab5, via the recruitment of its effectors, generates and maintains a spatially restricted and functionally specialised membrane domain on the early endosomes . The RabClathrin-dependent endocytosis and macropinocytosis are independent but interconnected pathways. Whereas macropinosomes and early endosomes remain segregated in EGF-stimulated A431 cells , they caStimulation of epithelial cells with either EGF or phorbol esters increases fluid-phase uptake while reducing internalisation via clathrin-mediated endocytosis . Our resIn addition to macropinocytosis in nonpolarised cells, we have found that Rabankyrin-5 regulates apical, non-clathrin-mediated pinocytosis in polarised epithelial cells. The role of apically stimulated pinocytosis is crucial for the physiology of various organs. For example, it plays an important role in the reabsorption of proteins from the glomerular filtrate in the renal proximal tubule . RabankyIn conclusion, the identification of Rabankyrin-5 opens new opportunities for investigating the molecular principles underlying macropinocytosis and its regulation by signalling molecules. The dual role of Rab5 and Rabankyrin-5 in endosome and macropinosome function argues for a role in coordinating these two different endocytic mechanisms. Future work is required to identify the molecular partners of Rabankyrin-5 and to establish the mechanisms responsible for its membrane targeting and its role in endocytic membrane dynamics.Phospholipids were purchased from Sigma  and Calbiochem . FITC- or Texas Red-conjugated dextran , rhodamine-conjugated EGF, and rhodamine-conjugated transferrin were purchased from Molecular Probes . Wortmannin and LY 294002 were from Calbiochem. A431 and NIH3T3 cells were grown in DMEM (high glucose) supplemented with 10% (v/v) heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. MDCK II cells were grown in MEM plus 10% FCS. MDCK II cells stably transfected for FcLR 5–27  were a gThe 130-kDa protein band was excised from gels and enzymatically digested. The tandem mass spectroscopy protein sequencing procedure was performed as described previously . Bovine Recombinant proteins of Rabankyrin-5 were expressed in SF plus insect cells according to the manufacturer's instructions using Rabankyrin-5 cDNA subcloned into pfastBAC vector or subcloned into pGEX-6P series and expressed in BL21 cells.Human anti-EEA1 serum (1:10.000) was a gift from Ban Hok Toh , whereas a mouse monoclonal EEA1 antibody, a sheep polyclonal anti-HRP antibody and an antibody against phosphotyrosine (α-4G10) were purchased from BD Bioscience , Abam , and Upstate Biotechnology , respectively. Fluorescently labelled secondary antibodies were purchased from Molecular Probes. Recombinant full-length Rabankyrin-5 was used to raise a polyclonal antibody in rabbit. Human full-length Rabankyrin-5 was cloned into pEYFP vectors containing an N-terminal tag . Recombinant adenovirus encoding full-length Rabankyrin-5 was generated according to the manufacturer's protocol (AdEasy). cDNAs encoding Rab5wt were fused to the amino termini of ECFP or EYFP, as previously described . The ant35S-Methionine–labelled proteins were transcribed and translated in vitro using a TnT-coupled transcription-translation kit . For Rab5 effector recruitment assays, in vitro-translated proteins were incubated with glutathione-sepharose beads complexed with GST-Rab5-GTPγS or GST-Rab5-GDP and eluted as described previously . After 16–24 h, transfected cells were washed twice with PBS and fixed with 3% paraformaldehyde and immunofluorescence labelling was performed according to standard procedures. Cells were mounted in moviol and examined on a confocal microscope  with a 100×/1.40 plan-Apochromat objective. Fluorescent images were collected using the Leica IM500 Image Manager and processed using Adobe Photoshop v5.5 .NIH3T3 cells grown on glass coverslips and transfected with plasmids encoding RN-tre were incubated with 3 mg/ml FITC-labelled dextran  in DMEM supplemented with 1% FCS and 20 mM HEPES , fixed, and stained with rhodamine-conjugated antirabbit secondary antibody. Dextran uptake was determined by quantifying grey values of thresholded, fluorescent images of at least 15 cells using MetaVue 6.1r3 and ImageJ (NIH).In vitro fusion assays were performed using early endosomes labelled with biotinylated transferrin or antitransferrin antibody as well as CCVs labelled with biotinylated transferrin prepared from HeLa cells as described previously . The conTime-lapse epifluorescence video microscopy was performed using an Olympus IX70  inverted microscope equipped with a polychrome II monochromator , a custom filter block for simultaneous visualization of YFP and CFP , a 100× oil-immersion objective  attached to a PIFOC z-SCAN , an incubation chamber (37 °C), and a 12-bit CCD digital camera IMAGO  (TILL Photonics), controlled by TILLvisION v3.3 software (TILL Photonics). Time-lapse video sequences of YFP and CFP images were merged as RGBs using TILLvisION v3.3. They were exported as single TIFF files and either further processed using Adobe Photoshop 5.5 and Illustrator 8.0 or converted into QuickTime movies using ImageJ (NIH).Anaesthetized mice were perfusion fixed with 4% PFA, 100 mM HEPES , and 0,2% sucrose either directly or 5 min after injection with 5 mg/g body weight HRP (Sigma) into the femoral vein. Kidneys were excised and postfixed 6 h at 4 °C. Some tissue pieces were cryoprotected in 2,3 M sucrose overnight at 4 °C, frozen in liquid nitrogen, and cryosectioned on a Leica UCT .2 and 1 mM MgCl2 (PBS++) containing 2 mg/ml BSA. Cell surface-bound transferrin was efficiently removed (95%) by three incubations over 10 min in IM . Extracellular HRP was quenched in the cold by incubating the cells for 20 min with 20 mM MESNA in 100 mM NaCl, 50 mM Tris , followed by two washes with ice-cold PBS and a further incubation for 10 min with ice-cold 50 mM iodoacetamide in PBS. For recycling experiments, cells were transferred again to 37 °C in fresh medium that was collected at the indicated time points. Thereafter, cells were washed once more with PBS and extracted for 15 min at 4 °C with 300 μl of lysis buffer . Total HRP activity present in the cells was determined in duplicates out of at least three independent experiments using the previously described enzymatic method . Ninety-six hours after seeding, cells were infected from the apical side for 4 h at 37 °C with adenoviruses in 250 μl of complete medium. After changing medium, cells were incubated for 18 h at 37 °C and then used for biochemical assays or microscopy. NIH3T3 and MDCK cells grown on coverslips in 24-well plates were infected in 500 μl of medium. HRP uptake was performed as described previously . In briec method  and stanc method .A431 cells were seeded in 24-well plates the day prior to transfection. Cells were transfected using oligofectamine  with either 300 ng/ml double- or single-stranded (control) esiRNA derived from the I.M.A.G.E. clone (IMAGE:258664) according to the protocol of Cells or tissues were fixed with 8% PFA in PHEM buffer and then processed for frozen sectioning according to published methods . Thawed Figure S1NIH3T3 cells were pretreated for 20 min with wortmannin (100 nM) and incubated for 25 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran , fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy. Scale bar represents 10 μm.(8.4 MB EPS).Click here for additional data file.Figure S2NIH3T3 cells were either microinjected with (A and D) preimmune or (B and C) affinity-purified polyclonal α–Rabankyrin-5 antibodies and subjected to a 30-min incubation at 37 °C with (A and B) 3 mg/ml FITC-conjugated dextran  or (C and D) 1 μg/ml rhodamine-conjugated transferrin. Cells injected with immune but not preimmune antibodies showed a significant reduction of the fluid-phase marker, while the uptake of transferrin was not significantly perturbed.p > 0.001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm.(D) Fluid-phase dextran quantification of single cells microinjected for indicated antibodies by measuring internalised fluorescence intensity ((44.0 MB EPS).Click here for additional data file.Video S1Images were taken every 3 s over a time period of 15 min. RGB stacks were converted to QuickTime format using ImageJ (NIH). The movie is played at 15 frames per second.(11.1 MB MOV).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink) accession numbers of proteins discussed in this paper are H-Ras (ID 3265), v-Src (ID 6714), Arf6 (ID 382), hVps34 (ID 5289), p110β (ID 5291), Rab34 (ID 83871), Rab5 (ID 5868), Ankhzn (ID 51479), Rab4 (ID 5867), Rab7 (ID 7879), Rab11 (ID 8766), Rabenosyn-5 (ID 64145), EEA1 (ID 8411), Caveolin-1 (ID 857), RN-tre (ID 9712), Transferrin (ID 7018), EGF (ID 1950), LAMP-1 (ID 16783), Prominin-1(ID 8842), PIKfyve (ID 200576), RhoA (ID 387), and PKC-α (ID 5578).The LocusLink (


PLoS Biology, volume 2, issue 3:In Table of ContentsPage iiiThis photograph was used on the March 2004 Table of Contents, where Adam Lazarus, who generously supplied the image, should have been acknowledged. We apologize for this omission.


Blocks of duplicated genomic DNA sequence longer than 1000 base pairs are known as low copy repeats (LCRs). Identified by their sequence similarity, LCRs are abundant in the human genome, and are interesting because they may represent recent adaptive events, or potential future adaptive opportunities within the human lineage. Sequence analysis tools are needed, however, to decide whether these interpretations are likely, whether a particular set of LCRs represents nearly neutral drift creating junk DNA, or whether the appearance of LCRs reflects assembly error. Here we investigate an LCR family containing the sulfotransferase (SULT) 1A genes involved in drug metabolism, cancer, hormone regulation, and neurotransmitter biology as a first step for defining the problems that those tools must manage.Sequence analysis here identified a fourth sulfotransferase gene, which may be transcriptionally active, located on human chromosome 16. Four regions of genomic sequence containing the four human SULT1A paralogs defined a new LCR family. The stem hominoid SULT1A progenitor locus was identified by comparative genomics involving complete human and rodent genomes, and a draft chimpanzee genome. SULT1A expansion in hominoid genomes was followed by positive selection acting on specific protein sites. This episode of adaptive evolution appears to be responsible for the dopamine sulfonation function of some SULT enzymes. Each of the conclusions that this bioinformatic analysis generated using data that has uncertain reliability (such as that from the chimpanzee genome sequencing project) has been confirmed experimentally or by a "finished" chromosome 16 assembly, both of which were published after the submission of this manuscript.SULT1A genes expanded from one to four copies in hominoids during intra-chromosomal LCR duplications, including (apparently) one after the divergence of chimpanzees and humans. Thus, LCRs may provide a means for amplifying genes (and other genetic elements) that are adaptively useful. Being located on and among LCRs, however, could make the human SULT1A genes susceptible to further duplications or deletions resulting in 'genomic diseases' for some individuals. Pharmacogenomic studies of SULT1Asingle nucleotide polymorphisms, therefore, should also consider examining SULT1A copy number variability when searching for genotype-phenotype associations. The latest duplication is, however, only a substantiated hypothesis; an alternative explanation, disfavored by the majority of evidence, is that the duplication is an artifact of incorrect genome assembly. Experimental and computational results estimate that 5–10% of the human genome has recently duplicated . These eAt the same time, the appearance of LCR duplicates may be an artifact arising from one of a number of problems in the assembly of a genome of interest. Especially when classical repetitive sequences are involved, it is conceivable that mistaken assembly of sequencing contigs might create in a draft sequence of a genome a repeat where none exists. In the post-genomic world, rules have not yet become accepted in the community to decide when the burden of proof favors one interpretation (a true repeat) over another (an artifact of assembly). Again, these rules will emerge over time through the study of individual cases.Through the assembly of many case studies, more general features of duplication and evolutionary processes that retain duplicates should emerge. Although each LCR family originates from one progenitor locus, no universal features explain why the particular current progenitor loci have been duplicated instead of other genomic regions. From an evolutionary perspective, duplicated material is central to creating new function, and to speciation. One intriguing hypothesis is that genes whose duplication and recruitment have been useful to meet current Darwinian challenges find themselves in regions of the chromosome that favor the generation of LCRs.Browsing a naturally organized database of biological sequences, we identified human cytosolic sulfotransferase (SULT) 1A as a recently expanded gene family with biomedically related functions. SULT1A enzymes conjugate sulfuryl groups to hydroxyl or amino groups on exogenous substrates (sulfonation), which typically facilitates elimination of the xenobiotic by the excretory system . Sulfonap-nitrophenol and α-naphthol .The July 2003 human reference genome (based on NCBI build 34) was queried with the ent tool , and seaent tool . Two disent tool  as the SSULT1A3 and ~500 kbp of sequence surrounding SULT1A4 were downloaded from NCBI and compared using PIPMAKER  (SULT1A3) and chimpanzee whole genome shotgun sequences [Genbank:AACZ01082721] (SULT1A1), [Genbank:AADA01101065] (SULT1A2), and [Genbank:AACZ01241716] (SULT1A3) using PIPMAKER exon analysis. These new SULT1A genes have been deposited with the GenBank Third Party Annotation database under accession numbers [Genbank:BK004887-BK004892]. DNA sequences were aligned with CLUSTAL W [The MASTERCATALOG was used for performing initial inspections of the SULT gene family and for delivering a non-redundant collection of SULT1A genes. Additional SULT1A ORFs were extracted from gorilla working draft contigs [Genbank:AC145177]  of two-fold redundant codons  where the amino acid had been conserved in pairs of aligned sequences, and the number of these codons where the third position was identically conserved (c) were counted by the DARWIN bioinformatics platform , where k is the rate constant of nucleotide substitution, t is the time separating the two sequences, and Eq. is the equilibrium state of the TREx value [k) was estimated as 3.0 × 10 -9 changes/site/year for placental mammals . These estimates were determined by sampling all pairs of mouse:rat and mouse:human orthologs in the public databases and following accepted placental mammal phylogenies and divergence times [k.Starting with aligned DNA sequences, the number  value . TREx vaEx value . The equce times ,83. TherHuman-chimpanzee genome alignments were inspected at the UCSC Genome Browser. Human-rodent genome alignments were examined with the VISTA Genome Browser ,51,84. VSULT1A3 were downloaded from NCBI UniGene [SULT1A3 or SULT1A4.All expressed sequences ascribed to  UniGene  and aligkbp (kilobase pairs); LCR (low copy repeat); Mbp (million base pairs); Ma (million years ago); ORF (open reading frame); SULT (sulfotransferase); TREx (transition redundant exchange); VISTA .M.E.B carried out the study and drafted the manuscript. S.A.B participated in designing the study and preparing the manuscript.Multiple sequence alignment of SULT1A genes.Multiple sequence alignment of SULT1A genes used in all phylogenetic analyses. Characters conserved in all sequences are indicated with asterisks.Click here for filen and c values for SULT1A genes. Pairwise n and c values between SULT1A genes. The names of the sequences are the row-headers and the column-headers. Lower triangular matrix contains n values, and upper triangular matrix contains c values.Pairwise Click here for file


Antidepressant prescribing has been increasing in England. Studies in other countries suggest that while this may be associated with reduced suicide rates, it may also be associated with increased fatal poisoning from antidepressant drugs. We therefore conducted an ecological study to assess the association between prescription rates for antidepressants and suicide or fatal antidepressant-related poisoning in England.The Office for National Statistics provided information on the number of suicides, antidepressant-related poisoning deaths and populations for England between 1993 and 2002. The Department of Health supplied data on prescriptions for all antidepressants dispensed in England. Associations between prescriptions and deaths were assessed using Spearman's rank correlation coefficient.s = -0.73, p = 0.02) and fatal poisoning involving tricyclic antidepressants . In contrast, increased prescribing of selective serotonin re-uptake inhibitors and other antidepressants was statistically associated with an increase in fatal poisoning involving these drugs .There were 46,747 suicides, 3,987 deaths involving tricyclic antidepressants and 430 involving selective serotonin re-uptake inhibitors and other antidepressants. Increased antidepressant prescribing was statistically associated with a fall in suicide rates (Spearman's rIncreased prescribing of antidepressants may indicate improved diagnosis and treatment of depression in primary care. Our analysis suggests that this was accompanied by lower suicide rates. A decrease in poisoning deaths involving tricyclic antidepressants may suggest a change in preference for using serotonin reuptake inhibitors and other antidepressant drugs for high-risk patients. This may also partially explain the increase in deaths involving these drugs. Due to the ecological nature of the design, we cannot say conclusively whether reduced suicide rates are a direct consequence of increased antidepressant prescribing rates. To confirm these associations, individual level data on prescribing and suicide is needed. Saving Lives: Our Healthier Nation sets a target to reduce suicide deaths by 20% by 2010 [Around 5000 people take their own lives in England every year . The Gov by 2010 . Treatme by 2010 . Hence, Studies in Sweden , DenmarkIn England, prescriptions for antidepressants have been increasing ,15. We cSuicide deaths were defined as deaths where the coroner has given a verdict of suicide or where an open verdict was reached in a death from injury or poisoning. This is because it is thought that most open verdicts are cases where the harm was self-inflicted but there was insufficient evidence to prove that the deceased deliberately intended to kill themselves . Open veThe Office for National Statistics has stored drug-poisoning mortality data for England and Wales from 1993 onwards in a dedicated database . The datDrug poisoning deaths were defined using the International Classification of Diseases codes shown in Table The Department of Health supplied data on prescriptions for all antidepressants dispensed in England between 1993 and 2002. Prescription information is derived from the Prescription Cost Analysis (PCA) system, which collects data from all prescriptions dispensed in the community . This inDirectly age-standardised rates for suicide and antidepressant-related deaths were calculated using the European Standard Population. Populations for England between 1993 and 2002 were interim revised estimates published by ONS in September 2003. Prescription rates were presented as number of prescriptions per 100 population. Statistical associations between prescription rates and directly age-standardised rates for antidepressant-related poisoning deaths and suicides were assessed using Spearman's rank correlation coefficient.In England between 1993 and 2002, there were 46,747 suicides. Suicide was twice as common in men as in women. There were 3,987 deaths involving tricyclic antidepressants  and 430 involving selective serotonin re-uptake inhibitors and other antidepressants . The number of antidepressant-related deaths was similar for men and women. Changes in age-specific rates were similar across all age groups for suicide and antidepressant-related deaths.Between 1993 and 2002, age-standardised mortality rates for suicide decreased from 98.2 to 84.3 per million population .A previous analysis found that about a third of antidepressant-related deaths also have other drugs or substances mentioned on the death certificate  . Hence, Increased prescribing of antidepressants was statistically associated with reduced suicide mortality rates. TCA-related poisoning deaths also decreased during the study period although the evidence for a statistical association with prescribing rates was weaker. Increased prescribing of SSRIs and other antidepressants was statistically associated with increased mortality rates involving SSRI & other-related poisoning, although the rates were much smaller than for TCA-related poisoning or suicide. Because our study uses population level data, we cannot conclude that these associations are necessarily causal.Although the ONS drug poisoning database is the most complete record of drug poisoning statistics available, about 10% of these deaths have no specific information about drug(s) taken . PrescriFurther bias may have been introduced due to prescriber and patient factors. Doctors may perceive SSRIs and other antidepressants to have clinical advantages over TCAs due to their lower toxicity, possibly making them more popular for treating newly diagnosed (an uncontrolled) depression , treatinAssessing the impact of increased antidepressant prescribing is difficult. This is because antidepressants are both a treatment and method for suicidal behaviour. Suicide is also closely linked to availability of methods , generatHowever, our study indicates that at a population level, increased antidepressant prescribing in England was statistically associated with lower rates of suicide and antidepressant-related poisoning. One explanation for our findings is that SSRIs and other antidepressants are being used in preference to TCAs to treat depression. The perceived side effects of TCAs means they are often prescribed or taken at sub-therapeutic doses ,27, leadNevertheless, the clinical benefits of antidepressant drugs for the management of depression and prevention of suicide are still unclear. A recent Cochrane review found little difference between antidepressants and active placebos on improvement of mood .  declare that they have no competing interests.OM designed the study, conducted the analysis and drafted the manuscript. CG participated in conducting the analysis and drafting the manuscript. AM participated designing the study and drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 . In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes.In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed.These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs. The CArG box is specifically bound by Serum Response Factor (SRF) [Quiescent cells exposed to growth factors respond by expressing a variety of immediate early genes (IEG) that do not need new protein synthesis for their expression . Serum oor (SRF) -4. Both or (SRF) . In addior (SRF) .Although the immediate early genes are so named because of their rapid inducibility after growth factor treatment, different kinetics of expression have been observed among the immediate early genes. Expression of the proto-oncogene c-fos peaks at around 30 minutes after stimulation whereas the peak expression of SRF mRNA occurs after 90–120 minutes ,9. Thus Activation of SRF by growth factors occurs through at least two mechanisms – the TCF and RhoA pathways ,11. SeruAn additional pathway that activates SRF is through activation of the small GTPase RhoA . ActivatThe use of pathway specific inhibitors has suggested the presence of two types of SRF target genes – those that are largely dependent on the Rho-actin pathway and those that are largely dependent on the MAP kinase pathway . Use of We and others have recently identified a family of SRF-specific transcriptional co-activators – the MKL family, comprised of MKL1 and MKL2, that function downstream of the RhoA pathway -26. MKL1Chromatin immunoprecipitation experiments have shown that MKL1 binds to the promoters of the cyr61, srf and vinculin genes but not egr-1 or c-fos after swinholide treatment which leads to the activation of the Rho-actin pathway . MoreoveAs a first step towards a better understanding of this differential target gene selection and to demonstrate the importance of the MKL coactivators in immediate early gene induction, we sought to identify the group of SRF target genes that were dependent on the RhoA-MKL pathway. We performed microarray analysis using cell lines expressing dominant negative MKL1 (DN-MKL1) to identify MKL-dependent and -independent serum-induced SRF target genes. The DN-MKL1 protein is effective in blocking both MKL1 and 2 activation of SREs, thus solving the problem of redundancy of these proteins in the cell ,24 as a dominant negative mutant. We previously found that this mutant can prevent activation of SRE reporter genes by MKL1 and MKL2 overexpression  containing 14,824 non-redundant probes were used for hybridization. All data points were done in triplicate and hierarchical clustering was performed using dChip software analysis. We found 229 genes that showed a significant variation across the 8 samples and used the results with these genes for unsupervised clustering [The hierarchical clustering analysis shows classes of differentially regulated genes which we have designated classes 1 to 10 Fig. . We wereTo analyze the serum-inducible genes more carefully, we further dissected the data using more stringent criteria to identify MKL-dependent genes at each time point. We filtered for genes that were serum-induced ≥ 2 fold in WT cells and whose expression in DN-MKL1 cells was at least 35% less than in WT cells Tables . A list To verify the results obtained from the microarray experiments, we checked seven genes by real time quantitative PCR using the SYBR Green method. There was a good, but not perfect correlation, of these methods Fig. . Four MKWe compared the serum inducible genes to known SRF target genes to identify those genes with SRF binding sites. Comparing to a list of SRF targets genes J.M. Mia. We furtWe also searched for promoters with more than one CArG box match since multiple SREs have been reported in several SRF target genes and this could provide a mechanism to distinguish responses. While there were more genes with greater than one CArG box match in the serum-inducible genes than in the DBTSS promoters, the numbers of MKL-independent vs. -dependent genes with greater than one match were too low to show a statistical significance Table .The search for SRF binding sites allowing a one base mismatch was more problematic since there were a high rate of matches in the DBTSS data set, an average of .98 hits per gene Table . There wThe increased frequency of the exact CArG box sequence in the upstream region of many of the MKL-dependent genes gives further confidence that SRF coordinately regulates many immediate early genes. However, the absence of a CArG box does not preclude a gene from being a direct target due to the flexibility of enhancer positioning. The regulatory site may be upstream or downstream of the 1200 bp we have analyzed. For instance, in the case of the jun B gene it has been shown that a CArG box downstream of the gene mediates its response to serum . In addiWe compared the CArG boxes more closely since previous work with altered SREs suggested that changes in the A/T core of the SRF can affect sensitivity to the TCF-independent (i.e. RhoA-MKL) pathway . The knoWe have compared the sequences of the known SRF target genes to determine whether flanking sequences can explain the differential sensitivity to DN-MKL1. We first compared sequence flanking the known CArG boxes. The MEME motif discovery tool was used to identify common elements in 30 bp flanking each side of the CArG boxes. There were no statistically significant matches in the sequences from MKL-dependent of -independent genes. We might have expected to see a TCF site, however this site is very short and flexible. TCF binds to a short site near an SRE and its binding is stabilized by binding to SRF. A consensus of (C/A)(C/A)GGA(A/T) was found, however the orientation and position relative to the SRE was flexible and only the GGA sequence was absolutely required . Visual We used two methods to compare the full promoter sequences of the MKL-dependent and -independent, serum-inducible genes for common elements that might be required for their induction. We first used the MEME tool to compare the promoters (-1000 to +200) derived from the DBTSS database. As in Table -4 to 6.9 × 10-3. These oligonucleotides were not enriched in a test case of 60 random promoter sequences. The related sequence GGGAGG, however, was similarly enriched in the MKL-dependent promoters albeit with less statistical significance (E = 0.8). It is intriguing that these oligonucleotides are similar to the TCF consensus binding site (C/A)(C/A)GGA(A/T). The most significantly enriched oligonucleotide in the 20 MKL-dependent promoters was CCGCGC with an E value of 0.078. This lower significance may partially reflect the smaller size of this data set but also may reflect the lower significance of this enrichment. Given the relatively small number of serum inducible genes and present computational tools, it appears that more careful experimental mapping of the sequence elements in MKL-dependent and -independent genes will be required to identify the elements that determine their sensitivity to the MKL pathway.A second method we used was to look for common regulatory elements was oligonucleotide analysis which has been used in yeast to identify regulatory sites . This meSerum inducible genes have far ranging effects in many physiological processes such as proliferation, wound healing, migration, and tissue remodeling . SRF hasCluster analysis of the microarray data identified different classes of genes that showed significant variation across the samples. Two classes of genes were either constitutively activated or constitutively repressed. These could be indirect targets of the SRF-MKL pathway or could suggest functions for MKL apart from its role as a transcriptional co-activator of SRF.Among the serum inducible genes, we identified a significant fraction, 28 of 150, that are MKL-dependent. This is not to say that the other 122 genes may not be sensitive to MKL under certain conditions. Rather, they must at least have additional mechanisms for serum induction in NIH3T3 cells. c-fos was identified as an MKL-independent gene although we previously found by RNase protection that its induction was significantly reduced in cells induced with serum for 60 minutes . We did Many of the serum-inducible genes identified here have been previously described as immediate early genes either as single genes or in microarray experiments ,47,48. T6GG, resulted in matches in only about 12% of the serum-inducible genes with similar proportions in the MKL-dependent and -independent classes. While some functional SRF binding sites contain a single base mismatch to the consensus, a broader search for CArG boxes allowing a mismatch resulted in a high number of matches in a broad promoter database. There was a modest, statistically significant increase in the matches in the MKL-dependent promoters suggesting that some of these sites are real SRF targets. Other SRF target sites may lie outside of the promoter regions we have searched (-1000 to +200). This still leaves the possibility that induction of many of the immediate early genes is independent of SRF such as we have found for the c-jun gene [We analyzed the promoter sequences of the MKL-dependent and -independent genes to identify elements that might determine this sensitivity. Exact matches to a simple CArG box consensus, CC(A/T)jun gene . MicroarPossible mechanisms for determining sensitivity to MKL are the sequence of the CArG box, flanking sequence or the context of other promoter elements. Our comparison of the known CArG boxes from MKL-dependent and -independent promoters did not show a strong difference. Similarly we were not able to identify differences in the flanking sequence. Analysis of the more full promoter sequences has also not yielded clear common regulatory elements to date. One set of oligonucleotides, containing the sequence GGAG, was found in both the MKL-dependent and -independent promoters at a significantly higher frequency than in the full promoter database. The significance of these sites remains to be determined though it is notable that they are similar to the TCF consensus site.One possible mechanism for distinguishing MKL target genes is the presence of a TCF site next to a CArG box. This would allow for TCF binding and activation by a MAP kinase pathway instead of the MKL pathway. Since the binding sites for TCF and MKL on SRF are similar , TCF binChromatin immunoprecipitation experiments have shown that MKL1 can bind to the SRF, vinculin and cyr61 promoters in cells treated with the actin inhibitor swinholide A . This isOur results indicate that a subset of serum inducible genes is dependent upon the MKL family for its induction. This genomic classification of MKL-dependent and -independent serum-inducible genes is a significant step for characterizing which pathways are required for induction of each cellular immediate early gene.E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen). The obtained double-stranded cDNA was then blunted by the addition of 20 units T4 DNA polymerase and incubation for 5 min at 16°C. The material was then purified by phenol:chloroform:isoamyl alcohol extraction followed by precipitation with ammonium acetate and ethanol. The cDNA was then used in an in vitro transcription reaction for 6 hours at 37°C using a T7 in vitro transcription kit (Affymetrix) and biotin-labeled ribonucleotides. The obtained cRNA was purified on an RNeasy column. The eluted cRNA was then fragmented by incubation of the products for 30 min in fragmentation buffer  at 95°C. The fragmented labeled RNA (15 μg) was hybridized to an Affymetrix mouse MOE430A Genechip at 45°C according to the manufacturer's protocol [NIH3T3 cells stably expressing dominant negative MKL1 (a.a. 1-630)(DN-MKL1) or containing the vector, pBabePuro, were previously described . The celprotocol  and staiThe percent of genes with significant expression  ranged from 50.4 to 56.5% for each microarray. The 3':5' ratio for a control actin gene ranged from 1.0 to 1.31 for each of the 24 microarrays scanned. The scaling factor to account for differences in probe labeling and general changes in hybridization in each microarray was scaled to a target intensity of 250 (using Affymetrix Microarray Suite 5.0 software) and ranged from 1.273–5.542.The dChip software ,53 was u2 distribution model with one degree of freedom [Expression values (MBEIs) at each time point were determined with a standard error of the mean. These values were used to calculate the fold change with 90% confidence intervals. The confidence intervals were derived from the standard errors and fold changes based on a χ freedom . For thet), the cycle at which a statistically significant increase in fluorescence occurs, was derived from the resulting PCR profiles of each sample. Ct is a measure of the amount of template present in the starting reaction. To correct for different amounts of total cDNA in the starting reaction, Ct values for an endogenous control  were subtracted from those of the corresponding sample, resulting in ΔCt. The ΔCt value of the serum starved wt sample was chosen as the reference point and was subtracted from the ΔCt value of all the other samples, resulting in ΔΔCt. The relative quantification value is expressed as 2-ΔΔCt giving the relative difference of the serum induced points compared to the serum-starved cells for expression of a particular gene. All real time PCR data sets shown are the results of two independent mRNA preparations and amplifications. Amplification of only a single species in each PCR reaction was determined by checking for a dissociation curve with a single transition.Total RNA was isolated from the DN-MKL1 and vector containing NIH3T3 cells as described for the microarray analysis and 1 μg was used for first strand cDNA synthesis  using oligo-dT primers according to the manufacturer's protocols. One fiftieth of the reverse transcription reaction was included in a 20 μl PCR reaction. For a quantitative analysis, SYBR green PCR technology was used (Applied Biosystems). Real-time detection of the PCR product was monitored by measuring the increase in fluorescence caused by the binding of SYBR green to double-stranded DNA with an ABI PRISM 7000 Sequence Detector. To calculate relative quantification values a threshold cycle ,54 was uThe CArG box sequences of the known SRE regulated genes were derived from the literature. The motif discovery tool MEME (Multiple EM for Motif Elicitation) ,56 was tPromoter sequences (-1000 to +200) of the serum inducible genes were also searched for common elements with MEME. Promoters for 20 MKL-dependent, 50 MKL-independent and 50 random promoters were searched. These promoter sequences were additionally searched by oligonucleotide analysis for enriched hexamers ,57 excepAS carried out all experimental sections of the paper. AS and RP conceived of the study and participated in its design and coordination. Both authors read and approved the final manuscript.List of serum inducible MKL-independent genesThis Microsoft Excel spreadsheet file contains a list of genes that were serum inducible (>2-fold) at either the 30, 60 or 120 minute time points and that satisfied the 90% confidence interval criteria for fold-change using the dChip software. The expression of these genes were reduced by less than 35% in the DN-MKL1 cells at each time point. The MKL-dependent genes (reduced by more than 35%) shown in Tables 1 to 3 were removed from this list to give 122 MKL-independent genes. The Affymetrix probe set information, the gene name, Genbank Accession number and Locus link identifier are shown for each of the genes. For each time point the fold induction in serum-treated wt NIH3T3 cells compared to serum-starved wt cells ('fold change') and its upper and lower confidence intervals are shown. The time points where serum induction was deemed significant for each gene are marked by asterisks (*) in the 'filtered' columns.Click here for file


The adrenal gland is one of the common sites of metastasis from primary lung cancer. Adrenal metastases are usually unilateral however bilateral adrenal metastases are seen in 10% of all lung cancer patients; of these 2–3% occurs at the initial presentation of non-small cell lung cancer. Secondary tumors can disrupt the structure and function of the adrenal. This can lead to adrenal hemorrhage, which constitutes a life threatening hazard for the patient.A 59-year-old male presented with persisting abdominal pain. His initial work-up revealed significant anemia, an invasive process in the right upper lobe of the lung and large masses of heterogeneous texture, with hemorrhagic and necrotic elements in both adrenal glands. A biopsy confirmed it to be a large-cell carcinoma of the lungs. The patient developed severe leukocytosis akin to the paraneoplastic syndrome and died suddenly five days after the administration of chemotherapy.Intratumoral hemorrhage is a rare but life threatening complication of adrenal metastases and should be treated as soon as it has been diagnosed. If adrenalectomy is not feasible, combination chemotherapy should be applied as in metastatic disease. For choosing the appropriate chemotherapeutic regimen it is important to accurately achieve the diagnosis. The adrenal gland is a common site for metastases in breast, lung and renal cell carcinomas, melanoma, and lymphoma . AdrenalA 59-year-old male, working in a quarry, was admitted to the hospital complaining of abdominal pain persisting for 45 days, afternoon fever, fatigue and 8 kg weight loss during the last month. Previous medical history was non contributory. He was a heavy smoker for 45 years.At first assessment, the patient presented with disseminated dry rales and decrease of vesicular murmur in the right upper lung field. Following abdominal examination, palpable smooth surface masses were discovered in both renal areas. Laboratory tests demonstrated a significant anemia with normocytic features .Chest X-ray revealed a consolidated area in the right upper lobe of the lung. Abdomen ultrasound demonstrated large masses with foci of hemorrhagic necrosis in both adrenal glands Figure .Computed tomographic (CT) -scan revealed an invasive process in the right upper lobe of the lung Figure  and larg1 and T2 weighted sequences. Radiological features were suggestive of adrenal metastasis from lung primary  was performed which showed the masses with disseminated foci of high, heterogeneous signal intensity and pathologic enhancement after intravenous (i.v.) administration of contrast agent in both T Figures , 5. HistAdrenal metastases in the natural history of a malignant neoplasm occur in 20 – 45% of cancer patients, depending on the localization of the primary site . Up to 4The differential diagnosis of an adrenal mass incidentally discovered during imaging examination of the abdomen in patients with non-small cell lung cancer, usually comprises one or more of the characteristics detailed in Table More than 90% of the cases comprising this involve a nonfunctional adenoma or an adrenal metastasis.et al, observed that CT-scan technology has low specificity and sensitivity, which culminates in false positive and negative readings, given that 21% of true adenomas can be mistaken for metastases (density > 10 Hounsfield units) and 11% of true metastases may be mistaken for adenomas (density < 10 Hounsfield units) [Nonfunctional adenomas can be distinguished from adrenal metastasis with precision using modern imaging techniques, especially with MR imaging, where adenoma presents a low intensity signals in T2 sequences compared to hepatic parenchyma. In contrast, the adrenal metastasis presents higher intensity T2 sequence signals than that of the adenoma, relative to hepatic parenchyma. In CT-scan adenoma presents low intensity signals, below 10 Hounsfield units, compared to higher intensity signals in adrenal metastasis. Porte d units) . CT-scand units) .In presence of adrenal metastasis the disease is staged as stage IV, which demonstrates the aggressive biological behavior and systemic dissemination of secondary lesions. These are treated as any other metastatic neoplasm and the appropriate chemotherapy is started. In select patients, where the primary lung site is surgically resectable , with no involvement or the involvement of only the peribronchial and portal lymph nodes  and where the adrenal metastasis constitutes the unique indication of the disease, simultaneous adrenalectomy along with resection of the primary can increase overall survival . TechnicThe most important complication of adrenal metastases is intratumoral hemorrhage and only nine cases have been reported previously . When heA less common complication of bilateral adrenal metastases is primary adrenal insufficiency, caused by the destruction of adrenal gland architecture and function due to the development of the tumor. The main symptoms resulting from the decreased production of steroid hormones often overlap with the general indicators caused by the primary site, hindering the proper treatment of these patients. Primary adrenal insufficiency is diagnosed when serum cortisol basal values are low (<5μg/dl), and particularly when the values fail to increase within six hours following the i.m. administration of 1 mg of tetracosactride, the synthetic analog of ACTH. The treatment comprises daily administration of glucocorticoid and mineralocorticoid for life.Bilateral adrenal metastases can be found in a small number of patients with non-small cell lung cancer. Early identification of these lesions is necessary, since adrenalectomy may increase survival in select patients. Intratumoral hemorrhage is a rare but life threatening complication and should be treated as soon as it has been diagnosed. If adrenalectomy is not feasible, combination chemotherapy should be applied.The authors declare that they have no competing interests.CK – conceived the study, did the literature search and coordinated the preparation of the manuscript for submissionAT – helped in literature search, reviewed and edited the manuscript for final submissionSM and KV – helped in literature search and drafted the manuscript for submissionAll authors read and approved the manuscript.


Wide field of view virtual environments offer some unique features that may be beneficial for use in vestibular rehabilitation. For one, optic flow information extracted from the periphery may be critical for recalibrating the sensory processes used by people with vestibular disorders. However, wide FOV devices also have been found to result in greater simulator sickness. Before a wide FOV device can be used in a clinical setting, its safety must be demonstrated.Symptoms of simulator sickness were recorded by 9 healthy adult subjects after they performed gaze shifting tasks to locate targets superimposed on an optic flow background. Subjects performed 8 trials of gaze shifting on each of the six separate visits.The incidence of symptoms of simulator sickness while subjects performed gaze shifts in an optic flow environment was lower than the average reported incidence for flight simulators. The incidence was greater during the first visit compared with subsequent visits. Furthermore, the incidence showed an increasing trend over the 8 trials.The performance of head unrestrained gaze shifts in a wide FOV optic flow environment is tolerated well by healthy subjects. This finding provides rationale for testing these environments in people with vestibular disorders, and supports the concept of using wide FOV virtual reality for vestibular rehabilitation. One out of three elderly persons and more than one out of five working adults report dizziness,2. ThereThe use of virtual reality (VR) has been explored in many areas of physical and mental rehabilitation -8. ViirrSecondly, people with vestibular disorders complain of what has been called "space and motion discomfort" (SMD) and "visual vertigo" ,18. SituWide field of view (FOV), screen-based projection devices  originated with the CAVE™ at the University of Illinois in Chicago . AlthougThe primary purpose of this paper is to present preliminary evidence for the ability of subjects to tolerate gaze shifting while situated in a wide FOV optic flow environment. We will demonstrate that healthy subjects were able to tolerate the environments without having a large incidence in simulator sickness. The incidence of simulator sickness depended strongly on how much experience the subjects had in the environment, and weakly on the duration of exposure within each visit.Nine adults  with no history of vestibular system pathology participated after providing informed consent. Subjects had a visual acuity of 0.3 LogMAR units or better without using corrective lenses, and contrast sensitivity greater than 1.8 (Pelli-Robson Contrast Sensitivity). The protocol was approved by the University of Pittsburgh Institutional Review Board.The Balance NAVE Automatic Virtual Environment (BNAVE), a wide field of view projection-based immersive display system, was developed to investigate the multi-sensory interactions in postural control . Three 2Several environments can be used for vestibular rehabilitation. One environment produces optic flow through the use of moving geometric patterns such as stripes or squares of alternating colors. Scene characteristics such as the spatial frequency, contrast, direction and speed of movement can all be independently prescribed. Furthermore, targets can be inserted into the environment that subjects are requested to look at. Consequently, for vestibular rehabilitation, we can ask a patient to perform unrestrained head gaze shifts to acquire moving targets while a background of moving stripes is moving past the patient, simulating the functional task of looking for a product while moving down the aisle of a grocery store. A virtual grocery store has also been developed Figure . This en2, respectively. During the low contrast conditions, the luminance of the stripes was 15 and 34 cd/m2. The low contrast condition was based on average measurements of luminance obtained from products sampled at a local grocery store, using a luminance meter . The spatial frequencies were set according to common sizes of soup cans  and cereal boxes  found in the local grocery store. The simulated velocity of the optic flow was 0.5 m/s. The central 25° of the display was masked by a solid region with a luminance of 15 cd/m2 in order to avoid aliasing in the display as the stripes became smaller in the distance.The ability of subjects to perform gaze shifts in response to moving targets superimposed on both static and moving backgrounds was examined. Eight different gaze tasks are performed on each visit Table . Each gaRests of 3 minutes were provided after each task, during which Subjective Units of Discomfort  was rated and the Simulator Sickness Questionnaire (SSQ) was completed . The SSQ2 statistics. Because of the large number of comparisons (5 dependent variables × 2 main effects), the significance level was set at α = 0.05/10 = 0.005.Five dependent variables of interest were examined: SUDS, total SSQ, and 3 SSQ subscales. The three subscales of the SSQ were computed by summing the scores for the component items of each subscale, and multiplying by an appropriate weighting factor  . The totGaze shifts performed by one subject in response to targets moving at least 80° in the yaw plane (Task G) are shown in Figure The tolerance of the subjects to the gaze shifts was assessed using Subjective Units of Discomfort (SUDS) and the Simulator Sickness Questionnaire (SSQ). For each of the measures, the majority of the responses were zero. Therefore, each of the measures was converted into a binary scale consisting of responses equal to zero or greater than zero. The SUDS rating was greater than zero only 25% of the time. For each of the SSQ subscales, a score greater than 0 was given if any of the 7 component items for the subscale was rated greater than 0. The SSQ:Oculomotor subscale had the most non-zero responses, at 29%. The SSQ:Nausea and SSQ:Disorientation subscales had 12% and 5% non-zero responses, respectively. Overall, the SSQ-Total had 31% non-zero responses.2 statistics. The effect of visit number was significant for SUDS, SSQ:Nausea, SSQ:Oculomotor, and SSQ:Total  device. HMD users frequently suffer from eyestrain, and blurred vision and short-term changes in binocular vision possibly due to alterations in the balance between the vergence and accommodation systems ,31. In oThe ability to move one's head and search for targets is a functional task that is often impaired in people with vestibular disorders. In vestibular rehabilitation, patients are encouraged to move their head during daily activities because movement is needed for adaptation and reweighting of the sensory signals. Using optic flow and/or virtual environments, this activity could be restored. Our preliminary analysis demonstrates that healthy people are able to coordinate their head and eye movements in the presence of stationary and moving backgrounds with few side effects. The next step is to perform similar experiments with people who have vestibular disorders.In addition, it is imperative to study if these movements can be tolerated while using narrower FOV HMDs. Head mounted displays will most likely be the desired display system of choice for vestibular rehabilitation because of the relatively modest cost and high portability. However, there are other characteristics of the display systems that need to be considered. For example, it is common for users of HMDs to complain of eyestrain, blurred vision, headache, and nausea ,31. In aThe performance of head unrestrained gaze shifts in a wide FOV optic flow environment is tolerated well by healthy subjects. This finding provides rationale for testing these environments in people with vestibular disorders, and supports the concept of using wide FOV virtual reality for vestibular rehabilitation.