Peer counselling is reported to increase breastfeeding rates. We evaluated an intervention consisting of mainly telephone contact peer counselling programme on breastfeeding duration and exclusivity.Peer counsellors (PCs) were mothers who had successfully breastfed and had received formal training. Following a postnatal visit, they provided scheduled telephone consultations  to PC group mothers (n = 100) who continued breastfeeding their infants after discharge. Control group mothers (n = 100) received routine care.After adjusting for mothers' previous breastfeeding experiences, mothers' working status and breastfeeding problems, no statistical differences in mothers' feeding methods  were noted at the three follow-up times for intervention and control mothers respectively . All differences between the groups were not significant. Also, there was no evidence to suggest that PC intervention prolonged breastfeeding duration.The lack of effect of our PC intervention may reflect the low baseline breastfeeding rate and low value placed on breastfeeding in our population, the type of PC intervention or group allocation biases.ISRCTN93605280. At the fifty-fifth World Health Assembly, the World Health Organization (WHO) recommended that optimal infant nutrition was exclusive breastfeeding for the first six months of life, followed by the introduction of nutritionally adequate and safe complementary feeding with continued breastfeeding for up to the age of two years or beyond . DespiteA number of strategies can significantly improve and sustain breastfeeding practice, including community-based breastfeeding peer counsellor (PC) programmes (mother-to-mother support) -8. TheseThe study was undertaken at one government hospital in Hong Kong that had active breastfeeding support and had recently started a PC volunteer programme. Mothers were eligible for inclusion in the study if they were Cantonese-speakers, healthy and had had a vaginal delivery of a full term healthy infant. It was required that mothers planned to stay in Hong Kong for six months postpartum, and that they expressed an intention to breastfeed upon admission to the postnatal unit.As part of the hospital's PC volunteer programme, mothers were visited by a PC postpartum on an ad hoc basis. The PC would provide mothers with information on the benefits of exclusive breastfeeding, breastfeeding during illness, basic lactation anatomy and physiology, positioning and "latching-on", common myths, problems and solutions, healthy breastfeeding patterns, maternal concerns, milk expression and storage, and sources of social and community support. If a mother was subsequently recruited into the PC intervention group, she would in addition receive seven regular telephone consultations from a PC . These contacts were discontinued at any time if the mother decided to completely stop breastfeeding her infant. The PCs were allowed to provide more frequent telephone support if necessary. The control group mothers would not receive a PC visit or any phone contacts. They would receive the usual postnatal care and breastfeeding advice which included antenatal education.This study was designed to assess whether a programme involving a postpartum PC hospital visit followed by PC telephone support after discharge could be an effective strategy to promote breastfeeding in Hong Kong by increasing the duration and exclusivity of breastfeeding.A total of 200 mothers were recruited into a PC intervention group (n = 100) and control group (n = 100). The hospital had information on the rate of any breastfeeding rate at discharge, and at one week and one month after discharge. These data showed that in 2001, 46% of mothers who initiated breastfeeding were continuing to mainly breastfeed at one month. A sample of 100 subjects in each group would demonstrate an increase in this "mainly breastfeeding" rate from 46% (control group) to 67% (intervention group) with alpha [confidence level] = 0.05 and power of 80%. We had assumed a more modest benefit of our PC intervention than those reported by Morrow (12% controls and 67% of a six-visit intervention group were exclusively breastfeeding at three months) and Haider (6% controls and 70% of a 15-visit intervention group were exclusively breastfeeding at five months) as these two studies had home visits as part of the intervention [The hospital's PC visiting programme was conducted on an ad hoc basis at a time convenient to the PC. In view of the ad hoc visiting schedule of the PCs it was decided for recruitment purposes to allocate eligible mothers on an alternating basis, and subject to bed availability, to one of two ward areas: a PC ward area or a control ward area. The PCs were asked to only see mothers allocated to the PC ward area during the study period. Although this was done to avoid the potential influence of placing a control mother next to mother receiving PC advice, the strategy had the effect of preventing a true randomisation of mothers as enrolment occurred after allocation to these ward areas. All potentially eligible mothers were screened by one of us (Esther Wong) on the day of discharge. PC group mothers needed to have had at least one PC hospital visit and they were recruited even if they had given up breastfeeding prior to discharge to avoid over-estimating any potential benefit of the PC intervention. As the numbers of potential control group mothers exceeded the number of potential PC mothers, computer generated random number lists were used for the selection of control mothers. The recruitment process is summarised in Figure Recruitment data were collected through a structured interview, after obtaining signed informed consent and included information on mother's obstetric and breastfeeding history, current feeding method, preparation for breastfeeding, lactation problems, breastfeeding support network, breastfeeding duration plan, breastfeeding confidence level, breastfeeding knowledge (pre-test), information on hospital practice postpartum related to the Ten Steps of the Baby Friendly Hospital Initiative, demographic information and infant formula supplementation (if any). Each study mother was followed for 6 months after they returned home. Follow-up interviews, regardless of whether mothers had changed their feeding methods, were scheduled at five days, three months and six months post-discharge. Information collected at these interviews included current feeding practices, feeding problems, baby's health, use of pacifier, infant formula advertisement exposure, and other factors likely to influence feeding choice. The Day five and Month three interviews were telephone interviews. The Month six interview was a face-to-face interview at the mother's local MCHC at the time of a routine visit for immunisation, or was conducted by telephone for those mothers who were not able to meet at the MCHC. Evaluation of the overall breastfeeding experience, evaluation of PC support (for PC group mothers only), breastfeeding knowledge (post-test), questions on breastfeeding plans and detailed questions on infant formula advertisements were only done at the Month six interview. Mothers who stopped breastfeeding at any stage during the study completed a termination questionnaire, which explored factors for early cessation of breastfeeding. Participating PCs completed an evaluation at the end of the study to obtain feedback and comment on the PC programme. The questionnaires were based on those used in the "Perth Aboriginal Breastfeeding Study" in Australia in 2000 , and on The PCs were recruited from 21 mothers who participated in the hospital's first PC training course that had been jointly organised with the Baby Friendly Hospital Initiative Hong Kong Association in 2001. These mothers were required to have breastfeeding experience and to be enthusiastic to promote and support other breastfeeding mothers. The 20-hour PC course was conducted by lactation consultants and medical staff with experience in managing breastfeeding problems and involved both lectures and practical sessions on the ward. The course syllabus was based on the 40 hours WHO/UNICEF course for health professionals. After completion of the course, the PCs agreed to provide 80 hours of volunteer work in the hospital. There were 16 of these 21 trained PCs who agreed to participate in the study.since the time of birth [The primary outcome measures, breastfeeding duration and exclusivity, were compared between two groups (PC mothers vs. control mothers). As these measures were self-reported by the participating mothers, memory aids and a diary were given to mothers to reduce possible recall bias. To classify breastfeeding during the study, we used Labbok's definitions namely full breastfeeding , partial breastfeeding  and token breastfeeding ,11. Howeof birth ,11; and of birth .The study was approved by the hospital's Ethics Committee and participating mothers were provided with written information about the study and signed informed consent.All data were presented as count, percentage, or mean . Demographic characteristics, breastfeeding characteristics and background information of the infants at the baseline between the PC and control groups were compared using t-test, Mann-Whitney test, Chi-square test or Fisher exact test as appropriate. Time to terminate any breastfeeding was analysed using survival analysis. Survival (continuing to breastfeed) was estimated by Kaplan-Meier method, and the log-rank test was used to compare the overall survival rates  between the two groups. Changes in proportions of breastfeeding practice at each of the follow-ups  relative to the baseline between the PC and control groups were assessed using mixed effects logistic regression models. Adjustment was made in the mixed effects logistic regression models for the following confounding factors: previous breastfeeding experience; primiparae or not; working status at baseline. The mixed effects models were fitted using MLwiN . All other statistical analyses were done using SPSS 11.5 . All statistical tests were two sided and p-value < 0.05 was considered statistically significant.Within the recruitment period, 15 November 2001 to 30 April 2002, 1587 mothers were admitted to the hospital's postnatal ward , fewer mothers in the PC group had had previous breastfeeding experience , among multiparous mothers in the PC group there was a shorter mean breastfeeding duration for their first child (9.8 (± 15.8) weeks vs. 20.2 (± 23.1) weeks, p = 0.02), and more mothers in the PC group reported breastfeeding problems at discharge   and at Day five follow-up, 46% of PC mothers and 53% of control mothers breastfed their infants exclusively or almost exclusively on breast milk (Aarts' definition) , frequent feeding (46%), and breastfeeding technique (36%). The main topics related to babies were sleepy baby (46%), information on general baby care (36%) and baby's stool pattern (36%). Key topics related to mothers were breast engorgement and mastitis (46%), emotional problems and handling techniques (18%) and maternal fatigue (18%). Using 10-point rating scales, PCs evaluated the efficiency of the PC program . The mean score (± SD) of the overall effectiveness of the program was 8 ± 1.3 (range 6 – 10), with the hospital visits being 8.8 ± 1.2 (range 7 – 10) and the telephone follow-up being 8.2 ± 1.9 (range 4 – 10). The mean score (± SD) for the adequacy of the numbers of scheduled telephone contacts for new breastfeeding mothers was 4.4 ± 3.2 (range 1 – 10). Four PCs rated their confidence at 9, three at 8, three at 7 and one at 5. The mean score (± SD) of the PCs level of enjoyment in supporting breastfeeding mothers through hospital visit and telephone consultations was 7.9 ± 1.4 (range 6 – 10) and 8.1 ± 1.4 (range 6 – 10) respectively. The aspects which participating PCs liked the most were the sense of being supportive (91%) of breastfeeding mothers and their babies, and a sense of satisfaction (73%). In contrast, the aspects which PCs disliked the most were being rejected when offering assistance (36%) and the strong misconceptions about breastfeeding (18%) by some mothers. Although, PCs were encouraged to give their phone numbers to study mothers for two-way contact, only three out of sixteen PCs did so. Although PCs acknowledged that "two-way contact" was likely to be of greatest benefit, most responded that the "one-way contact" model worked best for them in view of time constraints and other commitments. Some PCs noted that it was difficult to keep to the fixed schedule as mothers could not always be contacted. Three PCs responded that they would definitely take part in the program again if it was repeated, seven PCs reported that they probably would do so, and one PC said that she was not sure.There was no significant difference in the overall breastfeeding duration or the exclusivity of breastfeeding between mothers who received routine breastfeeding support and advice (control group) and mothers who received additional PC support consisting of a hospital visit and follow-up telephone support (PC group). These results contrast with many studies undertaken elsewhere ,8,13,14 Local perceptions of the value of breastfeeding could have prevented an effective PC programme from increasing breastfeeding duration or exclusivity. Any intervention alone may not be strong enough to overcome powerful prevailing cultural norms that favour infant formula feeding. One study that did not show any impact of a PC intervention was undertaken in Glasgow, Scotland . Hong KoThe second explanation for our results could be that the PC intervention itself was ineffective. Our PC programme provided at least one hospital visit and a total of seven telephone follow-up contacts at fixed time intervals after the mothers had returned home. Although the number of contacts were generally considered adequate by mothers, it was not always possible for PCs to stick to the schedule. Also such fixed schedules may not suit the individual mother's need for lactation support. Our programme was effectively a "one-way contact" in which PCs mainly initiated phone calls to breastfeeding mothers.Some PC interventions have started postpartum, whereas other programmes have arranged for the PC to meet the pregnant women during the antenatal period before the birth experience ,13-15,19Only mothers with vaginal births were recruited in our study. It is possible that our PC programme could have been beneficial to mothers experiencing a caesarean delivery who may have more problems initiating breastfeeding and who may need more support. Although our PC programme did not appear to prolong breastfeeding duration or increase breastfeeding exclusivity, it did improve the breastfeeding knowledge of those mothers who received the PC intervention (data not shown). The PCs also reported positive growth in their own personal life, in terms of knowledge of breastfeeding and a sense of achievement and self-confidence.Subjects were recruited prior to discharge after they had been allocated to different ward areas and after the PC group had received at least one PC visit. This recruitment strategy resulted in important differences between the PC and control groups Table . AlthougIn addition it is possible that even with good randomisation in a community setting, an effective peer counselling intervention could influence practice within the wider community in highly urbanised settings such as Hong Kong i.e. it may not be possible to prevent the control mothers from getting access to the intervention. Although a systemic review has shown lay support to be effective in reducing the cessation of exclusive breastfeeding, it is suggested that further trials are required to assess the effectiveness of this support in different settings, particular in those communities with low rates of breastfeeding initiation .A mainly telephone contact PC programme did not increase breastfeeding duration and exclusivity in our setting. It is possible that the somewhat limited PC intervention programme that we used and a low value placed on breastfeeding in Hong Kong society could help explain this result. Group differences could have also been a factor although these were controlled for in the analysis. It is possible that effective PC programmes in areas with prevailing "infant formula feeding" cultures may have limited impact unless these are part of more extensive programmes to implement all of the "Ten Steps" in entirety and other community measures to increase the cultural acceptability of breastfeeding. In future, consideration should be given to evaluating or implementing alternative PC programmes, such as those that link a PC to an individual mother antenatally.PC - Peer counsellor;WHO - World Health Organization;UNICEF - United Nations Children's Fund;MCHC - Maternal and Child Health centre.The author(s) declare that they have no competing interests.EHYW participated in design, acquisition, analysis and interpretation of data, drafting the manuscript and final approval of version to be published. EASN participated in design, interpretation of data, drafting the manuscript and final approval of version to be published. KCC participated in analysis and interpretation of data, drafting the manuscript and final approval of version to be published. KPW participated in design, help with acquisition of data, critical review of manuscript and final approval of version to be published. CI participated in design, help with acquisition of data, critical review of manuscript and final approval of version to be published. LCH participated in design, critical review of manuscript and final approval of version to be published.EHY Wong was supported by a research studentship from the Research Grants Council, Hong Kong. The funding body had no role in the study design; collection, analysis and interpretation of data; writing the manuscript; or decision to submit the manuscript for publication.


Previous reports have demonstrated that short durations of vibrotactile stimuli  effectively and consistently modify both the perceptual response in humans as well as the neurophysiological response in somatosensory cortex. The change in cortical response with adaptation has been well established by a number of studies, and other reports have extended those findings in determining that both GABA- and NMDAR-mediated neurotransmission play a significant role in the dynamic response of somatosensory cortical neurons. In this study, we evaluated the impact that dextromethorphan (DXM), an NMDAR antagonist, had on two distinct vibrotactile adaptation tasks.All subjects, both those that ingested 60 mg DXM and those that ingested placebo, were evaluated for their amplitude discriminative capacity between two simultaneously delivered vibrotactile stimuli both with and without 3 conditions of pre-exposure to adapting stimulation. The results demonstrated that the perceptual metrics of subjects who ingested 60 mg DXM were significantly altered from that of controls when the amplitude discrimination task followed one of the conditions of adapting stimulation. Without the condition of pre-exposure to an adapting stimulus (or stimuli), there was little difference between the observations obtained from the subjects that ingested DXM and controls. Peak impact on subject response occurred at 60 min post-ingestion, whereas the scores of controls who ingested placebo were not impacted.The results – that DXM blocks vibrotactile adaptation – is consistent with the suggestion that NMDAR-mediated neurotransmission plays a significant role in the perceptual adaptive response. This finding is also consistent with neurophysiological findings that report observations of the effects of NMDAR block on the SI cortical response to repetitive vibrotactile stimulation. In this study, we sought to observe perceptual correlates of these changes in cortical activity by characterizing the impact of NMDAR block on somatosensory adaptation.Although the effects of conditioning stimuli – or adaptation – have been well documented in a number of sensory modalities  values on this task ,6. AdditTwo separate 2AFC tracking protocols (see Methods) were used to obtain observations of the effects of DXM, an NMDAR antagonist, on the impact of adaptation on subjects' capacities for discrimination between the amplitudes of two simultaneously delivered vibrotactile stimuli. In the first protocol, one of the two stimulus sites that received the vibrotactile stimuli was pre-exposed to an adapting stimulus, and the tracking protocol targeted determination of the reduction in perceived intensity at the adapted stimulus site . In thesingle-site adaptation protocol for the placebo Control (n = 9) and DXM (n = 11) groups at times T0, T1, and T2 (see Methods for description). Repeated measures ANOVA shows that DLs were not different between the groups at T0 (p = 0.54 in Block 1 and p = 0.41 in Block 2). As previously reported [Figure reported , amplitu1 (60 min post-ingestion) DXM drug administration had a slight effect on amplitude discrimination – though not a significant one – as indicated by Block 1 data (p = 0.57). However, in the adaptation phase of the run (Block 2) the data from the DXM group demonstrated a significantly reduced impact of adaptation compared to the control group as shown in Block 2 (p < 0.01). The average post-adaptation DL for the control group remained at a level that was over 200% above the values obtained in block 1, yet the average post-adaptation DL for the DXM group was only approximately 67% above the block 1 values. Thus, as a group, subjects that ingested DXM experienced less adaptation, and subsequently performed better at the post-adaptation amplitude discrimination task than did the control group.At time T2), there was very little difference between the amplitude discrimination performance of the DXM and control group (as shown in Block 1 results). In the post-adaptation amplitude discrimination task (as shown in Block 2 results), the DXM group appeared to still be slightly outperforming the control subjects, although there were no significant differences observed between the two groups for this data set (p = 0.092), as it appears that the drug was nearing the end of its effectiveness.Two hours post ingestion  when the adapting stimulus duration was increased from 500 msec to 2000 msec (p < 0.01), and no difference in performance was observed between the groups for each of these two conditions .Subject performance on the 1) under both durations of adapting stimulation at  and approximately 105 μm with the 2000 msec adapting stimulus duration (compare 65 μm at T0 – approximately a 66% increase in the DL with DXM). At T2, or 120 minutes post-ingestion, these differences between the DXM and Control groups appear to be decreasing, although they remain significantly different (p < 0.01). The contrast between these measures, obtained with the dual site adaptation protocol and those obtained with the single site adaptation protocol, suggests that the results obtained with dual site adaptation may be more sensitive in detecting differences in the impact of the DXM on adaptation.The performance observed in the drug absent condition was significantly degraded 60 min after DXM ingestion , an NMDAR antagonist, on the ability of subjects to discriminate between the amplitude of two simultaneously delivered vibrotactile stimuli with/without pre-exposure to adaptation stimulation. The results show that the DXM drug administration had no effect on the subjects' performance on a standard amplitude discrimination task, but it significantly reduced the impact of adaptation on amplitude discrimination, and this reduction began to recover two hours after the ingestion of DXM.Under control conditions (placebo instead of DXM), the observed impairment in amplitude discrimination induced by adaptation is consistent with a previously published report, which demonstrated that a subject's ability to accurately discriminate differences in the amplitude of two simultaneously delivered vibrotactile stimuli was significantly degraded with prior exposure to an adapting stimulus at the test stimulus site . TommerdIt is important to note that the spatial and temporal patterns of responses in primary somatosensory cortex (SI) to repetitive vibrotactile stimulation are largely dependent on the complex interplay of GABA-mediated inhibition  and NMDASimilar observations in reduction of adaptation to vibrotactile stimulation have been observed in autism ,22. HoweIn short, without NMDAR mediated activity, the level of excitation required to evoke inhibitory responses becomes diminished, and the GABA-mediated activity required to diminish the cortical responses evoked by repetitive stimuli becomes weaker. Thus, with NMDAR block, although the overall cortical response is weaker, the change of the response caused by conditioning or adapting stimuli is significantly reduced. It should be noted that although DXM does have effects other than that of an NMDAR antagonist, the known effects of DXM as an NMDAR antagonist are consistent with the impact of NMDAR block on centrally mediated neurotransmission. The observation of weakened adaptation response – to repetitive stimulation in populations whose GABA- or NMDAR-mediated neurotransmission has been compromised – appears to strengthen the original proposal by Whitsel and colleagues that the adaptive changes observed in sensory perception at short stimulus durations (less than 5 seconds) are principally centrally mediated.The results – that DXM blocks vibrotactile adaptation – is consistent with the suggestion that NMDAR-mediated neurotransmission plays a significant role in the perceptual adaptive response. This finding is also consistent with neurophysiological findings that report observations of the effects of NMDAR block on the SI cortical response to repetitive vibrotactile stimulation.Twenty subjects (21–27 years in age) were studied who were naïve both to the experimental design and issue under investigation. The subjects consisted of 6 males and 5 females that received DXM during the testing session, and 5 males and 4 females that received placebo, all right-hand dominant. The study was performed in accordance with the Declaration of Helsinki, all subjects gave their written informed consent, and procedures were reviewed and approved in advance by an institutional review board.Two separate two-alternative forced choice (2AFC) tracking protocols were used to evaluate the effects of adaptation on amplitude discriminative capacity of each subject. Each subject fasted for 2 hours prior to the study beginning. Upon arrival, the subject was seated comfortably in a chair with the left arm resting on a table surface. The subject's left hand was placed under a dual-site portable vibrotactile stimulator . Two pr(T0) was conducted prior to ingestion of any drug medication or placebo. After the first session was complete, a subject ingested either 60 mg of NMDAR antagonist DXM or a placebo. The placebo drug was perceptually similar to DXM in shape, color, texture, and taste and the subjects ingested their respective capsules with eyes closed. The study was conducted in a double-blind fashion such that neither the subject nor the experimenter was aware of the actual drug (DXM or placebo) being administered. One hour post-ingestion (T1), each subject was re-tested using the same protocol. Finally, after one additional hour (T2), the subjects were tested a third time to observe the effects, if any, of recovery. Each individual subject session lasted approximately 15 min  and the subject performed desk work, unrelated to the study, between each session. Each session consisted of two separate 2AFC protocols – one tested the effects of single-site adaptation on dual-site simultaneous amplitude discrimination  was delivered to one skin site at the same time that a standard stimulus  was applied to the other skin site. The loci of the test and standard stimuli were randomly selected on a trial-by-trial basis. Stimulus duration was 0.5 sec, followed by subject response  and a 5 sec delay before onset of the next trial. Subjects were not provided any feedback during an experimental run. At the beginning of the experimental run, the test stimulus was 200 μm (peak-to-peak amplitude) and the standard was 100 μm. In the intial 10 trials, the difference in the amplitudes of the test and standard stimuli was adjusted on the basis of the subject's response in the preceding trial using a 1-up/1-down algorithm . After the initial 10 trials were completed, test stimulus amplitude was modified using a 2-up/1-down algorithm – in these trials two correct/one incorrect subject response(s) resulted in a decrement/increment, respectively, in the amplitude difference between the test and standard stimuli. This approach was selected because it enabled rapid determination ("tracking") of each subject's minimally detectable difference in the amplitudes of two-site skin flutter stimulation [During the first run of each session, a tracking protocol, 40 trials total, was implemented that consisted of 2 sequential blocks , delivery of the test and standard stimuli was preceded by adapting stimulation at one of the stimulus sites. Specifically, a 25 Hz 100 μm adapting stimulus at the location of the test stimulus was delivered for 1 sec prior to the presentation of the test and standard stimuli. By presenting the adapting stimulus to the same site as the test stimulus, it was possible to quantify the effect of reduced perceived intensity due to adapting stimulation. The duration of adapting stimulation delivered was 1 sec. A 2-up/1-down algorithm was used in Block #2 to track the subject's ability to determine the most intense stimulus, and the initial conditions of Block #2 were the final conditions of Block #1.A separate 2AFC tracking protocol was also administered during each session  contributed to the design and conduct of the experiment. VT and SEF contributed to the data analysis and the initial draft of the manuscript. All authors contributed to manuscript preparation.


Human immunodeficiency virus-1 (HIV-1) has a minimal genome of only 9 genes, which encode 15 proteins. HIV-1 thus depends on the human host for virtually every aspect of its life cycle. The universal language of communication in biological systems, including between pathogen and host, is via signal transduction pathways. The fundamental units of these pathways are protein protein interactions. Understanding the functional significance of HIV-1, human interactions requires viewing them in the context of human signal transduction pathways.Integration of HIV-1, human interactions with known signal transduction pathways indicates that the majority of known human pathways have the potential to be effected through at least one interaction with an HIV-1 protein at some point during the HIV-1 life cycle. For each pathway, we define simple paths between start points (i.e. no edges going into a node) and end points (i.e. no edges leaving a node). We then identify the paths that pass through human proteins that interact with HIV-1 proteins. We supplement the combined map with functional information, including which proteins are known drug targets and which proteins contribute significantly to HIV-1 function as revealed by recent siRNA screens. We find that there are often alternative paths starting and ending at the same proteins but circumventing the intermediate steps disrupted by HIV-1.A mapping of HIV-1, human interactions to human signal transduction pathways is presented here to link interactions with functions. We proposed a new way of analyzing the virus host interactions by identifying HIV-1 targets as well as alternative paths bypassing the HIV-1 targeted steps. This approach yields numerous experimentally testable hypotheses on how HIV-1 function may be compromised and human cellular function restored by pharmacological approaches. We are making the full set of pathway analysis results available to the community. Human immunodeficiency virus-1 (HIV-1) is the causative agent for acquired immune deficiency syndrome (AIDS), a world-wide epidemic that according to statistics from the world health organization has resulted in more than 2 million deaths; 33 million people are living with HIV, including 2 million at the age of 15 years and under. While current antiviral medication has dramatically improved the life expectancy of AIDS patients, the medicines are not available to everyone and drug resistance and side effects are increasingly recognized problems. Thus, novel avenues to anti-HIV-1 drug discovery are needed. The availability of large-scale transcriptomic  and phenThe general function of these interactions is for the virus to subvert the human cellular machinery for its purposes. Multiple mechanisms are employed by the virus, including manipulation of endo- and exocytosis, nuclear transport, transcription and translation, protein degradation and autophagocytosis. In all cases the virus must communicate with the host cell, and this communication typically takes place through interactions between HIV-1 and human host proteins. Communication through protein interaction is the universal language of cells; chains of interactions referred to as signal transduction pathways allow cells to respond to the environment. The virus simply needs to intercept these chains at one or more positions to "talk" to the cell. To systematically generate viable hypotheses on causal relationships between the phenotypic information of which proteins are critical for virus function and the protein interactions, a coherent picture of cellular pathways has to be developed in which the position of the proteins important for HIV-1 function are highlighted. Within this global picture, it should be possible to identify the points of interception by the virus through specific physical interactions with human proteins. Towards this goal, we recently presented an integration of the literature-reported  and predIn this paper, we propose a novel approach to analyze the HIV-1 intercepted pathways based on identification of paths of connected interactions with directionality within the pathways. The conceptual basis for the approach is as follows. The major features of cellular communication networks are their efficiency and robustness brought about by the use of hubs and redundancy, respectively. In contrast, the virus has to be a minimalist in order to survive and will therefore target the hub proteins, and this is what we (data not shown) and others  have obsGroup 1 are the most likely direct, physical known interactions based on reported literature [Group 2 refer to more likely indirect interactions reported in the literature [Expert validated targets 1 refer to those interactions where at least one expert annotated the interaction as real. Expert validated targets 2 refer to those interactions where all experts agree on their label. Finally, we included all of our predicted interactions[To identify HIV-1 targeted and alternative paths, we first determined the intersection between the HIV-1, human interactome and the gathered signal transduction pathways . This reterature , while Gterature . Group 1terature ). In an eractions. These dAn overview of the result of integrating these five interaction networks with known signal transduction pathways (see Methods) are shown for 453 pathways analyzed in Figure To obtain an indication as to whether these interactions are possible based on actual translation of the gene transcripts, we investigated their presence in HIV-1 susceptible tissues (see Methods). The number of unique proteins in all pathways combined is 3039. This corresponds to 2956 genes, indicating that in the vast majority of genes, it is only one isoform that has been studied with respect to its interaction with HIV-1 proteins. This set of proteins gives rise to 15161 unique HIV - UniProt pairs, out of which 689 are observed in pathways. 396 of these pairs are targeted by HIV-1 according to group 1. Most of these interactions are possible based on the expression of the human proteins in HIV-1 susceptible tissues: of the 3039 proteins in the pathways, only 85 are expressed in tissues that are not susceptible to HIV-1, while 2728 are in tissues that are susceptible to HIV-1 (for 175 there was no information available and for 51 a corresponding gene could not be identified). The information whether a gene is expressed in a HIV-1 susceptible tissue is provided in Additional File To analyze the integrated pathway maps, for each of the 453 pathways, we define simple paths that start with a protein that does not receive an input from another protein and end with a protein that does not induce a change in any other protein. Searching for simple paths was carried out by a breadth first search algorithm (see Methods). Within these paths, we identify which contain proteins that interact with HIV-1 proteins, and then find alternative paths between the same start and end points that do not traverse any protein that can interact with an HIV-1 protein. We supplement the combined map with functional information, namely which proteins are known drug targets  and whicTwo of the top-most frequently HIV-1 targeted (according to Group 1 definition) pathways are shown in Figure The pathways described above are those targeted most extensively according to the Group 1 definition. To demonstrate the utility of also including predictions of novel HIV-1, human protein interactions, we selected the cholesterol biosynthesis pathway, shown in Figure Cholesterol efflux and cholesterol synthesis are different pathways, having in common that they link cholesterol to HIV-1 pathogenesis. Thus, our pathway analysis suggests that HIV-1 not only takes over control of cholesterol concentrations inside cells, but also cholesterol synthesis. This would not be surprising, since cholesterol is needed for HIV-1 virion formation, docking and fusion. Thus, one novel anti-HIV-1 drug treatment strategy suggested by our result would be to inhibit cholesterol synthesis, and drugs to test this hypothesis already exist (since the enzymes encoded by the LSS and SQLE genes are known drug targets).We created a mapping of HIV-1, human interactions to human signal transduction pathways to link interactions with functions. We proposed a new way of analyzing virus-host interactions by identifying HIV-1 interaction partners in pathways as well as alternative paths bypassing the HIV-1 targeted steps in human signal transduction pathways. Due to spatial and temporal constraints, the fact that there is a human host factor in a pathway does not necessarily imply that this pathway is altered during infection. In particular, the strength and lifetime of an interaction between a human and an HIV-1 protein will determine what pathways may really have an effect that is lasting and transforming. Many proteins targeted by HIV-1 are hub proteins with a high degree of interactions. These interactions will not occur at the same time and in the same complex composition, so the effect that the interaction with the HIV-1 protein would have will vary over time and with regulation of each respective complex by posttranslational modifications, protein activity and localization. Thus, taking the view that interaction with an HIV-1 protein means that the signal transduction pathway is targeted is clearly simplistic. However, despite the use of these simplifying assumptions, the observation that a pathway contains a putative HIV-1 binding partner at least provides us with a testable hypothesis that HIV-1 interferes with normal cellular functioning via the respective pathway.Future work may include taking the hierarchy of nodes in pathways into account in the analysis. This would allow us to study in detail where in the logical progression of a pathway the HIV-1 targets are located so as to gage to what extent putative drug targets are located up-or down-stream of these targets. This may influence how pharmacologically effective a treatment would be. For example, treatment strategies based on downstream targets may have fewer side-effects than up-stream targets. On the other hand, up-stream targets may be more amenable to pharmacological intervention, especially if they include cell surface receptors. Other definitions of input and output nodes can also be evaluated, such as linking them to molecular functions such as receptors (input) and transcription factors (output). Another approach could be to view the known drug targets as putative input nodes. While these are many viable future directions, using our current definition, the approach already yields numerous experimentally testable hypotheses on how HIV-1 function may be compromised and human cellular function restored by pharmacological approaches. We described some pathways in this paper, such as the cholesterol biosynthesis pathway, and our analysis provides proof-of-concept that we can find druggable pathways that may hold promise for anti-HIV drug discovery. There are many other pathways that may hold equal or more promise for drug discovery and we make available as a resource to the community the full set of HIV-1 path and alternative path statistics on pathways, as well as the lists of the points of HIV-1 interception of pathways. This resource will allow researchers to investigate these pathways in detail and derive complementary approaches to understand the mechanisms of and circumvent HIV-1 takeover of the cell.We have collected hundreds of pathways from the National Cancer Institute (NCI) and Reactome protein pathway databases ,15. At tTo determine the intersection between the HIV-1, human interactome and the gathered signal transduction pathways, we need to define the interactome. We used the following 5 definitions: Group 1 are the most likely direct, physical known interactions based on reported literature , while GFor each pathway, we define simple paths that start with a protein that does not receive an input from another protein and end with a protein that does not induce a change in any other protein. Searching for simple paths in signal transduction pathways was accomplished as follows. Simple paths are those from a start node to an end node which have no repeating vertices, where a start node is defined as a node which has all its edges directed away from it and an end node is defined as one which has all its edges directed into it. The following example illustrates the way paths are counted. Assume you have the pathway consisting of interactions X → Y → Z, X → A → Z, B → A → Z, C → D → E, where Y is the HIV-1 targeted protein. According to our definition X, B, C are valid input nodes and Z, E are valid output nodes. Four is the total number of paths in this pathway. Then, the paths containing the HIV-1 targeted proteins are removed. Counting again, we would have 3 alternative paths left . This number answers the question how many of the complete paths  are still "active" after HIV-1 infection. These paths are referred to as alternative paths throughout the text.To find these simple paths in the full network graph of each signal transduction pathway we use a breadth first search algorithm , where wWe supplement the combined map of signal transduction pathways with HIV-1, human protein interactions with functional information, namely which proteins are known drug targets ,18 and wAIDS: Acquired immune deficiency syndrome; HIV-1: human immunodeficiency virus type 1; EGFR: epidermal growth factor receptor. NIAID: National Institutes of Autoimmune and Infectious Diseases.The authors declare that they have no competing interests.Sivaraman Balakrishnan collected the pathway and drug target data and conducted the study. Oznur Tastan collected and predicted HIV-1, human protein interaction data along with siRNA data and participated in the analysis of the results presented here. Jaime Carbonell supervised computational aspects of this work. Judith Klein-Seetharaman proposed the alternative path idea, designed this study, analyzed the data collected and wrote the paper. All authors participated in the editing of this paper.BMC Bioinformatics Volume 10 Supplement 15, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Bioinformatics, available online at http://www.biomedcentral.com/1471-2105/10?issue=S15.Other papers from the meeting have been published as part of Table S1. This file lists the human proteins in each pathway, along with the information if and if yes which HIV-1 proteins interact with them, if they are siRNA genes or if they are drug targets.Click here for fileTable S2. This file contains statistics on number of paths, HIV-1, siRNA genes, and drug targets for pathways from the NCI PID and the Reactome databases.Click here for file


S. enterica, but pre-treatment with oral aminoglycosides makes them susceptible to Salmonella colitis. However, genetically susceptible ItyS mice  die from systemic infection before they develop diarrhea, so a new model is needed to study the pathogenesis of diarrhea. We pretreated ItyR mice (Nramp1G169) with oral kanamycin prior to infecting them with virulent S. Typhimurium strain 14028s in order to study Salmonella-induced diarrhea. We used both a visual scoring system and the measurement of fecal water content to measure diarrhea. BALB/c.D2Nramp1 congenic started losing weight 5 days post-infection and they began to die from colitis 10–14 days after infection. A SPI-1 (invA) mutant caused cecal, but not colonic inflammation and did not cause diarrhea. A phoP- mutant did not cause manifestations of diarrhea in either normal or NADPH-deficient (gp91phox) mice. However, strain 14028s caused severe colitis and diarrhea in gp91phox-deficient mice on an ItyR background. pmr A and F mutants, which are less virulent in orally infected BALB/c mice, were fully virulent in this model of colitis.Mice do not get diarrhea when orally infected with S. enterica must be able to invade the colonic epithelium and to persist in the colon in order to cause colitis with manifestations of diarrhea. The NADPH oxidase is not required for diarrhea in Salmonella colitis. Furthermore, a Salmonella phoP mutant can be cleared from the colon by non-oxidative host defenses. Salmonella gastroenteritis is a global health problem, even in industrialized nations. The Center for Disease Control (CDC) estimates that there are nearly 1.4 million foodborne Salmonella infections annually in the USA Salmonella intestinal infections are self-limited, the illness usually lasts 3–5 days and is estimated to cost $1.4 billon/year in lost wages, expense of recalls, and medical costs S. enterica serovar Typhimurium  S. enterica required hospitalization, emphasizing that even gastroenteritis can be a serious disease S. enterica are invasive enteric pathogens, but the pathogenesis of diarrhea in Salmonella gastroenteritis is poorly understood. Many investigators have focused on mechanisms of cell invasion using cultured epithelial cells. This led to the discovery of a type 3 secretion system (TTSS) that is encoded by a Salmonella pathogenicity island (SPI-1). The TTSS mediates epithelial cell invasion by injecting bacterial products into host cells that rearrange the host cytoskeleton, causing localized membrane ruffling where the bacteria enter Salmonella also triggers a complex transcriptional response in epithelial cells that results in expression of many NF-κB regulated genes. Since most of those genes encode pro-inflammatory cytokines and chemokines this response has the potential to initiate inflammation, and inflammation is a prominent feature of Salmonella gastroenteritis. The induction and basal secretion of chemokines by polarized epithelial cells occurs rapidly Salmonella -induced changes initially observed in cultured cell lines have been confirmed in vivo in bovines and in human intestinal xenografts in SCID mice Salmonella induce additional epithelial responses that are not apparent in vitro because cell lines do not recapitulate all of the properties of the native intestinal epithelium.S. enterica, which has greatly hampered the study of the pathogenesis of intestinal infection. However, a mouse model of Salmonella colitis in streptomycin-pretreated mice was developed more than fifty years ago by Bohnhoff Salmonella infection, and that alterations in the normal intestinal microflora are responsible for their increased susceptibility. Indeed, germ free mice are also susceptible to Salmonella colitis S. Typhimurium S. Typhimurium (invJ and invG ) did not produce colitis, though they still could infect the cecum as well as wild type (WT) Salmonella, and they were also invasive as measured by CFU in the mesenteric lymph nodes et alSalmonella colitis. Thus, they established the physiological relevance of the model.Mice do not develop diarrhea when orally infected with Salmonella colitis in mice have been done with G169DNramp1 mutant mice (ItyS) that are so susceptible to systemic Salmonella infections that they die 4–5 days after oral infection. Perhaps because of that, most studies of Salmonella colitis in mice have focused on cecal inflammation Salmonella colitis, inflammation and gross pathology are focused on the cecum with some inflammation in the contiguous colon. Nramp1 is encoded by a gene on chromosome 1 that is now called Slc11a1Salmonella colitis in ItyR (NrampG169) mice. This paper reports our results with this model of colitis, a model of pan colitis with features of diarrhea.Nearly all reported experiments on S. Typhimurium in kanamycin-treated BALB/c and BALB/c.D2Nramp1 mice. As expected, all the BALB/c mice were dead by 5 days after infection and they had 106–107 CFU/spleen at the time of death. We expected the BALB/c.D2Nramp1 congenic mice to survive and to spontaneously clear the infection, but instead they started to lose weight by day 5–6 after infection  Salmonella in their ceca remained fairly constant throughout this time period. Even though BALB/c.D2Nramp1 congenic mice could not clear the intestinal infection, colony counts of Salmonella in the spleen were well below the numbers that were found in genetically susceptible BALB/c mice at the time of death. On day 2 after infection inflammation was limited to the cecum (not shown), but by 8 days after infection in the pathologic process had progressed to involve the entire colon with many mucosal ulcers scattered throughout . However, by day 8 after infection there were approximately 10 times more WT bacteria than invA mutants in ceca, and the differences were even greater in the feces, the mid and distal portions of the colon  and there was no significant difference between their diarrhea scores or in the numbers of Salmonella that we recovered from their ceca (phox mutation had full blown diarrhea by day 7 (mean diarrhea score 4.5) as happens in ItyR mice, and the 3 remaining males with CGD all had diarrhea scores of 5 . Because they expressed Nramp1 in their macrophages, they were better able to inhibit the growth of virulent Salmonella in their tissues, which allowed the mice to survive an infection with S. Typhimurium 14028s for at least 10 days. However, colitis progressed during that time and the mice developed more signs of diarrhea. Diarrhea in humans is often defined as an increase in the number and/or the volume of bowel movements per day, or the passage of unformed stools that assume the shape of the container. When it has been measured, the water content of diarrheal stools is greater than do solid stools We used a mouse model of Citrobacter rodentiumBecause the diarrhea score was somewhat subjective, we sought to validate it by measuring the water content of feces in the distal 3 cm of the colon, using a modification of the method of Guttman et al, who measured fecal water content from the distal colon as part of a study of the pathogenesis of diarrhea induced by S. enterica infections. S. enterica are a common cause of infectious diarrhea, but the mechanism of diarrhea in this infection is not well understood. Prior studies of the mouse model of Salmonella colitis have focused primarily on the pathogenesis of inflammation We used this mouse model to begin to investigate pathogen and host factors that contribute to diarrhea due to Salmonella colitis in mice we infected them with SPI-1 (invA) or phoP mutants. SPI-1 is required for invasion of cultured epithelial cells et al used GFP expressing Salmonella with mutations in either SPI-1 or SPI-2 to localize WT and mutant bacteria in the cecum, and they found that WT Salmonella were in both epithelial cells and the lamina propria, while SPI-2 mutants were found only in the epithelial layer. Two laboratories recently reported that the SPI-2 pathogenicity island also contributes significantly to cecal inflammation et al reported that the residual inflammation caused by infection with a SPI-1 mutant was reduced significantly if mice had a mutation in MyD88, which abrogates most signaling through TLRs Salmonella can induce colonic inflammation in two ways. SPI-1 promotes epithelial invasion, which stimulates those cells to make chemokines and pro-inflammatory cytokines To begin to assess the role of bacterial virulence factors in invA) invaded the cecum and caused significant inflammation, but we found that WT Salmonella infection spread distally to infect and inflame most of the colon by day 8/9 after infection, while the SPI-1 (invA) mutant remained largely confined to the cecum, as did the pathology. Most importantly, the diarrhea scores and the water content of the feces from mice infected with the invA mutant were significantly lower than the scores produced by WT Salmonella  that S. enterica use to modify the negative charge on their LPS to increase resistance to antimicrobial peptides such as polymyxin B pmrA and pmrF mutants for their ability to cause colitis, and they were fully virulent. Thus, the profound attenuation of a phoP/Q mutant in the colitis model is not due to lack of expression of any of the pmrA-coregulated genes. PmrA/B control only a subset of the genes that regulate modifications of the LPS, and pmrA mutants remain resistant to some antimicrobial peptides such as cathelicidin. phoP mutants are more susceptible to cathelicidins, which may explain why phoP mutants are avirulent in this model PhoP/Q regulates many genes and it is not clear which of them are necessary for virulence. Among the genes that are indirectly regulated by PhoP/Q are Salmonella gastroenteritis, we reasoned that inflammatory mediators made by the host may contribute to diarrhea. We assessed the contribution of one host factor, reactive oxygen species (ROS), by infecting gp91phox -deficient mice, which cannot generate ROS. Since the gp91phox mutation is on a B6 genetic background, and gp91phox is an X-linked gene, we were able to breed gp91phox -/- mice on an ItyR genetic background by crossing homozygous female B6.gp91phox -/- mice with male TF6 (Nramp1G169 transgenics). We then compared male F1 mice that had CGD with female littermates, who were carriers of the mutation and did not have CGD because of the Lyon effect. We found that male mice with CGD developed severe diarrhea when infected with 14028s, as severe as, or possibly worse than the controls. These results show that ROS from the host are not necessary to produce diarrhea. Not surprisingly, the male mice with CGD were also much more susceptible to Salmonella infection than were their sisters. These results extend the observations of Shiloh et al who reported that B6 mice with CGD are more susceptible to systemic Salmonella infections phox -/- mice to determine whether CGD mice were susceptible to oral infection with the phoP mutant, and they were not. This result implies that ROS are not required to kill phoP mutant Salmonella in vivo, and that there are non-oxidative killing mechanisms in mice that are an effective defense against phoP mutants but not WT Salmonella.The above results suggest that inflammation was connected with diarrhea. If inflammation is somehow responsible for diarrhea in Salmonella colitis, though the analysis presented was only of the cecal pathology Nramp1 congenic mice that we studied did not die of infection. We are still uncertain why BALB/c.D2Nramp1 congenic mice died, but they probably died from colitis and not from overwhelming systemic Salmonella infection. In addition to Nramp1, there are a number of polymorphic genes in mice that influence resistance to systemic Salmonella infections Nramp1 congenic mice and the strains studied by Stecher et al.While we were doing these experiments Stecher et al reported that 2 strains of genetically resistant mice(ItyR) developed a severe form of Salmonella colitis that mimics a severe form of human Salmonella gastroenteritis that is manifested as dysentery Salmonella that are unable to invade epithelial cells, nor phoP mutants that cannot survive in macrophages are able to cause diarrhea, and they caused much less inflammation in the colon than do wild type Salmonella. Diarrhea occurred even in the absence of the host's oxidative burst.In summary, we have developed a model of phox -/- mice were purchased from Jackson Laboratory . B6. Nramp1G169 transgenic mice (TF6) Nramp1 congenic mice phox -/- with male B6. Nramp1 transgenic mice in order to breed F1 males that carried the mutant B6.gp91phox allele, and therefore have chronic granulomatous disease (CGD) on a Nramp1G169 genetic background, since the WT version of Nramp1 is dominant. The female F1 mice are carriers of the B6.gp91phox mutation and served as the controls for the male mice.C57BL/6 (B6) and B6.gp913 CFU of S. Typhimurium 14028s in 0.1 ml of 0.1M NaHCO3. Mice were allowed free access to food and water. They were weighed daily in some experiments. At necropsy we exposed the abdominal cavity and scored the severity of the diarrhea before we disturbed the tissues. We processed the tissues for culture as previously described 2S positive colonies that grew on the HE-kanamycin plates were assumed to be Salmonella. Three cm long segments of the colon were processed similarly before they were cultured. If mice died before the time of sacrifice we assigned them the same number of CFU that we recovered from the most intensely infected living mouse, a conservative estimate. If we could not obtain feces from a mouse because of diarrhea we did not include that mouse in our calculations. Feces were weighed before we processed them and we calculated CFU/gram of feces.We administered oral kanamycin (40mg) in 0.1 cc of saline by gavage. The following morning the mice fasted for 4 hours before we infected them by gavage with ∼5×10C. rodentium-infected mice The scoring system that we used to assess the severity of the diarrhea is shown in spv operon in the virulence plasmid. This S. enterica serovar Typhimurium 14028s KanR strain S. Typhimurium 14028s invA::aphT has been described previously phoP::Tn10 allele pmrApmrFWe constructed derivatives of Typhimurium strain 14028s containing a kanamycin resistance cassette (kanR) inserted in a genetically silent region downstream from the We filled the entire colon with Bouin's solution, tightly coiled the colon around a wooden applicator stick, and immersed the coiled colon in Bouin's solution. After embedding in paraffin we made 5 mm thick sections that we stained with hematoxylin and eosin (H&E). Each slide was coded and read blind by the PI.Results were analyzed using GraphPad Prism version 4 . Multiple groups were compared using a one way ANOVA and the differences between two groups were examined with the Kruskal-Wallis statistic. To compare two groups of results we used the non-parametric Mann-Whitney U test. P values of <0.05 were considered significant.


Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of Dovo increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.Measurement of food intake in the fruit fly  Drosophila melanogaster is a key model organism for discovery of evolutionarily conserved biological mechanisms, which include the control of nutrient sensing Drosophila are therefore often needed. However, quantification of food consumption in the fly poses challenges. In mammals, food ingestion can be directly quantified by weighing the food before and after feeding has taken place. However, flies consume volumes of food that are too low to weigh accurately, and feed by extension of their proboscis into the food medium, prohibiting direct observation of the volume of food ingested. One method has overcome this problem by measuring the food consumed in liquid form in a capillary feeder (CAFE) Drosophila feed on microorganisms, particularly yeast, on the surface of fruit The fruit fly Drosophila, which increases the capacity of the crop Drosophila, a time period during which disturbance of the flies affects their feeding behaviour. There is thus a requirement for a method of measuring feeding in undisturbed conditions.To overcome the problems of measuring food intake when flies feed on gelled media, several studies have made indirect measures of food uptake after marking the food, either with a visible dye Previously, we have reported that direct observations of fly proboscis-extension onto the food surface chico1takeout1ovoD1In this study we tested the accuracy of the method, by measuring the volume of food ingested using a dye food label in parallel with observing the number of proboscis-extensions. We compared flies that had either known or suspected differences in food intake, such as males versus females, flies subjected to dietary restriction Drosophila is often achieved by dilution of the food medium, and complete records of food intake are needed to determine if flies compensate for the reduced nutritional content of food by increasing the total amount of food they consume. Measurement of food intake is also needed to determine if other interventions, such as sensory perception of food chico, which extends the lifespan of DrosophilaDietary restriction (DR) in In nature and in the laboratory, fruit flies feed on the food surface, by extending their proboscis into contact with the food and drawing it in. The amount of proboscis-extension onto the food surface was measured by making periodic observations of groups of flies. The number of observations of proboscis-extension was then expressed as a proportion of the total number of observations Volume of blue food found in the fly and the proportion of feeding events Observed (V/O) . The gradient of this relationship represents the ingestion ratio of the flies, as it describes the volume of blue accumulated per proboscis-extension. To test for non-linearity , we added a quadratic term to the statistical model. The quadratic term was not significant (P = 0.62), indicating the V/O relationship is indeed linear over the timespan we measured . The gradients of these relationships were not found to be significantly different (P = 0.9871), indicating that the ingestion ratio did not differ between the sexes. However, the intercept of the male relationship was significantly lower than that for females (P<0.001) and suggested males across all observations contained a lowered basal level of blue dye content than in females (P = 0.54), indicating that a linear V/O relationship exists. In spite of the sexes sharing the same ingestion ratio, females were found to have fed more than males over the 30-minute period because they spent a greater proportion of time with the proboscis extended (2.8-fold more on average) than males . This suggested it is possible for flies to increase their food intake by feeding at a greater frequency rather than by consuming in greater volume, and it is possible to detect such differences in food intake.Next, we tested whether the sexes differed in ingestion ratio (gradient of the V/O relationship) by repeating the combined assay with 7-day-old mated males and females , with no significant differences in the gradient and intercept of this relationship between the two different diet regimes .We then extended the use of this method to examine the effect of other factors that could determine the physiology and behaviour of feeding flies. The nutritional environment may be such a factor, and is particularly important in the context of DR experiments where dietary dilution is employed to restrict access to nutrients. We therefore performed the combined assay with 7-day-old mated females that were fed either DR or full fed control diet 1chico, is a null mutation in the single fly insulin receptor substrate in the insulin/insulin-like growth factor-1 signalling (IIS) pathway, a pathway suggested to affect foraging and feeding in larvae 1chico and their genetic control (Dahomey) , with no significant differences in the gradient or intercept between 1chico heterozygotes and control flies .Finally, we also tested whether the ingestion ratio or feeding frequency were altered by genetic mutations known or suspected to affect feeding. The first mutation, 1takeout, is in a gene reported to regulate the circadian rhythm and to increase food intake prior to starvation in Drosophila1takeout flies and their genetic control (Canton-S)  with gradient and intercept not significantly different between the two genotypes . The flies thus elevated their nutrient-intake by feeding at a greater frequency, rather than by increasing the volume of intake per proboscis-extension.The second mutation, ovoD1, causes female sterility and has been reported to induce a reduced feeding frequency whiteDahomey) ; however, the gradient and the intercept for the relationship differed between the two genotypes . The V/O gradient for ovoD1 was steeper (205.52 versus 14.46 in whiteDahomey) and the intercept greater (56.40 versus 28.65 in whiteDahomey) than for whiteDahomey controls , as well as a greater basal level of blue dye. However, no significant difference in the proportion of time spent feeding between ovoD1 females and whiteDahomey controls was recorded . This indicated that D1ovo flies elevated their received nutrition by increasing the volume of intake per proboscis-extension rather than by feeding at a greater frequency.The final mutant studied, P<0.0001, linear regression model), but neither the gradient (P = 0.0961) nor the intercept (P = 0.649) changed with age. The volume of intake per proboscis-extension was thus unaffected by the age of the flies.We also analysed the effect of age upon the ingestion ratio. Dahomey females were subjected to the combined blue dye and proboscis-extension assay at 4 different ages . The food intake of the flies thus varied widely. Despite the variation in the overall amount of feeding, there was no significant variation in the ingestion ratios except in one genotype, DrosophilaWe investigated other variables that could affect food intake during undisturbed conditions. The circadian rhythm is reported to alter feeding in P<0.001 for both group size and time of day, GLM), while the interaction between these two was not significant (P = 0.88). The lowest feeding proportion was observed in the morning for flies housed singly (0.15 of the time spent feeding), and this increased to approximately 0.50 in the afternoon and evening for flies feeding in groups of 5 or more. Both the afternoon and evening feeding proportions were significantly higher than those in the morning . There was no significant difference in feeding proportions between flies during the afternoon and evening . The lowest proportion of feeding was observed for flies housed singly 0.15–0.22 (depending on time of day), and this significantly increased to 0.18–0.31 (depending on time of day) when flies were housed in pairs . The proportion of flies feeding was found to nearly double when the number of flies was increased to 5 per vial , and did not increase further when flies were housed at 10 per vial , in the afternoon (4 hours after lights-on), and in the evening (8 hours after lights-on) using 4 different group sizes . As previously reported, flies exhibited significantly lower feeding frequency when the concentration of yeast extract was increased in the CSYExtract diet . In contrast, the feeding frequency of flies was unaffected when altering the yeast concentration of the SYBrewer's diet .Finally, we tested the response of 7-day old female flies to two different yeast-based diets, one made with water-soluble yeast extract (CSYExtract) The proboscis-extension method allows repeated feeding assays to be performed with the same cohort of flies, an advantage over methods that sacrifice flies during measurements. As far as we are aware, no publication to date has studied either the feeding frequency of a cohort of flies throughout their lifespan or measured how much food flies consume throughout their lives. This is especially important when monitoring the effects of dietary restriction on lifespan, as the short-term probability of death as revealed by mortality analysis is rapidly affected by changes in nutritional conditions P<0.001, GLM). No overall difference was found in average feeding frequency (0.17 in both cohorts) for the course of the lifespan. However, flies on a DR diet fed in a greater proportion of observations than full fed flies early in life, while this reversed later in life when full fed flies fed more than DR flies (between day 31 and day 50), after which the feeding became similar on the two diets. Preliminary studies showed that the feeding frequency of flies on both diets were low at the beginning of the proboscis-extension assay but gradually increased to a steady state over 30 minutes . Overall observed feeding proportions also did not differ significantly from wild type controls .We also compared the feeding frequency of wild type and long-lived Drosophila (measuring proboscis-extensions) by combining it with a direct method (measuring food intake with a food dye). Despite considerable variation in feeding between replicate groups of flies and between experiments performed on different days, the volume of food ingested per proboscis-extension (ingestion ratio) did not significantly differ between females and males, flies of different ages, flies subjected to DR and flies with mutations in chico or takeout. Only ovoD1 females ingested more dye per proboscis-extension.In this study, we validated an indirect method of measuring food intake in The observation data revealed that males feed less than females. The higher food intake of female flies is presumably related to their high nutrient-usage in egg-production ovoD1 females exhibited a greater ingestion ratio than any of the other genotypes tested. This finding is surprising, because egg development is arrested in D1ovo flies before the major nutrient investment occurs ovoD1 flies, it could be that they expend more energy through a higher level of activity than fertile flies. This could partially explain why we found a higher ingestion ratio in these mutant flies. ovoD1 has been reported as feeding less frequently during long-term undisturbed conditions et al. (2008), because our data were obtained from the first 30 minutes after transferring to blue-labelled food, and we found no differences with feeding frequency, only with ingestion ratio.Sterile The combined assay is not suitable for long-term, undisturbed feeding experiments because the assay requires that flies are transferred to dyed food, which disturbs fly feeding behaviour. Frequencies of proboscis-extension were observed to be lower than in steady-state conditions, and only reached a constant level by the end of the 30-minute observation period. However, the indirect method alone is accurate for measuring fly feeding during long-term, undisturbed, experimental conditions once assessed by the combined assay for any differences in the ingestion ratio.Data from the undisturbed steady-state studies suggested that flies exhibit marked diurnal differences in feeding behavior when feeding in groups and earlier in life. The effect of fly group size may reflect the role of aggregation pheromones, which act as communication signals between flies on breeding substrates, with feeding and oviposition rates increasing with the level of aggregation pheromone Caenorhabditis elegans, as well as Drosophila and the mouse Drosophilachico in Drosophila both extends lifespan hico-heterozygotes using the proboscis-extension assay and found total food intake was not reduced in the mutants. The increased survival of 1chico mutant flies compared to controls can therefore not be explained by a reduction in food intake 1chico mutants Mutations in the IIS pathway have been shown to extend the healthy lifespan of the nematode worm et al. (2005), we saw feeding frequency decrease as the concentration of CSYExtract in the medium was increased. In contrast, but consistent with previous reports DR in flies can be imposed by dilution of their food source, which is available in excess. Flies could therefore adjust their feeding frequency to compensate for the reduction in nutritional value, thus reducing or eliminating the effect of food dilution on nutrient-intake. The literature on this topic is conflicting, with some reports that flies can partially compensate for the food dilution An important element of studies into ageing is the longitudinal effects of lifespan-altering interventions. Previously, we have reported that flies subjected to DR do not alter their feeding frequency on day 7 of adult life DrosophilaDrosophila to be measured during conditions that reflect either the experimental set-up they are normally housed in or their feeding frequency in undisturbed conditions. We established that the proboscis-extension method fulfils these criteria.In recent years, various methods have been proposed to measure food intake in et al. (2007) 1chico allele is maintained as a balanced stock that has been backcrossed to the Dahomey outbred laboratory population as described in Clancy et al. (2001) wsn, 506ry, 1to (takeout) flies were a gift from Brigitte Dauwalder. All flies were maintained at 25°C, 65% humidity, on a 12h∶ 12h light∶ dark cycle. Unless stated otherwise, all assays used mated females at day 7 after eclosion. Day 7 was chosen because the flies are still young, but several early adult developmental processes have been completed 2 anaesthesia, into 30 mL glass vials containing 7 mL food.Wild type Dahomey flies were housed and maintained as described in Bass The DR food medium contained 100 g autolysed Brewer's yeast powder , 50 g sugar, 15 g agar, 30 ml nipagin (100 g/L), and 3 mL propionic acid made up to 1 litre of distilled water. The full fed food contained 200 g autolysed yeast powder, 50 g sugar, 15 g agar, 30 ml nipagin (100 g/L), and 3 ml propionic acid made up to 1 litre of distilled water For DR lifespan experiments, flies were maintained 5 per vial at 25°C, 65% humidity, on a 12h∶ 12h light∶ dark cycle. Proboscis-extension assays were performed for 60 minutes at 5-minute intervals, 4 hours after lights-on at 21 separate days across the lifespan experiment.For undisturbed observations of feeding, 7-day-old mated flies of the same sex, were transferred to new food at a density of 5 per vial on the evening before the assay. Flies were maintained in a pooled population, 100 flies per bottle, and a subset was collected and returned before and after the assay. Different measurements on different days were therefore considered to be independent of each other. Vials were coded and placed in a randomised order in rows on viewing racks at 25°C overnight. The assay occurred with minimal noise and physical disturbance to the flies. To avoid recording disturbed fly feeding behaviour, 30 minutes was allowed between the arrival of the observer and commencement of the assay. Observations were performed “blind” the next day for 90 minutes, commencing one hour after lights-on. In turn, each vial was observed for approximately 3 seconds during which the number of flies feeding was noted. A feeding event was scored when a fly had its proboscis extended and touching the food surface while performing a bobbing motion. Once all vials in the experiment had been scored in this way, successive rounds of observations were carried out in the same way for the whole 90 minutes of the assay, which, depending on the size of the experiment meant that each vial was observed once every 2 to 5 minutes. At the end of the assay, the vial labels were decoded and the feeding data expressed as a proportion by experimental group . For statistical analyses, comparisons between experimental groups were made on the totals of feeding events by all flies within a vial, to avoid pseudoreplication.Groups of five 7-day-old mated flies were transferred onto fresh food medium as indicated containing 2.5% (w/v) blue food dye (F D & C Blue Dye no. 1). Vials were scored approximately every 2 minutes for proboscis-extension and after a total of 30 minutes were transferred to eppendorf tubes and snap frozen in liquid nitrogen.Flies were homogenised in 200 µL of distilled water. A further 800 µL of distilled water was added and the suspension passed through a 0.22 µm Millex filter  to remove debris and lipids. The absorbance of the liquid sample was then measured at 629 nm [Hitachi U-2001 Spectrophotometer ]. Age-matched flies exposed to non-dyed food were used as the baseline during spectrophotometry. The amount of labelled food in the fly was calculated from a standard curve made by serial dilution in water of a sample of blue food.Statistical analyses were performed using R, v2.2.1 F-tests for analysing significance). The generalised linear models incorporate information on the sample sizes and use weighted regression analyses. Significance among factor levels (e.g. among the 4 different group sizes) was determined by model simplification, where we evaluated whether combining >1 factor level into a single level led to a significant increase in deviance of the model, using F-tests To compare the effect of time of day, group size and dietary composition on feeding frequency, we used generalised linear models (with binomial error structure and logit link function, the deviances were scaled to correct for over-dispersion, and using


With the conditional approach, SNPs that reach Bonferroni-adjusted significance at the first stage are tested in pairwise combinations with all SNPs in the data set. We compared the performance of those strategies by using Replicate 1 of the simulated data set of the Genetic Analysis Workshop 15 Problem 3. Most interactions detected resulted from SNP pairs within 1000 kb of each other. The remaining were false positives involving SNPs with excessively strong marginal signals. Our results highlight the need to account for locus proximity in the evaluation of interaction effects and emphasize the importance of marginal signal strength in logistic regression-based interaction modeling. We found that modeling additive genetic effects alone was sufficient to capture underlying dominance interaction effects in the data.Large genetic association studies based on hundreds of thousands of single-nucleotide polymorphisms (SNPs) are a popular option for the study of complex diseases. The evaluation of gene × gene interactions in such studies is a sensible method of capturing important genetic effects. The number of tests required to consider all pairs of SNPs, however, can lead to a computational burden, and efficient strategies to reduce the number of tests performed are desirable. In this study, we compare two-stage strategies for pairwise SNP interactions testing. Those approaches rely on the selection of SNPs based on the single-locus test results obtained at the first stage. In the  Genetic association is an increasingly popular method to identify genetic determinants of common diseases. Traditional single-locus association tests evaluate the marginal effects of each marker. It is to be expected, however, that the genetic susceptibility of complex traits would result from the interplay of several factors, including gene × gene interactions. As such, analytical approaches that consider single-nucleotide polymorphism (SNP) interaction effects have the potential to provide more power, especially when susceptibility genes have small or undetectable marginal effects.n) of SNPs in a study, following n(n - 1)/2, and testing for the entire probability space for SNP × SNP interaction can become computationally unfeasible or cumbersome. Some promising two-stage approaches have been proposed , and the p-value of the interaction coefficient iaa was used in the additive-only model [Eq. (4)]. Bonferroni correction was used by considering the total number of valid interaction term tests. Valid interaction tests refer to those for which the problem of quasi-separation does not occur when using the logistic regression model. We also used an interaction model in which the additive effects are considered without covariates (results not shown), in this case, the p-values of the one interaction term (iaa) was used.SNPs were selected in the first stage for marginal significance levels up to 0.1 and 0.05 for the simultaneous design and up to the Bonferroni adjusted threshold for the conditional method. In the second stage, the p = 0.1, and 930 SNPs below the threshold of p = 0.05. The tests for all possible pairwise combinations involved (1361 × 1360)/2 = 925,480 interaction tests for the threshold of 0.1 and (930 × 929)/2 = 431,985 tests for the 0.05 threshold. For the conditional design, Bonferroni correction left 443 significant SNPs: 428 of which are located on chromosome 11, 14 SNPs on chromosome 6, and 1 SNP on chromosome 18. These results correspond to the detectable marginal SNP effects simulated in the data set ], we obtained 1361 SNPs that fell below the threshold of ], we obtp = 0.1, and 894 SNPs below the threshold of p = 0.05. Using the global Bonferroni correction, 398 SNPs were found significant, of which, 395 were on chromosome 11 and 3 SNPs were on chromosome 6.For the logistic model with adjustment for sex, smoking, and DR alleles [Eq. (2)], we considered only the additive effects. 1319 SNPs fell below the threshold of idd) (Table iaa). The same model detected 57 interactions with the iaa term, and the logistic model for additive effects-only (not shown) detected 7 more interactions, due to the reduction in the multiple testing correction. Based on this result, we proceeded to conduct the analysis with adjustment for covariates by using only the additive interaction model [Eq. (4)]. The use of either thresholds at p = 0.05 or p = 0.1 for the simultaneous design provided exactly the same interaction results for the full model without covariates [Eq. (3)], but a few more were found with the additive model with covariates [Eq. (4)] when using the threshold of p = 0.1.When using the full model with dominance and additive genetic terms [Eq. (3)] with the simultaneous design, five significant interactions were found by the dominance interaction term (d) Table , but allp-values < 10-200), which we believe to be responsible for generating false-positive interaction signals ] reveals that 129 different SNP × SNP interactions were found with either the conditional or simultaneous design. After eliminating the 52 interaction results between SNP < 1000 kb, 77 interactions remained, 75 of which include a chromosome 11 SNP around Locus F, 6 of those are proximity interactions on chromosome 11, and 69 are false positive between chromosome 11 and 1. This chromosome 11 region had excessively high marginal signals . According to the simulation schema of Problem 3, a simulated genetic interaction effect between Locus A on chromosome 16 and Locus C on chromosome 6 was modulated by the DR alleles. In Stage 1, we were able to detect the simulated Loci C on chromosome 6, D on chromosome 6, and E on chromosome 18, which passed Bonferroni correction in both logistic regression models with adjustment for covariates and without adjustment for covariates ] and a p-value of 0.0001 with the additive model without adjusting for covariates, which was insufficient to be detected after multiple testing adjustment. We mention here that we used only the first replicate of GAW15 Problem 3 simulated data due to computational constraints, but the analysis of more replicates may have provided different results.The simultaneous method optimizes the search for interaction effects between SNPs that provide some indication of marginal associations. This situation is most appropriate for disease models with additive polygenic effects. The advantage of the conditional design, on the other hand, is that it can capture interactions with loci that have no marginal effects, and as such is expected to be valid over a wider range of disease models. With our data, neither approach was able to capture the real underlying interaction between Loci A and C. Posterior testing for interaction between these loci revealed a An important advantage of the conditional design in a real data set, is that Step 1 should provide only a small number of globally significant SNPs , which are then tested in pairwise combination with the full complement of SNPs in the study. As such, the conditional design should generate fewer interaction tests than the simultaneous design. In the GAW15 simulated data set, however, over 400 SNPs remained significant after adjustment for multiple testing in Stage 1 and caused a special situation in which the conditional design exploded into many interaction tests and lead to the physical proximity interaction effects that we detected with both approaches. It is not clear why there were so many significant results on chromosome 11 and it would be interesting to validate those results by testing the other replicates. The use of logistic regression for the detection of interactions has the disadvantage of performing poorly with high dimensionality, which can lead to false positives and decreased power. In the present study for instance, the conditional approach with additive and dominance effects required the estimation of 16,279,364 parameters from 32,154,500 observations. Another potential problem with logistic regression is the phenomenon of the separation or quasi-separation of data. This occurs in the fitting process of the logistic model when the likelihood converges while at least one parameter estimate diverges to infinity. Quasi-complete separation of data, however, was only observed in 0.3% of the tests that we performed with the additive model with covariates in this study. High correlations between predictors, referred to as multicollinearity, may also decrease the power of the logistic regression. This may occur with SNPs that are in high LD. Multicollinearity due to linkage disequilibrium could, however, be addressed statistically by combining SNPs that are in high LD into haplotypes.iaa). We conclude from our results that using a model with only the additive genetic effects of two SNPs should capture most of the underlying interactions and will reduce the multiple testing otherwise imposed by the four interaction terms. A large portion of the interactions that we detected resulted from SNP pairs that were within 1000 kb of each other. Our results clearly highlight the need to include an additional step or procedure to account for locus proximity in the evaluation of interaction effects. All remaining interactions without exception were false positives that included a SNP with exceptionally high marginal signals from chromosome 11 or chromosome 6  are likely to be responsible for the false-positive signals. This finding draws attention to the importance of directing further work toward the evaluation of the impact of strong marginal SNPs in interaction modeling.The significant interactions found by the full logistic regression model with both dominance and additive effects [Eq. (3)] were all captured by the additive effect coefficient (The author(s) declare that they have no competing interests.


Members of the dynamin super-family of GTPases are involved in disparate cellular pathways. Dynamin1 and dynamin2 have been implicated in clathrin-mediated endocytosis. While some models suggest that dynamin functions specifically at the point of vesicle fission, evidence also exists for a role prior to fission during vesicle formation and it is unknown if there is a role for dynamin after vesicle fission. Although dynamin2 is ubiquitously expressed, dynamin1 is restricted to the nervous system. These two structurally similar endocytic accessory proteins have not been studied in cells that endogenously express both.The present study quantitatively assesses the dynamics of dynamin1 and dynamin2 during clathrin-mediated endocytosis in PC12 cells, which endogenously express both proteins. Both dynamin isoforms co-localized with clathrin and showed sharp increases in fluorescence intensity immediately prior to internalization of the nascent clathrin-coated vesicle. The fluorescence intensity of both proteins then decreased with two time constants. The slower time constant closely matched the time constant for the decrease of clathrin intensity and likely represents vesicle movement away from the membrane. The faster rate may reflect release of dynamin at the neck of nascent vesicle following GTP hydrolysis.This study analyses the role of dynamin in clathrin-mediated endocytosis in a model for cellular neuroscience and these results may provide direct evidence for the existence of two populations of dynamin associated with nascent clathrin-coated vesicles. One population of dynamin (70%) decreased in fluorescence at the same rate as clathrin or internalized cargo, consistent with being the result of movement of the vesicle out of the excitatory evanescent field. A second population (30%) decreased much faster (20–40 fold) and may represent dynamin dissociated from the vesicle. Similar results were observed for dynamin1 and dynamin2. Thus, imaging individual events of endocytosis provides evidence for two dynamin populations associated with clathrin-mediated endocytosis: one that is rapidly released as well as one that internalizes associated with the nascent clathrin-coated vesicle.All constructs were the same as we employed in previous studies 2. Cells were plated onto MatTek 35 mm glass bottom dishes  coated with Mouse Collagen Type IV , according to the supplier's directions. Cells were transfected using Lipofectamine 2000  according to the supplier's directions. All imaging was performed 48 hours post-transfection.PC12 cells were obtained from American Type Culture Collection  and were cultured in DMEM  supplemented with 10% horse serum and 5% fetal bovine serum in a humidified 37°C incubator with 5% COTIR-FM was performed at 37°C using an Apo 60× 1.45 NA microscope objective , as previously described After subtraction of extracellular background, 12-bit dual-color TIR-FM image streams were aligned in MetaMorph. On the basis of single-fluorophore control experiments, green-to-red bleed-through corrections of 10% for were employed; following background subtraction and alignment, 10% of the EGFP signal was subtracted from dsRed images.Paradigms determined from our previous studies in this area were applied to the current data sets The criteria for identification and analysis of dynamic spots from image stacks were based upon that for static co-localization studies. However, spots were omitted if any laterally mobile spots, or other separation or coalescence of spots, interfered with identification, tracking or quantification. Furthermore, if the dynamin signal appeared to show multiple flashes, only spots where the first flash was the brightest were analyzed – In order to be able to clearly identify the point at which disappearance began. Regions around spots to be analyzed were drawn to maximize the spot fluorescence and minimize the contribution of local background fluorescence. “Temporal alignment” and normalization of fluorescence intensity data was performed as in our previous studies Clathrin and dynamin tagged with fluorescent proteins have been previously employed in numerous live-cell imaging studies without evidence of perturbation of the endocytic system Very similar observations were obtained when clathrin and dynamin2 were analyzed by the same methods . HoweverIn order to test whether differences between dynamin1 and dynamin2 during clathrin-mediated endocytosis could be observed, each was imaged simultaneously with clathrin by live-cell TIR-FM in PC12 cells. When individual spots were tracked over time, the clathrin fluorescence in individual puncta was observed to disappear, while the fluorescence at neighboring sites remained unchanged  decreased with a time constant of 17 seconds, which was the same as the time constant for clathrin disappearance. However, the rest of the dynamin1 decreased with a much faster time constant of 0.89 seconds. The time constant for clathrin decrease at sites of dynamin2 recruitment equals 21.5 seconds. While ∼70% of dynamin2 signal decreased with a time constant of 15.8 seconds, ∼30% decreased with a much faster time constant of 0.36 seconds.Previously, we, and others, have employed total internal reflection fluorescence microscopy (TIR-FM) to analyze the dynamics of proteins involved in clathrin-mediated endocytosis Our results demonstrate that both dynamin1 and dynamin2 significantly co-localise with clathrin in PC12 cells. The observation that ∼70% of the clathrin puncta are positive for dynamin1 and ∼80% of the clathrin puncta are positive for dynamin2 suggests that a substantial percentage of the clathrin puncta are positive for both dynamins. Additionally, the clathrin puncta that were disappearing, and thus endocytosing, were equally labeled with dynamin1 and dynamin2. These results do not provide evidence for a functional difference between dynamin1 and dynamin2 in these cells. Thus, for some endocytic events they may have redundant functions and, at least in these non-stimulated PC-12 cells, under the growth conditions used, the minor differences discerned in individual static images may not reflect a functional disparity.Prior to internalization, the dynamin was observed to increase in parallel with clathrin. However, just before the clathrin puncta internalized the fluorescence intensity of either dynamin1 or dynamin2 increased over a second by 50–65%. As the complete duration of dynamin ‘burst’ only lasted ∼2 seconds, this demonstrates the need for rapid sampling when performing live-cell imaging studies of the dynamics of endocytosis. This increase in the fluorescence could result from the recruitment of additional dynamin. Alternatively, it may not reflect an increase in the number of dynamin molecules, but may reflect a physical movement of dynamin molecules to the neck of the vesicle. This would move them closer to the cover slip and increase their fluorescence excitation. If we assume that the evanescent field decays with a space constant of 70 nm and the vesicle has a radius of 35 nm then the fluorescence should increase 58%. The larger the radius of the vesicle, the greater the predicted increase of fluorescence (see table). This calculation assumes that that dynamin initially is evenly distributed over the membrane, and then 100% of the dynamin moves to the interface between the vesicle and the plasma membrane. If less than all of the dynamin moves, the predicted increase would be less. However, the calculation exists that the increase could be sufficient to account for the observed increase.While the behaviors of dynamin1 during clathrin mediated endocytosis in PC12 cells were similar to those observed in other cells lines that lacked dynamin 1, the dynamics of dynamin2 were different than seen in some previous studies Previously we have observed within each endocytic puncta, other endocytic markers such as epsin and endocytic cargo such as transferrin leaves the evanescent field with a time course that is indistinguishable from clathrin