Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization.Q69L, a mutant locked in the GTP-bound state, is less highly concentrated in the nucleus and associates with nuclear pore complexes within the nuclear envelope. During mitosis, GFP-RanQ69L is excluded from chromosomes and localizes to the spindle. By contrast, GFP-RanT24N, a mutant with low affinity for nucleotides, interacts relatively stably with chromatin throughout the cell cycle and is highly concentrated on mitotic chromosomes.Here, we have characterised the localisation and mobility of Ran coupled to green fluorescent protein (GFP) during the cell cycle in live human cells. Ran-GFP is nuclear during interphase and is dispersed throughout the cell during mitosis. GFP-RanThese results show that Ran interacts dynamically with chromatin, nuclear pore complexes and the mitotic spindle during the cell cycle. These interactions are dependent on the nucleotide-bound state of the protein. Our data indicate that Ran-GTP generated at chromatin is highly mobile and interacts dynamically with distal structures that are involved in nuclear transport and mitotic spindle assembly. Ran is a member of the Ras superfamily of small GTPases and has multiple roles in coordinating essential nuclear and mitotic processes in eukaryotes . Like otRan plays a central role in the nucleocytoplasmic transport of many macromolecules during interphase ,4. The hXenopus egg extracts and live mammalian cells [Xenopus egg extracts, a gradient of Ran-GTP concentrations decreasing away from metaphase chromosomes arranged on the spindle is observed [Xenopus egg extracts [Since the activity of Ran is determined by both its guanine nucleotide-bound state and its localisation, it is of interest to determine the relationship between these factors. This differential concentration of Ran-GTP across the nuclear envelope (NE) has been visualised indirectly using FRET (fluorescence resonance energy transfer) probes derived from Ran-GTP-binding domains in both an cells -13. In Mobserved . In mammIn fixed cells, Ran has been localised to specific structures including the NE and the mitotic spindle by either indirect immunofluorescence or tagging Ran with naturally a fluorescent protein -16. HoweQ69L, which is defective in GTP hydrolysis and therefore is predominantly in the GTP-bound form [T24N, which has reduced affinity for nucleotides and forms a stable complex with RCC1 [Here, we have determined the localisation of Ran expressed as a fusion with green fluorescent protein (GFP) at high resolution in living, unfixed cells in interphase and during progression through mitosis. To investigate the role of the GTP-GDP cycle of Ran in determining its localisation, we have also localised two mutants of Ran expressed as GFP-fusions, Ranund form ,19, and In order to study the sub-cellular localisation and dynamics of Ran during the division cycle, wild-type (WT) and mutant Ran proteins were expressed as GFP-fusions in cultured human cells. To assess the localisation of these fluorescent proteins with respect to nuclear or mitotic chromosomes, co-transfections were performed with a plasmid expressing red fluorescent protein (RFP) fused to histone H2B (RFP-histone). The localisation of these fluorescent proteins was then observed during stages of the cell division cycle by fluorescence microscopy using a DeltaVision Spectris microscopy workstation. Data is shown for U2OS cells, but consistent results were also obtained using HeLa and HEK293 cells.WT localised predominantly to the nuclei of live interphase cells, with low levels of cytoplasmic signal  showed that the fluorescence signal was rapidly lost throughout the nucleoplasm, while the punctate NE signal persisted  for the GFP-proteins  experiments in GFP-Ran-transfected cells to determine the recovery half-times  not significantly different from than that in the cytoplasm (0.19 s), and only marginally greater than that for GFP-GST in both compartments (0.07 s). By comparison, the FRAP t1/2 for RFP-histone H2B, a protein expected to be very stably associated with chromatin, was determined as being 50-60 s. In contrast, the mean FRAP t1/2 for nuclear GFP-RanT24N (~ 2.5 s) was nearly 20-fold greater than that for nuclear GFP-RanWT and GFP-RanQ69L, and for cytoplasmic GFP-RanT24N. During progression through mitosis the mobility of GFP-RanT24N decreased considerably, with the mean FRAP t1/2 rising to 3.85 s in metaphase, and 5.35 s in telophase.We carried out multiple such experiments on interphase cells expressing sufficient GFP-Ran in both the nucleus and the cytoplasm for photobleaching in both compartments Figure . BecauseThe functions of Ran in different processes during the cell cycle are determined by both the guanine nucleotide-bound state and the localisation of the protein. Two divergent models have been hypothesised for the interplay of these conditions, particularly with regard to mitosis in animal cells when the controlled boundary between the chromosomes and cytoplasm is broken down : one modHere, we have used live-cell imaging techniques to determine the localisation of Ran in somatic human cultured cells. We find that wild-type Ran, which can freely convert between GDP and GTP-bound states depending upon its interaction with localised regulators, when expressed as a fusion with GFP is dispersed in the nucleus during interphase and also localises to NPCs. In mitosis, GFP-Ran is dispersed throughout the cell and no localisation to specific structures could be discerned, even using FLIP techniques to bleach mobile GFP-Ran molecules. Nevertheless, GFP-Ran was not excluded from chromosomes during mitosis, suggesting that it does interact transiently with chromatin.Q69L may also be sequestered by stable binding to components of the NPC, such as RanBP2 via its Ran-GTP binding domains. RanQ69L alters the conformation of NPCs formed in vitro in Xenopus egg extracts [WT to NPCs, albeit less pronounced, indicates that this is a normal if transient interaction for Ran. However, we found that GFP-RanT24N is also associated with NPCs in live cells, consistent with previous immunofluoresence of this mutant in fixed cells , indicating that Ran can interact with NPCs independently of its GTP-bound state. This would be consistent with the ability of Ran to also interact with the NPC when in the GDP-bound conformation [To investigate the relationship between localisation and the nucleotide-bound state of Ran, we also used GFP-Ran stabilised in different nucleotide-bound states by mutation. A mutant that is locked in the GTP-bound state, Q69L, was less concentrated in the nucleus and showed prominent localisation to NPCs within the NE during interphase. The lack of concentration of the mutant into the nucleus is consistent with its inability to interact with the import factor NTF-2 even when it has GDP bound . GFP-Ranextracts , suggestormation .Q69L and chromatin means that it is unlikely that Ran-GTP forms stable interactions with chromatin-bound proteins either during interphase or in mitosis. However, we show that Ran-GTP in the form of GFP-RanQ69L localises to the mitotic spindle where it may interact with Ran-GTP-binding proteins. Concentration of GFP-RanQ69L on the spindle strongly suggests that Ran-GTP normally interacts transiently with this structure and may be released with GTP hydrolysis induced by RanGAP1.The lack of association between RanQ69L since it is does not form interactions dependent on the GTP-bound conformation of Ran. RanT24N probably exists in cells mainly in a nucleotide-free form that makes a stable complex with the chromatin-associated guanine nucleotide exchange factor for Ran, RCC1. RanT24N stabilises the interaction of RCC1 with chromatin [T24N is very similar to that of RCC1 throughout the cell cycle [T24N associates with chromatin much more stably than GFP-RanWT, although it is worth noting that its interaction is still dynamic, has kinetics similar those of RCC1 fused to GFP and is similarly stabilised during progression through mitosis from metaphase to telophase [T24N therefore is likely to represent the sites of generation of Ran-GTP by RCC1.The T24N mutant of Ran does not simply correspond to a GDP-bound conformation, since it is defective in binding either GTP or GDP , but it hromatin . Indeed,ll cycle . We findelophase . The locWT and GFP-RanQ69L (only the latter is excluded from mitotic chromatin) may indicate that Ran-GDP interacts with chromatin in cells, as has been found in vitro in Xenopus egg extracts [Unfortunately, no mutation has been identified that would stabilise Ran specifically in the GDP-bound conformation and so the localisation of Ran-GDP cannot yet be determined directly by this approach. The difference in chromatin interaction of GFP-Ranextracts , suggestOur results are consistent with the interplay between the nucleotide-bound state of Ran and its dynamic localisation, whereby soluble Ran-GTP is generated at chromatin by RCC1 and then rapidly diffuses throughout the compartment until it interacts with specific structures where it carries out localised functions. During interphase, Ran is concentrated in the nucleus by active import where it is predominantly GTP-bound, but it does interact with NPCs. During mitosis, Ran is dispersed throughout cells, but interacts with chromatin and the spindle. The interaction of Ran with structures within the cell may allow localised nucleotide cycling linked to the assembly/disassembly of immobilised protein complexes and control their function specifically at that site . Such a Xenopus egg extracts. In this system, chromatin recruits Ran, initially in its GDP-bound state [Localisation of Ran to structures such as chromatin may alternatively be thought of as a mechanism to simply increase its concentration above a certain threshold, which when exceeded triggers complex formation, ensuring spatial organisation of the functions of Ran. Such a mechanism has been proposed for the function of Ran in nuclear envelope assembly in nd state . The recnd state  and can nd state . The sufnd state .In a general model for the role of Ran-GTP in intracellular spatial organisation, soluble Ran-GTP concentrations provide an indicator of the presence of chromosomes, with a homogenous Ran-GTP concentration throughout the nucleoplasm during interphase. In mitosis, the high concentration of Ran-GTP in the vicinity of chromosomes relative to the rest of the cell provides a low-resolution spatial signal to organise spindle assembly around chromosomes. However, the local action of Ran is determined by its interaction with intracellular structures such as the NPC in interphase, centrosomes and the spindle during mitotic spindle formation, and chromatin at telophase. The relative importance of the gradient in soluble Ran-GTP concentration in providing a spatial signal as opposed to the action of Ran at pre-defined sites is not yet certain and may differ between cell types. It may also change during progression through mitosis, for instance when the function of Ran switches from microtubule organisation at a distance from chromatin in prometaphase to nuclear envelope formation on chromatin at telophase.We have analysed the dynamic localisation of Ran fused to a fluorescent protein in live human cells. Using mutants of Ran stabilised in either the GTP-bound or nucleotide-free state, we show that Ran interacts dynamically with specific subcellular structures depending on its nucleotide-bound state. We infer that Ran-GTP generated at chromatin is highly mobile and interacts with distal structures that are involved in nuclear transport and mitotic spindle assembly. These results support a model for the spatial action of Ran in which Ran-GTP provides a low-resolution signal of the presence of chromosomes while its localised sites of action are specified by the subcellular structures with which it interacts.WT, RanT24N, RanQ69L and GST constructs in the pEGFP-1 expression vector (Clontech) were as previously described [Ranescribed . These vQ69L, where at least 10 cells were observed for each experiment.Human cells were cultured on coverslips, transfected with plasmids, and assembled into a live-cell chamber as previously described . The cha1/2) and mobile fraction (MF) values. At least 10 cells were analysed for each condition and t1/2 values are expressed ± 1 standard deviation.For fluorescence recovery after photobleaching (FRAP) experiments, three images of the cell were captured prior to bleaching, then the laser was focussed to a diffraction-limited spot and spot bleaching performed with a single 20 ms stationary pulse. Sixty post-bleach images (50 ms exposure) were captured during the subsequent recovery phase. For faster-recovering cells, a 'fast-as-possible' protocol was employed, in which the camera shutter remained open and fluorescence images were sampled every 50 ms. For slower-recovering cells, the four-phase sampling protocol was employed, as previously described . AnalysiFor fluorescence loss in photobleaching (FLIP) experiments, three pre-bleach images of each cell were taken, then one hundred cycles of laser-bleaching (20 ms) and imaging were performed. Image data were exported from softWoRx as MPEG files, from which frames were imported into Improvision Openlab software as separate layers for analysis.To quantitate the loss of fluorescence, signal intensities within the defined region of interest (nucleus or chromatin) at different time points were determined, and expressed as percentages relative to the first pre-bleach image. The ratio module of Openlab was used to divide selected grayscale images from the FLIP timecourse by a pre-bleach image, generating a set of grayscale ratio images. These were then re-coloured to make a series of ratio images that correspond to a spectrum.JRAH generated and analysed the data on localization, FRAP and FLIP. WJM made GFP-fusion constructs, and generated and analysed FLIP data. PRC was the principal investigator and was responsible for supervision of the study and writing the manuscript together with JRAH. All authors read and approved the final manuscript.Q69LFLIP analysis of GFP-Ran. Images of a cell expressing nuclear GFP-RanQ69L and after successive cycles of photobleaching within a defined spot within the nucleus. The cell was subjected to 100 cycles of laser-bleaching, each of 20 ms duration. Image data were exported from softWoRx as MPEG files, from which frames were imported into iMovie and Quicktime software.Click here for file


Web-based self-help interventions for problem drinking are coming of age. They have shown promising results in terms of cost-effectiveness, and they offer opportunities to reach out on a broad scale to problem drinkers. The question now is whether certain groups of problem drinkers benefit more from such Web-based interventions than others. We sought to identify baseline, client-related predictors of the effectiveness of Drinking Less, a 24/7, free-access, interactive, Web-based self-help intervention without therapist guidance for problem drinkers who want to reduce their alcohol consumption. The intervention is based on cognitive-behavioral and self-control principles. We conducted secondary analysis of data from a pragmatic randomized trial with follow-up at 6 and 12 months. Participants (N = 261) were adult problem drinkers in the Dutch general population with a weekly alcohol consumption above 210 g of ethanol for men or 140 g for women, or consumption of at least 60 g (men) or 40 g (women) one or more days a week over the past 3 months. Six baseline participant characteristics were designated as putative predictors of treatment response: (1) gender, (2) education, (3) Internet use competence (sociodemographics), (4) mean weekly alcohol consumption, (5) prior professional help for alcohol problems (level of problem drinking), and (6) participants’ expectancies of Web-based interventions for problem drinking. Intention-to-treat (ITT) analyses, using last-observation-carried-forward (LOCF) data, and regression imputation (RI) were performed to deal with loss to follow-up. Statistical tests for interaction terms were conducted and linear regression analysis was performed to investigate whether the participants’ characteristics as measured at baseline predicted positive treatment responses at 6- and 12-month follow-ups.P = .05, 2R = .11). At 12 months, females displayed modest predictive power in both imputation models . Those with higher levels of education exhibited modest predictive power in the LOCF model only . At 6 months, prior help for alcohol problems predicted a small, marginally significant positive treatment outcome in the RI model only : 47285230; http://www.controlled-trials.com/isrctn47285230 (Archived by WebCite at http://www.webcitation.org/5cSR2sMkp). Problematic alcohol use is not only a pervasive individual problem; it also imposes serious health and social burdens on the general population ,2,3. ThiIt is well known that treatment response is not influenced by treatment alone . A numbed = 0.40, 95% CI 5.86 - 18.10; P < .001). At 12 months, the difference between the groups had faded , mainly due to a further decrease in alcohol consumption in the control group. Results of this pragmatic randomized trial have been reported elsewhere [We therefore investigate here whether specific baseline characteristics can be identified as predictors of a positive treatment outcome for problem drinkers in the Dutch population who completed a Web-based self-help intervention called Drinking Less. On the basis of predictors already reported in the literature, we hypothesized that six putative baseline characteristics—(1) female gender, (2) higher education, (3) Internet competence, (4) a moderate level of problem drinking, (5) prior professional help for problem drinking, and (6) high expectancy for positive results from a Web-based intervention—would interact with Drinking Less to predict a more favorable treatment outcome at follow-up. We conducted a secondary analysis of our Drinking Less trial data  to examilsewhere .To the best of our knowledge, this is the first article that uses randomized trial data to assess predictors of short- and longer-term outcomes in Web-based self-help for problem drinkers in the general population.Data were retrieved from a pragmatic randomized trial with two parallel groups using block randomization stratified for gender, with follow-up at 6- and 12 months . In brieWe kept our exclusion criteria to a minimum to facilitate a low-threshold inclusion strategy consistent with the nature of self-help interventions without therapeutic guidance. We therefore did not conduct diagnostic interviews. After screening and baseline assessment, participants were randomly assigned to the experimental condition (the Drinking Less intervention) or to the control condition  . We sele                    Participants in the experimental condition received access to the Drinking Less intervention . Drinkin                    Our choice of baseline participant characteristics as putative predictors was based on theoretical assumptions and results from previous prediction studies -22. We sThe outcome measure was defined as the individual differences between baseline (T0) mean weekly alcohol consumption and the mean level of consumption at posttreatment  and at follow-up  in the total group. Alcohol consumption was assessed with the Dutch version of Weekly Recall (WR) ,33. It rt-tests, chi-square tests, and logistic regression to assess whether the randomization had resulted in two comparable groups at baseline and whether any differential loss to follow-up had occurred. We then performed intention-to-treat (ITT) analysis, using last-observation-carried-forward (LOCF) data and regression imputation (RI) to deal with loss to follow-up. Overall loss to follow-up was high . Values on the WR scale were transformed into a variable distinguishing moderate problem drinking  from severe problem drinking (> 35 or > 50 units women/men). Categorical variables with more than two categories were recoded into two meaningful categories: (1) education: high/low ; (2) Internet competence: experienced/beginner; (3) prior professional help for alcohol problems: yes/no; and (4) expectancies of Web-based intervention: high/low. We then applied regression analyses to ascertain whether these particular groups benefited more from the intervention than others. We assessed the interactions between the above-baseline attributes and the Drinking Less intervention modality, and then the effects of those interactions on treatment outcome. In this model, the standardized individual change scores (pre- to post-intervention effect sizes) served as the dependent or outcome variable. The interaction terms of each participant characteristic with the intervention dummy  served as independent predictor variables, along with their constituent main effects.P < .05. Independent-samples t-tests were used to analyze differences between the conditions in terms of problem drinking outcome at T1 and T2. This technique permitted us to test for the differential effects of the predictors in interaction with the Drinking Less treatment. It also enhanced the power to detect effects. If neither of these interaction terms proved significant, then the effect of the predictor was deemed not to be modified by Drinking Less. That is, the effect of Drinking Less on drinking outcome could not be explained by the predictor’s modifying effect on the relationship between treatment and outcome.We next calculated the product of the intervention dummy and each of the dummy variables describing the participants’ characteristics ,37. The We subsequently repeated this procedure in completers-only analyses on those participants who completed the follow-up questionnaire at 6 months (n = 151) or at 12 months (n = 163) to verify whether the results of the two ITT analyses would be sustained. Finally, we used descriptive statistics to illustrate the changes in alcohol consumption over time in terms of the identified predictors. The sample size provided 24 participants per variable at 6 months and 26 per variable at 12 months . All anaP < .10 to ensure against marginal differences that could affect results). This indicated that the randomization was successful. At baseline, all 261 participants (100%) were exceeding the mean number of weekly alcohol units set by the Dutch guideline for sensible drinking for healthy adults. Mean weekly alcohol intake was 43.6 standard units (SD = 21.6). More than half the sample belonged to the category of moderate, as opposed to severe, problem drinkers . The female-to-male ratio was almost 1:1. Two-thirds of participants had high educational backgrounds . Most participants considered themselves experienced Internet users . Almost half had positive expectations of the intervention . The large majority of participants  were in the contemplation stage of change, meaning that they wanted to reduce their alcohol consumption in the near future [The demographic and clinical characteristics of participants at baseline are shown in r future ,40. MostP > .10; 2                    1 = 1.71, P = .19). At 12 months, loss to follow-up was 37% (n = 98) and was greater in the experimental condition  than in the control condition . Non-responders at 12 months had a higher baseline mean weekly alcohol intake as measured by WR  than non-responders .Participants who did not return the questionnaire 6 months after baseline did not differ from posttreatment responders in terms of the characteristics assessed at baseline  and the completers-only model. We therefore present here only the intention-to-treat models. Results of the completers-only analysis are available from the first author.P = .05, 2R = .11). At 12 months, female gender predicted successful alcohol reduction in both analysis models , while the LOCF model showed a less strongly significant impact and a lesser amount of explained variance . High education level was identified as an additional predictor of successful outcome at 12 months. The LOCF analysis  showed a significant but modest effect and accounted for a small fraction of the variance in treatment outcome, but the effects in the RI model were not significant.Analyses of predictor-by-treatment interaction effects in terms of a successful reduction of mean weekly alcohol use found no significant effects for the putative predictors at 6 months , with ths models . RI indiWe compared the mean weekly alcohol consumption at 6 and 12 months for the two conditions as shown by the intention-to-treat and completers-only analyses. The last-observation-carried-forward (LOCF) model appeared to be the most conservative estimation method for the total group, as it returned the highest alcohol intake in both conditions—thus suggesting less improvement. We therefore chose these more cautious LOCF results to report outcomes for the two main predictors identified in our analysis. Detailed information about the other two models can be obtained from the first author.                                        At 6 months, the more highly educated Drinking Less  participants had achieved the greatest reduction in both absolute and relative terms  as compared to other categories . Althoug                    The aim of this study was to determine whether some groups would benefit more than other groups from Drinking Less, a Web-based self-help intervention for problem drinkers, when assessed at 6 and 12 months. We investigated six characteristics of the participants at baseline as putative predictors of treatment response: (1) female gender, (2) high level of education, (3) high Internet experience, (4) moderate as opposed to severe level of problem drinking, (5) prior professional help for alcohol-related problems, and (6) high expectancies for Web-based intervention.At the 6-month follow-up, we could not convincingly establish predictive value for any of these putative predictors, with the possible exception of prior help for alcohol problems, which was only marginally significant under the regression imputation model. Some other studies have likewise identified prior professional help as a predictor of positive client-by-treatment interaction leading to successful outcomes . An explAt 12 months, we found a modest prognostic value for female gender and for higher education; both variables were associated with better treatment response to the Drinking Less self-help intervention. Women who completed the intervention were found to have reduced their alcohol consumption to a significantly greater extent than men or than control group participants. Comparable results for female gender as a predictor of a successful brief intervention outcome in general population samples were reported by Sanchez-Craig and colleagues  and, to Higher levels of education also had modest predictive power and explained a small amount of variance at 12 months in combination with Drinking Less. This finding is consistent with results from other studies that identified high education as interacting with treatment interventions to produce favorable outcomes ,45. LikeThe other characteristics investigated were not found to act as predictors in our study. A moderate baseline level of problem drinking  did not predict better outcomes than a severe level. This contrasts with the many studies that assume brief interventions to be better suited to moderate problem drinkers . One expWe did not find any predictive value for the two remaining putative predictors, Internet experience and positive expectancies of treatment efficacy, in contrast to some other studies that did ,48. ExplThis study has several limitations that are important to acknowledge. We conducted secondary analysis of data from our pragmatic randomized trial .The overSecondly, we conducted a prespecified subgroup analysis and hence cannot rule out false-positive or false-negative predictors resulting from multiple testing ,54. GiveWe were also limited by the data in the number of predictors we could investigate. That prevented us from studying self-efficacy, a potentially important predictor . Nor couOur study has a number of strengths as well. The study on which the analysis is based was one of the first pragmatic randomized trials on the effectiveness of Web-based self-help interventions without therapeutic guidance for problem drinkers in the general population. The data also enabled us to examine short- and longer-term relationships. Because we had anticipated a high overall loss to follow-up when we first selected the trial sample, we included enough participants to ensure the statistical power to detect differences between the experimental and control conditions and between subgroups .Female gender and a high level of education were found to have interacted with the Drinking Less self-help intervention to predict a somewhat better treatment response one year after the start of the intervention. This suggests that Web-based self-help without therapeutic guidance may hold a special attraction for problem drinkers with greater fears of stigmatization, including women or more highly educated people—population segments that might otherwise be difficult to reach with face-to-face brief interventions . The nonAt the same time, the effects of the predictors identified here offer only a very partial explanation for how client characteristics interact with treatment to affect outcome. Other baseline attributes such as self-efficacy may also play a role . In addiOur findings could enhance public health strategies that use stepped-care approaches to curb problem drinking in the general population. Since none of the groups we identified stood out conspicuously against others as deriving benefit from Drinking Less, we would argue that Web-based self-help is well suited to a broad, heterogeneous group of problem drinkers. It may therefore serve well as an initial intervention in a stepped-care model, suitable for matching to a large and varied group of problem drinkers in the general population and not just at more individual levels ,59. The Our results add to the knowledge already gained from prediction studies in that we tested the role played by individual baseline attributes in the effectiveness of Web-based self-help for problem drinkers in the general population. The scope of future prediction research now needs to be extended to include the contributions of within-treatment progress variables, such as dose-response relationships and the time required to initiate positive behavioral change, and of posttreatment variables like social support. Replication of our study is needed in view of the novelty of Web-based interventions for problem drinkers and the related prediction research.


Cimex lectularius) mount any large, newly fed individual in an attempt to mate. Last instar nymphs overlap in size with mature females, which make them a potential target for interested males. However, nymphs lack the female's specific mating adaptations and may be severely injured by the abdominal wounding. We, therefore, hypothesized that nymphs emit chemical deterrents that act as an honest status signal, which prevents nymph sexual harassment and indirectly reduces energy costs for males.Abdominal wounding by traumatic insemination and the lack of a long distance attraction pheromone set the scene for unusual sexual signalling systems. Male bed bugs (E)-2-hexenal, (E)-2-octenal 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal, from their dorsal abdominal glands. We report that the aldehydes and 4-oxo-(E)-2-hexenal are detected by olfactory receptor neurons housed in smooth and grooved peg sensilla, respectively, on the adult antennae, at biologically relevant concentrations. Behavioural experiments showed that application of 4-oxo-(E)-2-hexenal or the two aldehydes at a nymph-emitted ratio, to a male/female pair during mounting initiation, decreased mating frequency to a rate comparable to that of a male/nymph pair.Behavioural mating assays showed that males mount nymphs significantly shorter time compared to females, although initial mounting preference was the same. In support of our hypothesis, nymphs experienced the same percentage of mating with sperm transfer as females if they were unable to emit (By combining behavioural and sensory studies, we show that the nymph-specific alarm pheromone plays an important role in intra-specific communication in the common bed bug. Alarm pheromones are commonly looked upon as a system in predator/prey communication, but here we show that alarm pheromones may be used as multipurpose signals such as decreasing the risk of nymphal mating by males.http://www.biomedcentral.com/1741-7007/8/117See commentary:  Cimex lectularius) has received increased attention in recent years as bed bug infestations have increased worldwide. The occurrence of alarm pheromones has been known since the 1970 s . The behavioural role of these nymph-specific compounds, or the significance of the reversed ratio of the two aldehydes, has not yet been established.It is well established that stressed bed bugs of all stages and sexes release an alarm pheromone, causing the dispersal of conspecifics ,11. Howe-octenal ,13, both-octenal . Female n nymphs . In addioctenal , did not affect the percentage of mating: 78.1%  and 72.5% , respectively, compared to control (84.2%).Transforming the females into silenced females by blocking their metathoracic glands did not affect male mounting behaviour -2-hexenal:(E)-2-octenal in a nymph-specific ratio [2:5] and 4-oxo-(E)-2-hexenal, which all elicit ORN responses at biologically relevant concentrations, are sufficient to deter males from copulating with the manipulated female -2-hexenal produced specifically by the nymphs is detected by specific sensilla on the male antennae and may contribute on its own to a repellent effect on males. This double effect may give a stronger protection to the nymphs against mating and piercing mistakes. The chemical communication system of the bed bugs is only just unfolding, and further analyses on longevity costs to nymphs as well as males who have been pierced is a high priority to fully understand the picture of traumatic insemination.The unusual mating behaviour of the bed bug, with traumatic insemination, gives new insights to their chemical communication systems. Like males avoiding homosexual mistakes , nymphs in vitro feeding system as described by . The three ratios of aldehydes, the oxo-aldehyde and a hexane control were tested on four different groups of male/female pairs, and observations were conducted in the same manner as described above.Prior to the observation, the methathoracic glands of an unfed female were covered by nail polish to prevent emission of alarm pheromones. In order to mimic a male/nymph interaction, odours were mechanically puffed onto a male/female pair as soon as the male mounted the silenced female. The couple was exposed to a single puff of stimulus delivered via a Pasteur pipette equipped with a 5 mL rubber bulb as a manual release system. The Pasteur pipette contained a filter paper strip (~5 × 15 mm) impregnated with 10 μL of a stimulus solution. The chemicals used in the behavioural experiments were diluted singly in hexane (99%), down to a 100 ng/μL level  was calculated by dividing the number of mated partners by the number of mounted partners.The Kruskal Wallis test was applied to compare general differences in sample groups larger than two. It was followed by the Mann Whitney U-test in order to statistically compare the proportions between two samples. The time of mounting was log transformed in order to reach normality (Kolmogorov Smirnov test) and as the variance was statistically similar between the two groups (Levene test), one way analysis of variance was then applied. All the statistical analyses were performed with SPSS software .ORN: olfactory receptor neuron; F: females; SN: silenced nymphs; N: controls; STN: sham-treated nymphs.VH participated in the design of the study, carried out the electrophysiological study, contributed in the behavioural studies, performed the data and statistical analysis and drafted the manuscript. CR conceived the behavioural design of this study, helped in the data interpretation and drafted the manuscript. RI helped in the data interpretation and edited the manuscript. All authors read and approved the final manuscript.


Intentional self-harm is common, through out the world; however, there is scanty data from India.To study the sociodemographic and clinical profile of subjects with “intentional self-harm” referred to consultation-liaison psychiatric services for evaluation in a tertiary care hospital.Retrospective chart review.For this study, the consultation-liaison register of Department of Psychiatry was screened to obtain data of all patients who were referred to psychiatry referral services and were diagnosed as “intentional self-harm” while they were admitted in Nehru Hospital, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh during the period of 2000-2005. The case notes of these patients were reviewed for obtaining the sociodemographic profile and clinical profile for the current study.Majority of the subjects were married (61%), educated beyond matriculation (75%), were employed or retired (53.6%), belonged to Hindu (87%), nuclear family (64.5%) of middle socioeconomic status (85%) and came from urban background (53%). Most common reasons/precipitating events prior to intentional self-harm were interpersonal problems with family members (39.2%), followed by interpersonal problems with spouse (16.9%). The most common method of intentional self-harm used was consumption of insecticides (44.6%), followed by use of corrosives (17.5%). Half of the sample (48.2%) did not fulfill criteria for any axis-1 or axis-2 psychiatric diagnosis at the time of assessment and most common psychiatric diagnosis was depression (30.7 %).Nearly half of the subjects who present to a tertiary care hospital with intentional self-harm do not have diagnosable psychiatric illness. Various terms like “attempted suicide,” “deliberate self-poisoning,” “deliberate self-injury,” and “parasuicide” have been used interchangeably, to defin26The psychosocial milieu of the developing countries is different from that of the developed countries. Indian society is nontolerant to suicide, views it as an act of cowardice and betrayal. ComparedIn many countries including India, subjects who harm themselves frequently present to hospital emergency for medical complications arising as a result of self-harm. Hence, they form an important group to understand the psychosocial profile of patients who harm themselves. With this background, aim of our study was to study the sociodemographic and clinical profile of subjects with “intentional self-harm” referred to consultation-liaison psychiatric services for evaluation in a tertiary care hospital.The study was carried out at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, providing services to a major part of North India. PGIMER is a multispecialty tertiary-care teaching hospital; there is extensive cross-referral among the various departments of the Institute for specific problems. Department of Psychiatry of the Institute runs a round-the-clock, three-tier system for providing emergency psychiatry cover for the entire hospital including its Emergency Medical and Surgical outpatients. This system was initially run on written calls. Later, this system was supported by the paging system of the Institute. All the referred cases are initially evaluated by a trainee psychiatrist (junior resident), under the supervision of a qualified psychiatrist (senior resident). The cases are evaluated for the presence of psychiatric morbidity, and if it is present, it is assessed whether it is attributable to a physical illness or not. The diagnoses are made as per the ICD-10. The caseIn addition to this, information of all the calls received from various wards is also summarized in a referral register under the following headings - age, sex, source of referral, diagnosis of the physical condition, reason for psychiatry referrals, psychiatric diagnosis, management done, and outcome. This register is monitored for completeness weekly by the designated Consultant Incharge of Psychiatry Referral Services.Whenever a patient is admitted to hospital with intentional self-harm/suspected self-harm of any kind, the psychiatric referral team is consulted for detailed psychiatric evaluation. Besides the documentation of sociodemographic profile, the detailed psychiatric evaluation of such patients involves obtaining a psychiatric history, immediate precipitating event prior to self-harm, method used, family history of psychiatric disorders including suicide or suicide attempts, current mental status examination, risk of future attempt, etc. After the initial evaluation, these patients are subsequently followed up in the inpatient setting till they are physically stable. After this, depending on the mental status examination and risk of future attempt, these patients are either transferred to psychiatry ward or are followed up in psychiatry OPD. The psychiatry management usually involves treatment of axis-1 diagnosis by pharmacotherapy and psychotherapy.For this study, the referral register was screened to obtain data of all patients who were referred to psychiatry referral services and were diagnosed to have “intentional self-harm” (as per ICD-10) while tDuring the study period (2000-2005), a total of 3092 cases were referred to psychiatric referral services, out of which 166 cases were of intentional self-harm, which gives a prevalence figure of 5.36% of intentional self-harm cases in psychiatry referrals.There was nearly equal distribution of males (53%) and females (47%) in the sample. Majority of the subjects were married (61%), educated beyond matriculation (75%), were employed or retired (53.6%), belonged to Hindu (87%), nuclear family (64.5%) of middle socioeconomic status (85%) and came from urban background (53%).The mean age at intentional self-harm of the sample was 29 years (SD - 12.11), with a range of 12-76 years. There was no significant difference between males and females in terms of mean age at intentional self-harm. When the age at intentional self-harm was analyzed in terms of groups, nearly half of subjects (48%) were in the age group 20-30 years and one-fifth (19%) were aged between 10 and 20 years.n = 130) had not consulted any psychiatrist in the past. Among the diagnosed cases, the most common diagnoses were affective disorders (39%). There was history of previous intentional self-harm in 9% of cases. When the subjects were assessed after the intentional self-harm, nearly half (52%) of them were diagnosed to have some psychiatric disorder, with depressive disorder being the most common [More than three-fourth of the sample .Majority of the subjects (78%) had a precipitating event prior to the intentional self-harm, most common of which was interpersonal problems with family members other than spouse (38%), followed by interpersonal problems with spouse (16.9%). The most common method of self-harm was consumption of insecticides (44.6%), followed by use of corrosives (17.5%) and use of psychotropic drugs (12.7%). About 17% of patients had harmed themselves after consuming alcohol. Out of the psychotropics, sedative-hypnotic group was the commonly used drug for overdose . Use of Comparisons based on the type of method used showed that majority of subjects who took overdose of psychotropics were suffering from depression whereas subjects who used corrosives and organophosphorous did not have a diagnosable psychiatric illness at presentation. In more than half of the cases (56.6%), intentional self-harm did not lead to any severe medical complications. Sixteen percent of the patients developed delirium, 10% developed postcorrosive esophageal ulceration and 3.6% developed hypoxic ischemic encephalopathy as a complication to the methods used for intentional self-harm.Eight patients died during the inpatient stay. Mortality was associated with methods like burns (four patients), poisoning (n=2), stab injuries (n=1) and hanging (n=1).t value= 2.50*, DF = 164; P < 0.05). In females, the intentional self-harm was more frequently preceded by depressive cognition  and this difference was statistically significant . Further, any axis-I or axis-II diagnosis was more frequently seen in females  and the difference was statistically significant .On comparison, it was found that female subjects who indulged in self-harm behavior were significantly more educated than males (P < 0.01), whereas subjects without any axis-I or axis-II diagnosis more frequently used insecticide .There was no statistically significant difference in the sociodemographic profile between those with and without any axis-I or axis-II diagnosis. There was difference in the immediate reason/precipitating event proceeding intentional self-harm. As expected subjects with a psychiatric diagnosis more frequently reported depressive cognition, whereas subjects without any axis-I or axis-II diagnosis more frequently reported interpersonal problems leading to intentional self-harm. Patients with psychiatric diagnosis more frequently used methods like psychotropic overdose (Chi square value 9.95**, DF = 1; et al.[et al.[The present study was a retrospective chart review for the sociodemographic and clinical profile of subjects with intentional self-harm presenting to a tertiary care hospital referred to psychiatry consultation-liaison services for psychiatric evaluation. The sociodemographic profile of our sample was similar to that in other studies from India.13 Majori667et al. Similar l.[et al. in whichl.[et al. The nuclThe commonest method employed to execute self-harm was insecticide poisoning. Similar findings have been reported from elsewhere in India718 and o7Studies from the West20 as wel15et al.[A significant proportion of our sample did not meet criteria for any axis-I psychiatric diagnosis. Similarly, Parker et al. also repDiagnosis of depressive episode or adjustment disorder in 40% subjects matches the existing literature from India. It is toOur study has some limitations. Our sample cannot be considered as truly representative of the population as all cases who present with intentional self-harm are not referred for psychiatric consultations; some are discharged prior to assessment and in some cases families do not disclose the facts to the treating agencies due to legal consequences. Another limitation of the study is the retrospective nature of the same. Hence, the findings should be interpreted in this background. Our study also did not look at the suicidal intent of the subjects at the time of intentional self-harm.Young age group represents the most vulnerable group in need. Half of the patients were diagnosed with psychiatric illness at presentation, which clearly argues for need of early, prompt diagnosis and treatment of such cases so as to preempt such attempts. Depression remains the most common diagnosis; its early identification and proper treatment can lead to reduction in suicide attempts/intentional self-harm and perhaps completed suicides. Our findings also suggest that intentional self-harm is also used by so-called normal persons as a coping mechanism under stress to communicate their needs and distress. Hence, promoting healthy coping mechanism and reduction in stress is required to reduce self-harm. As is evident from the study, modifying the interpersonal relationship problems in the family might help in preventing many of suicide attempts/intentional self-harm. There is also need to develop clear policy for sale and possession of insecticides.


Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive.We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA)-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication.Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase. Polyketides comprise a large class of natural products synthesized by unrelated organisms, such as bacteria, protists, plants, fungi and animals. These compounds are often found in organisms living in mutualistic associations, such as symbiotic bacteria of fungi , insects, and sponges Polyketide synthases (PKSs), among other enzymes, are involved in the biosynthesis of polyketides. PKSs are multifunctional enzymes, which are related to fatty acid synthases (FAS) The horizontal movement of genetic material between distantly related organisms, horizontal gene transfer (HGT), has played an important role in the evolution of prokaryotes In the current study we infer the evolutionary history of a clade of fungal type I PKS genes which is closely related to bacterial PKSs. Gene products of PKSs in this clade are small monocyclic or polycyclic aromatic compounds, which are precursors of fungal antibiotics, such as patulin An additional reason for our interest in this clade is the occurrence of sequences from lichenized fungi The aim of this study was to establish the phylogenetic origin of the enigmatic fungal 6-MSAS-type PKS biosynthetic gene in a comparative phylogenetic framework. Our results provide statistical support to the hypothesis that this PKS was transferred from an actinobacterial source into ascomycete fungi during an ancient HGT event. We report the finding of 6-MSAS-type PKS genes in a variety of lichen-forming fungi, and speculate about the possible role of lichen symbionts in the evolution of this gene.Ochrolechia yasudae and Pertusaria corallina). Sequence divergence between the two copies is 54% in Ochrolechia yasudae and 41% in Pertusaria corallina. We also retrieved two copies from three herbarium specimens.We generated 24 new 6-MSAS-type PKS sequence fragments from lichen-forming fungi . In fiveMycobacterium, such as phenolphthiocerol.Our initial phylogenetic analysis of 165 KS sequences confirms that the fungal 6-MSAS-type clade is more closely related to bacterial than to other fungal PKSs . This reMycobacterium. Most closely related to these two groups is an unsupported clade of iterative bacterial PKSs. The products of these genes are small aromatic polyketides, such as 6-MSA and orsellinic acid , Eurotiomycetes , Lecanoromycetes , and Sordariomycetes (Xylaria). This PKS has not been found in the most basal groups in Ascomycota, such as Saccharomycotina and Taphrinomycotina, or the most basal filamentous fungi (Pezizomycetes). Thus we hypothesize that the PKS was gained through an ancient HGT event that happened before a radiation in Leotiomyceta, which gave rise to the extant crown group of Ascomycota. Subsequently, this horizontally inherited gene was lost in the majority of extant Leotiomyceta. Within some lineages the gene experienced at least one duplication event, as demonstrated by the presence of different copies in some lichenized fungi.Our results are in agreement with previous studies in suggesting HGT as a likely mechanism in the evolution of fungal 6-MSAS-type PKS genes. Using comparative phylogenetic methods we demonstrated that the origin of this gene is likely to be found in the Actinobacteria. Our analyses suggest that fungal 6-MSAS-type PKS genes came from the ancestor of a group including bacterial iterative PKSs and a small group of bacterial modular PKSs. The wide occurrence of fungal 6-MSAS-type PKSs within the crown group of Ascomycota suggests that the gene was acquired through an ancient HGT event. Fungal 6-MSAS-type PKSs are shared by representatives of different classes within the higher Ascomycota (Leotiomyceta) The rare occurrence of introns in fungal 6-MSAS-type PKSs provides additional support to the argument of horizontal transfer from a bacterial source. While we only found a single spliceosomal intron in a 6-MSAS-type PKS from a lichenized fungus, these introns were omnipresent in other types of PKSs from lichenized fungi There are many examples of intimate bacterial-fungal interactions in nature The fact that 6-MSAS type PKS genes are only found in a small number of ascomycete genomes suggests that the gene has been lost in most ascomycete species during evolution. This is in agreement with the assumption that secondary metabolite gene clusters, once acquired, are only maintained, if natural selection favors their presence The base unit of depsides and depsidones, which are the most common extrolites found in lichens, is orsellinic or β-orsellinic acid. Considering the structural relatedness of orsellinic acid and 6-MSA , it seemHere we could support previous studies suggesting HGT of 6-MSAS-type PKS genes between bacteria and fungi using an extended sampling of DNA sequences. This allowed us to identify actinobacteria as the most likely source for this type of PKS genes in ascomycete fungi. Using comparative phylogenetic analyses, we were able to reject HGT from fungi to bacteria.Material used in this study included mycobiont cultures and field collections of lichens . We extrOchrolechia yasudae . These sequences were used as query for a BLASTx search in GenBank in April 2008. All sequences which had matches greater than 200 score bits were included in the alignment, and redundant sequences from the two searches were discarded. Additionally, we included five representatives of reducing fungal PKS clades Using the LC3/5c primers th tree. The run was terminated at 6.723.300 generations, when the average standard deviation of split frequencies between the two runs was <0.01, and examination of the p-files in the program Tracer 1.4 (http://tree.bio.ed.ac.uk/software/tracer/) showed that the two runs had converged. The initial 7234 trees were discarded as burn-in of the chain, and the remaining 120.000 (2×60.000) trees were summarized in a 50% majority rule consensus tree. A cartoon summary of this tree is given in The alignment was analyzed in a Bayesian phylogenetic framework using MrBayes 3.1 th tree into a file. The program Tracer 1.4 was used to determine whether the preset burn-in of 100,000 generations  was appropriate. The phylogeny resulting from the analysis of alignment 3 is given in The tree resulting from this analysis was used to determine the PKS clades most closely related to the fungal 6-MSAS group. The fungal 6-MSAS plus the four most closely related bacterial PKS clades are here referred to as “ingroup”. This subset of sequences was aligned based on amino acid sequences and reverted back to nucleotide sequences. To evaluate potential problems with outgroup selection th tree  to avoid autocorrelation www.evolution.rdg.ac.uk.) to reconstruct ancestral character states in a maximum likelihood framework at five selected nodes in the phylogeny.For ancestral character state reconstructions we used 492 post burn-in trees from the B/MCMC analyses. We sampled every 200Table S1PKS sequences from GenBank included in the alignment. All blastx hits greater 200 score bits were included .(0.26 MB DOC)Click here for additional data file.


The enhanced interaction of T-1249 with cell membranes correlates with its higher efficacy, as it can increase and accelerate the drug binding to gp41 in its pre-fusion state.Enfuvirtide and T-1249 are two HIV-1 fusion inhibitor peptides that bind to gp41 and prevent its fusogenic conformation, inhibiting viral entry into host cells. Previous studies established the relative preferences of these peptides for membrane model systems of defined lipid compositions. We aimed to understand the interaction of these peptides with the membranes of erythrocytes and peripheral blood mononuclear cells. The peptide behavior toward cell membranes was followed by di-8-ANEPPS fluorescence, a lipophilic probe sensitive to the changes in membrane dipole potential. We observed a fusion inhibitor concentration-dependent decrease on the membrane dipole potential. Quantitative analysis showed that T-1249 has an approximately eight-fold higher affinity towards cells, when compared with enfuvirtide. We also compared the binding towards di-8-ANEPPS labeled lipid vesicles that model cell membranes and obtained concordant results. We demonstrated the distinct enfuvirtide and T-1249 membranotropism for circulating blood cells, which can be translated to a feasible  This engagement triggers the exposure of the hydrophobic N-terminal region of another envelope glycoprotein, gp41. This fusion peptide anchors to the membrane of the host cell, enabling the two gp41 helical heptad repeat domains, the C-terminal (CHR or HR2) and the N-terminal (NHR or HR1), to fold into each other to form a hairpin-like structure (6-helix bundle). This approximates the cell and the viral membranes, facilitating their fusion and the release of the viral content into the cell The Human Immunodeficiency Virus type 1 (HIV-1) is a highly pathogenic, evasive and difficult to eradicate agent that causes Acquired Immunodeficiency Syndrome (AIDS). This discovery in the early 1980s triggered major international scientific efforts in antiviral drug discovery and development LAIBesides maraviroc, a recently approved CCR5 antagonist blocking gp120 co-receptor engagement in vitroSince the first appearance of enfuvirtide, the search for peptide drugs against HIV has been a growing field of research and several candidates proved to be efficient sn-glycero-3-phosphocholine) with partition constants  enfuvirtide showed residual association, while T-1249 binding was significant, despite also presenting a low partition (membrane insertion) Membrane binding/tropism emerged as an important factor to take into account concerning the mode of actions of these peptides. Previous studies evaluated the preferences of enfuvirtide and T-1249 to membrane model systems. Partition of these peptides to fluid phase and cholesterol-rich lipid vesicles was followed by intrinsic tryptophan fluorescence present in both peptides ants Kp of 1.6×1In this study, we aim to understand the nature of HIV-1 fusion inhibitors-membrane interactions using human blood cells, namely erythrocytes and PBMC . We made use of an indirect reporter, the fluorescent lipophilic probe di-8-ANEPPS, as direct measure of the peptide intrinsic tryptophan fluorescence was impracticable with cells. This probe incorporates in the outer leaflet of the cell membranes and is sensitive to the local dipole potential by shifting its excitation spectrum Isolated human erythrocytes and PBMC membranes were successfully labeled with di-8-ANEPPS. Confocal microscopy images clearly show the delineated labeled membrane . ImportaIncubation of the labeled erythrocytes and PBMC with enfuvirtide or T-1249, lead to changes in spectral properties of the membrane-bound di-8-ANEPPS. Excitation spectra were deviated to the red meaning that a decrease in the dipole potential occurs. Differential spectra can be used in order to help the visualization of the spectral shift . In thisR  =  I455 nm/I525 nm), that correlates with the dipole potential variations. After the addition of each peptide, we observed a decrease on the dipole potential within the first minutes for erythrocytes  for each peptide , accounting for a higher preference for the cell membrane. The relative behavior of the peptide towards the erythrocyte and PBMC membrane seems to be maintained: the ratios Kd,ENF/Kd,T-1249 are similar for both cell type .In order to quantify the affinity of the peptides to the cell membranes, we set to investigate the variation of the dipole potential as a function of peptide concentration . The rat peptide . For botAs stated earlier, interaction of enfuvirtide and T-1249 with lipid vesicles has been previously studied by means of their tryptophan residues fluorescence. For the sake of comparison with the data obtained for the interaction with blood cells membranes and in order to validate the comparison with previous data, we used di-8-ANEPPS labeled lipid vesicles to assess the interaction of these peptides, reported by changes on membrane dipole potential . The lipin vivo. We made use of a dipole potential membrane probe, di-8-ANEPPS, to indirectly assess the membrane binding of enfuvirtide and T-1249 to erythrocytes and mononuclear leukocytes. These peptides decreased the membrane dipole potential of the two cell types, indicating that an interaction is occurring. Quantitatively, T-1249 was found to have approximately 8 times more affinity towards these cell membranes than enfuvirtide. Furthermore, studies with di-8-ANEPPS labeled POPC vesicles corroborated this by showing higher binding of T-1249 in comparison with enfuvirtide. Concerning POPC/cholesterol vesicles, the relative binding affinity between the two peptides was maintained. The presence of cholesterol reduced the binding extension of enfuvirtide and had an opposite effect in the case of T-1249, in agreement with our previous results that followed the interaction by tryptophan fluorescence Designing peptide drugs based in gp41 sequence to prevent viral entry into host cells has been in focus in the antiretroviral research field. Several factors related to their mode of action must be taken into account in order to optimize efficacy. For the first time, we addressed the interactions of HIV-1 fusion inhibitors with human cell membranes to better understand their membranotropic behavior LAI 628–635) that binds to a hydrophobic groove in the NHR region The membranotropism of these peptides can be explained in molecular terms by their structure and aminoacid sequence. It has been identified in the CHR and adjacent regions specific motifs that account for different roles in the interaction with NHR and membrane environment. Some of these regions also translate to the CHR derived peptides. Towards the N-terminus of CHR, there is a pocket binding domain  via the complement receptor 1 (CR-1), that binds C3b or C3bi opsonized virus et al.The relevance of peptide binding to erythrocytes is not to be diminished. In fact, HIV can be found associated with erythrocytes In a therapeutic context, several factors can influence the availability of the antiretroviral peptides in circulation. The drug is distributed to different components of the blood, namely, dissolved in the plasma, bound to plasma proteins  and, as we established with this study, bound to cell membranes. The membrane-bound fraction would enhance the availability towards the molecular target in the virus, as explained earlier in this section, opposing to the fraction bound to plasma proteins, a factor that enhances peptide clearance. In conclusion, the dynamics between the peptide interaction among the different cell populations and with the virus, along with emerging new perspectives of HIV entry are complex factors that should the taken in consideration when rationally designing the next generation of fusion inhibitor peptides.3EDTA anticoagulant tubes . For erythrocytes, the blood samples were centrifuged at 1200 g during 10 min to remove plasma and buffy-coat. The erythrocytes were washed at least two times with working buffer . For labeling, they were prepared at 1% hematocrit in working buffer supplemented with 0.05% (m/V) Pluronic F-127 (Sigma) and di-8-ANEPPS 10 µM . PBMC were isolated by density gradient using Ficoll-Paque Plus , accordingly to manufacturer's instructions. Cells were counted in Cell-Dyn 1600 analyzer  and were prepared at 3000 cells/µL in Pluronic supplemented buffer with di-8-ANEPPS 3.3 µM. The suspensions of erythrocytes or PBMC with probe were incubated at room temperature with gentle agitation for 1 h. Unbound di-8-ANEPPS was removed by two wash cycles, with centrifugations at 1500 g for 5 min.Human blood samples were obtained from healthy volunteer donors at the public blood bank Instituto Português do Sangue , with their informed written consent. These samples were obtained under an institutional agreement between IPS Lisboa and Instituto de Bioquímica from Faculdade de Medicina da Universidade de Lisboa. This study was approved by the Ethics Committee of Faculdade de Medicina da Universidade de Lisboa. Donors were asked to give up to 10 mL of blood for research purposes additionally to their common blood donation of 450 mL to the blood bank. The blood collection was made anonymously as the data treatment resulting from the experiences made with these samples. The samples were drawn to KLarge unilamellar vesicles (LUV) with ∼100 nm diameter, composed of pure POPC  or POPC with 33% mol cholesterol (Sigma), were obtained by extrusion of multi-lamellar vesicles (MLV) as described elsewhere The HIV-fusion inhibitors enfuvirtide (T-20) and T-1249  were prepared in working buffer at maximum stock concentration of 250 µM. These peptides were incubated at different concentrations with labeled erythrocytes (0.02% hematocrit), PBMC (100 cells/µL) or lipid vesicles  during 1 h for cells or 90 min for vesicles, with gentle agitation before measurements.Erythrocyte microscopy images were obtained in a laser scanning confocal microscope Zeiss LSM 5 Live  using a water immersion objective Zeiss C-Apochromat 20× . PBMC images were obtained in a Zeiss LSM510 Meta using an oil immersion objective Zeiss Plan-Apochromat 63× . Di-8-ANEPPS was excited by a 488 nm laser . For the PBMC, Hoescht dye  was also used for nucleus labeling, excited by a 405 nm laser (LP 420 filter).The membrane probe di-8-ANEPPS assesses dipole potential by shifting its excitation spectrum. Differential spectra for detecting these shifts are obtained by subtracting the excitation spectrum of labeled cells in the presence of peptide from the spectrum in its absence. Before subtraction the spectra were normalized to the integrated areas to reflect only spectral shifts. The differential spectra are waveform shaped, which amplitude directly correlates with the peak shifting magnitude, and hence, with the dipole potential variation. Blue-shifts will result in differential spectra in which a maximum comes at a lower wavelength than the maximum, and the opposite for red-shifts.R for this case as I455 nm/I525 nm . It was already established that an increase in the membrane dipole potential leads to a blue-shift in the membrane incorporated di-8-ANEPPS and, consequently, to an increase in the ratio To qualitatively define a value for the spectral shift and hence, the dipole potential, a ratio was established from fluorescence intensities at two wavelengths on the sides of excitation spectrum peak R with the peptide concentration was analyzed by a single binding site model R values normalized for R0, the value at zero peptide concentration. Rmin defines the asymptotic minimum value of R and Kd is the dissociation constant.The variation of R values were measured through temperatures from 15°C to 50°C, with emissions at 580, 600 and 670 nm. The slope was lower for 670 nm, meaning a lower interference of fluidity effect at this wavelength. Excitation and emission slits for all measurements were 5 nm and 10 nm, respectively.All the fluorescence spectroscopy measurements were made in a Varian Cary Eclipse  fluorescence spectrophotometer. Membrane-bound di-8-ANEPPS excitation spectra and ratios were taken with emission at 670 nm in order to avoid membrane fluidity effects, as previously tested for lipid vesicles


Plasmids are extrachromosomal elements that replicate autonomously, and many can be transmitted between bacterial cells through conjugation. Although the transcription pattern of genes on a plasmid can be altered by a change in host background, the expression range of plasmid genes that will result in phenotypic variation has not been quantitatively investigated.Pseudomonas resinovorans CA10 and the transconjugant P. putida KT2440(pCAR1) during growth on either carbazole or succinate as the sole carbon source. We identified the operons in pCAR1, which consisted of nearly identical transcription units despite the difference in host background during growth on the same carbon source. In accordance with previous studies, the catabolic operons for carbazole degradation were upregulated during growth on carbazole in both hosts. However, our tiling array results also showed that several operons flanking the transfer gene cluster were transcribed at significantly higher levels in the transconjugant than in the original host. The number of transcripts and the positions of the transcription start sites agreed with our quantitative RT-PCR and primer extension results.Using a microarray with evenly tiled probes at a density of 9 bp, we mapped and quantified the transcripts of the carbazole catabolic plasmid pCAR1 in its original host Our tiling array results indicate that the levels of transcription for the operons on a plasmid can vary by host background. High-resolution mapping using an unbiased tiling array is a valuable tool for the simultaneous identification and quantification of prokaryotic transcriptomes including polycistronic operons and non-coding RNAs. Prokaryotic transcriptomes change not only in response to physiological parameters but also to genetic rearrangements mediated by mobile elements. Plasmids are extrachromosomal genetic elements that replicate autonomously, and many can be transmitted between different strains through conjugation. Plasmids provide benefits to their hosts, such as resistance to antibiotics or degradation of recalcitrant aromatic compounds ; howeverPseudomonas resinovorans CA10, encodes the degradation pathway of carbazole, a nitrogen-containing recalcitrant aromatic compound  was designed using the CustomExpress Array Program . The array contained a total of 88,460 probes consisting of pairs of 25-mer perfect match (PM) probes overlapping at 9-nt offsets, and corresponding mismatch (MM) probes with a one-base mismatch at the thirteenth nucleotide.A high-density oligonucleotide microarray covering all 199,035-bp of the pCAR1 plasmid  and P. resinovorans CA10 [GenBank:AB047273]. All reactions were performed a minimum of three times, and the data were normalized using the average of the internal standard.Quantitative RT-PCR was performed using the ABI 7300 Real-Time PCR System  as described previously . The priPrimer extension analysis was performed using a Li-Cor model 4200L-2 auto-DNA sequencer as described previously  as templ) and are accessible through GEO Series accession number GSE10862.The data were deposited in NCBI's Gene Expression Omnibus (GEO, MM conceived the study, carried out the experiments, and drafted the manuscript. MM, HNi, and MS analyzed the data. HNi, MS, HY, and HNo assisted in the preparation of the manuscript. All authors read and approved the final manuscript.Primers used for quantitative RT-PCR. Nucleotide sequences of primers used for quantitative RT-PCR.Click here for file


The central paradigm of ecological immunology postulates that selection acts on immunity as to minimize its cost/benefit ratio. Costs of immunity may arise because the energetic requirements of the immune response divert resources that are no longer available for other vital functions. In addition to these resource-based costs, mis-directed or over-reacting immune responses can be particularly harmful for the host. In spite of the potential importance of immunopathology, most studies dealing with the evolution of the immune response have neglected such non resource-based costs. To keep the immune response under control, hosts have evolved regulatory pathways that should be considered when studying the target of the selection pressures acting on immunity. Indeed, variation in regulation may strongly modulate the negative outcome of immune activation, with potentially important fitness consequences.Here, we experimentally assessed the survival costs of reduced immune regulation by inhibiting an anti-inflammatory cytokine (IL-10) with anti-IL-10 receptor antibodies (anti-IL-10R) in mice that were either exposed to a mild inflammation or kept as control. The experiment was performed on young (3 months) and old (15 months) individuals, as to further assess the age-dependent cost of suppressing immune regulation. IL-10 inhibition induced high mortality in old mice exposed to the mild inflammatory insult, whereas no mortality was observed in young mice. However, young mice experienced a transitory lost in body mass when injected with the anti-IL-10R antibodies, showing that the treatment was to a lesser extent also costly for young individuals.These results suggest a major role of immune regulation that deserves attention when investigating the evolution of immunity, and indicate that the capacity to down-regulate the inflammatory response is crucial for late survival and longevity. Costs and benefits of the immune response have attracted considerable attention in the last years among evolutionary biologists [1–3 for reviews]. Ecological immunology considers the immune response as a trait whose expression should be optimized by natural selection as to maximize fitness. Therefore, the central paradigm of ecological immunology follows an economical perspective and postulates that immune defences are beneficial, but also costly, and that natural selection should minimize the ratio between costs and benefits Since an over-reacting immune response, or a response failing to make a distinction between self and non-self, potentially generates substantial fitness costs, organisms have evolved regulatory mechanisms based on cells and effectors whose function is to control the immune system and dampen the negative consequences of its activation. Therefore, in addition to the selection pressures acting on the allocation pattern of energy/resources to immune organs and cells, it seems plausible to expect strong selection on the regulatory mechanisms that prevent the system to get out of control. However, in spite of its importance, immune regulation has been neglected in studies devoted to the evolutionary forces shaping immune functioning. Here, we wished to assess the cost of suppressing the regulatory mechanisms underlying the control of the inflammatory response.Inflammation is a non-specific process characterized by the delivery of effectors from the blood into infected tissues, and whose function is to fend off invading pathogens. At the systemic level, the inflammatory response induces fever, an increased number of leucocytes recruited for defence, and elevated levels of pro-inflammatory cytokines driving the microbicidal activity of phagocytic cells To protect their structures from inflammatory injury, hosts have evolved regulatory mechanisms based on specific cytokines  controlling the resolution of inflammation. There exists a polymorphism in genes that code for such regulatory mechanisms vs. rat IgG1 antibodies (IgG)], stimulation of the inflammatory response [injection of an E. coli lipopolysaccharide solution (LPS) vs. phosphate buffered solution (PBS) injection], age (3 vs. 15 month old).In this study, we inhibited an anti-inflammatory cytokine in order to assess the cost of reduced immune regulation in mice. We used a full factorial design with three factors: suppression of an anti-inflammatory cytokine [injection of anti-IL-10 receptor antibodies (anti-IL-10R) The experiment has been conducted in compliance and has received the agreement of the Animal Care and Ethical Committee of the Université de Bourgogne, Dijon (protocol # 6904).ad libitum.Sixty young (3 months old) and sixty old (15 months old) virgin male C57BL/6 mice were purchased from Janvier . Upon arrival at the laboratory, animals were individually housed in Plexiglas cages and kept in an air-conditioned room  with a 12-hours light/dark cycle. Pellet food and tap water were provided Escherichia coli  at a dose of 0.05 mg/kg, whereas the other half were injected with a phosphate buffered solution (PBS) for control. This LPS dose is more than 500-fold and 30-fold lower than the 50% lethal dose of LPS in 2-month and 24 month old mice, respectively Young and old mice were randomly assigned to one of four groups . At day 0, the group “anti-IL-10R” received an intraperitoneal injection of 20 µg of monoclonal anti-IL-10 receptor antibodies , whereas the group “IgG” received an intraperitoneal injection of 20 µg of rat IgG1 antibodies (Sigma) for control. At day 1, half of the males in each group were intraperitoneally injected with a solution of lipopolysaccharides (LPS) from Blood samples were obtained by retroorbital puncture immediately before the first injection (day 0) and 24 hours after the last injection (day 8). Body mass (±0.1 g) was measured at day 0, 2 and 8. Mortality rate was recorded every day for the duration of the experiment.Serum amyloid A (SAA), a protein of the acute phase response, was quantified using an immunoassay kit . Coloration of the antibody complexes was measured with a spectrophotometer  at 450 nm.Differences in survival rate were analyzed with a Log-rank test. We also used a log-linear model where in addition to the treatment we included changes in body mass as a potential predictor of survival rate. Two 15-month old male died during the first injection and were therefore removed from the survival analysis.Generalized linear mixed models were used to assess the effect of the treatments and age on changes in body mass and SAA, with individual identity declared as a random variable. We started with the full model and then we dropped all non significant three- and two-way interactions. Degrees of freedom were adjusted using the Satthertwaite method. All the analyses were done with SAS (2001) P = 0.0012). The analysis of the effect of the treatment on the survival was, therefore, restricted to aged mice. Old individuals treated with anti-IL-10R and with LPS suffered a substantially higher mortality rate compared to the other three groups , whereas 9 out of 58 old mice died  individuals had stable body mass . On the Both treatments had a strong effect on the production of the serum amyloid A (SAA), a protein of the acute phase response . The antvs. anti-inflammatory profile Here, we explored the fitness consequences of inhibiting an anti-inflammatory cytokine both under a mild inflammatory challenge and control conditions. Our findings show that suppressing the anti-inflammatory regulation leads to high mortality. Interestingly, however mortality only occurred in conjunction with the activation of the inflammatory response and in aged individuals. Although the treatment was repeated three times during the course of the experiment, mortality of LPS/anti-IL-10R treated mice only occurred following the first injection, suggesting that the treatment selected individuals with a particular pro- While most ecological immunology studies implicitly assumed that evolution has shaped immune defences through resource-based trade-offs with other competing crucial traits Our results show that age is a very important factor affecting the cost of inhibiting immune regulation. Immunosenescence refers to the age-associated changes in immune functioning that are observed in humans and animal models One of the central tenets of ecological immunology is that natural selection shapes the optimal investment into immune defences as to maximize fitness. Over investment into immune defences might be maladaptive if this diverts resources from other vital functions; similarly, a too small investment might make hosts too vulnerable to parasite exploitation. We think that another dimension has to be added to this simple trade-off. In addition to the total investment into immunity, selection is likely to strongly act on how well this function is regulated. Ignoring immune regulation might, therefore, provide a misleading picture on the real target of selection. For instance, an individual with a low total investment into immunity but with poor regulation might pay higher cost of immune activation than an individual with a huge allocation into immune defences and a very effective immune regulation. Clearly, ecological immunology needs to explicitly take into account immune regulation to better assess the selection forces acting on immunity.Cost of reduced immune regulation, as shown in this study, suggests that natural selection has the potential to mold this trait. However, the strength of natural selection progressively declines with age, because the fraction of old individuals in a given population is much smaller than of young animals. We used for our study young adults (3 month old) and old (15 month old) mice. Fifteen month old mice can be considered old Plasmodium chabaudii and whose IL-10 was experimentally neutralized experienced shorter time to death compared to infected/IL-10+ controls, showing that immunopathology substantially contributes to the overall cost of infection in this system In this study we focused on one anti-inflammatory cytokine (IL-10). IL-10 has been extensively studied during the last decade To conclude, we report here experimental evidence showing that immune regulation is crucial for survival in aged individuals under a mild inflammatory challenge. To our view, this result opens a novel area of research on the fitness consequences of inter-individual variation in immune regulation.