The synergies among biology, computing and other formal disciplines continue to produce a unique blend of domain-specific and methodological advances that is shaping the very fabrics of the new scientific method. Among the many examples of this phenomenon, the one offered by the unrelenting growth of bioinformatics and computational biology is unique in that nowhere else is the native lexicon of a natural science more directly conducive to digital representation and manipulation.Research in Computational Molecular Biology Tenth Annual International Conference on (RECOMB 2006), which was held in Venice, Italy, on April 2–5, 2006. The RECOMB conference series was started in 1997 by Sorin Istrail, Pavel Pevzner and Michael Waterman. The previous meetings were held in Santa Fe, NM (USA); New York, NY (USA); Lyon, France; Tokyo, Japan; Montréal, Canada; Washington, DC (USA); Berlin, Germany; San Diego, CA (USA); and Boston, MA (USA). RECOMB 2006 was hosted by University of Padova in the Venice Convention Center at Cinema Palace, Venice Lido, Italy.The research articles contained in this Supplement originate from posters presented at the The Tenth Edition of RECOMB was special in several ways. For one, the Program Committee, consisting of 38 specialists of the highest distinction in the field, included all past Committee and Conference Chairs as well as the Members of the Steering Committee. The Committee selected 40 papers out of the received submissions of well over 200. Some of the accepted papers were further expanded and refereed in order to appear in the special issue traditionally devoted to the Conference. For the Tenth Edition, however, in view of the high quality of the contributions submitted for poster presentation, it was felt that another Special Issue, devoted to expanded and duly refereed versions of poster submissions was also warranted.Thus, the present Supplement constitutes one more innovation brought about by the Tenth Anniversary of RECOMB. The eight enclosed papers emerged at the outset of a rigid selection and review and represent a vivid snapshot of work mature enough to be reported within vibrant frameworks still in the making. We hope that they will start one more tradition for RECOMB.This Issue was made possible thanks to the effort of many, in particular, the special task force set up for handling posters, which consisted of Luca Bortolussi , Giovanni Ciriello , Matteo Comin , Claudio Garrutti , Giosué Lo Bosco , Sabrina Mantaci  Cinzia Pizzi , Simone Scalabrin , and Nicola Vitacolonna .We are also grateful to the external reviewers, the members of the Steering Committee and other colleagues who helped in the process. Finally, we express our thanks to the institutions and corporations who provided financial support for the conference: Broad Institute of MIT and Harvard, USA; College of Computing, Georgia Tech., USA; Department of Energy, USA; Department of Information Engineering, University of Padova, Italy; IBM Corporation, USA; ISMB, International Society for Computational Biology; AICA, Italian Association for Informatics and Automatic Computation; National Science Foundation, USA; University of Padova, Italy.


The role of alcohol as a risk factor for motor vehicle crashes is long known. Yet, reports on the prevalence of drinking and driving suggest values between 20%–30% when the adult driving population is interviewed. We wondered whether these values hold true among European educated citizens and whether there are any significant differences in prevalence by age, gender, type of profession and other lifestyle indicators.Cross-sectional analyses of baseline data from a cohort of university graduates in Spain (SUN study). Answered questionnaires contained items on current drinking and driving practices, together with data on socio-demographic characteristics and lifestyle habits. Chi square, Fisher test, and multivariate logistic regression were used to investigate the impact of several variables on drinking and driving practices. Analyses were stratified by gender.Almost 30% of the participants reported "sometimes" drinking and driving. This percent increased to 47% when "almost never" was also included as a positive answer to the drinking and driving practice question. These percentages varied significantly by gender, with up to 64% of men reporting "sometimes" or "almost never" vs. 36% of women doing so. Drinking and driving practices also differed by overall alcohol consumption habits, smoking, use of safety belts, and notably, type of profession.Our findings are amongst the first on the high prevalence of drinking and driving among Spanish. Particularly worrisome is the fact that health professionals reported this habit even at higher rates. Multidisciplinary interventions  are needed to reduce this serious health risk. Motor vehicle crashes constitute one of the leading mechanisms of death and disability around the world. The WHO estimates that 1.2 million people die on a yearly basis in relation to these crashes. They further estimate that between 20 and 50 million people are injured or disabled because of non-fatal motor vehicle injuries  when he/she has had any alcohol". Answers to this question could be "I do not drive", "absolutely never", "yes, sometimes", and "almost never".Other questions of relevance were related to average kilometres driven per year, use of safety belt, and lifestyle indicators such as weight and height  , smokingFor our analysis we used all baseline questionnaires of participants who had joined the cohort prior to February 2006.Since alcohol consumption, exposure to motor vehicles and involvement in motor vehicle crashes are so closely related to gender, we conducted stratified analyses by gender. Fisher 2-tail tests, Pearson chi-square tests, and chi square tests for trend were used for the comparisons of proportions between groups. Multivariate logistic regression models were created to evaluate the role of several socio-demographic and lifestyle characteristics in the probability of reporting drinking and driving.Last, since enrolment in the cohort is open, we investigated whether subjects enrolling in different years reported differences in the practice of drinking and driving.Statistical significance was defined at a p < 0.05. SPSS version 12.0.1 was used for analyses Data were available from 16,171 university graduates who had enrolled in the cohort at the time of this analysis and their data had been cleaned and processed, 59.4% of them were women . The average age of participants was 38 years old (SD 12.4). The vast majority of participants were drivers (92.4%), although this percent differed significantly by gender, with a larger proportion of men being drivers . The average alcohol intake was 6.9 g/d (SD 11.2), although men had a higher average alcohol intake than women . Almost a third of the participants reported on occasional binge drinking, although men reported higher prevalence of such practice . Although 5% of participants reported ever requiring hospital admission in relation to a motor vehicle crash, this prevalence differed also by gender, being 6.6% among men and 4.3 among women (p < 0.001). Slightly more than half of the participants (52.8% or 8531) reported to never drink and drive. This differed by gender, with the percent being 35.8% among men and 64.2% among women (p < 0.05). Among the participants reporting drinking and driving, 4,691 indicated to do so "sometimes" . However, among women other covariates showed an independent significant increased likelihood of reporting drinking and driving. Being a nurse was associated with an OR 1.23 (95%CI 1.05–1.43) as compared with having a non health-related profession. Not always wearing safety belts had an OR of 1.19 (95%CI 1.03–1.38) as compared to always using it. Presenting a history of traffic injury had an OR of 1.65 (95%CI 1.33–2.05) as compared to not having it. Being an ex-smoker (for at least one year), had an OR of 1.24 (95%CI 1.10–1.40) as compared to never smoking.We repeated the same multivariate models, this time including in the outcome those reporting drinking and driving "almost never", only to find very similar results to those described above (data not shown). Last, we repeated these models excluding cohort participants who were not drivers. Table Adding cohort enrolment year to these multivariate regression models to investigate whether the prevalence of drinking and driving among subjects enrolling in recent years produced the results summarized in Figure Our findings reveal an extremely high prevalence of self-reported drinking and driving among a high education segment the Spanish participants in the SUN cohort study. Only slightly more than half our participants (53%) reported "never" drinking and driving, and up to 30% of the participants acknowledged to drink and drive "sometimes". Although our cohort is not expected to be representative of the Spanish educated population, this high prevalence seems to be also spreading to the general Spanish population if one is to use the 2002 SARTRE-3 unpublished findings or studies from other countries ,12,14-19In particular, and even though our sample is not comparable on educational level to those in some US studies ,12,14 ouMore importantly, the strengths of cohort studies reside on the investigation of relationships between variables and the follow-up data that they can provide. Our study found that drinking and driving was also related to other unsafe practices. Not surprisingly, binge drinking and daily drinking were also more common amongst those who reported drinking and driving, as seen in US studies , where 8The elevated prevalence rates of drinking and driving of the educated population included in our study may seem in conflict with other studies, where high income and education levels have proved to have a protective effect on alcohol-related fatal motor vehicle crashes . HoweverNever reported before is our finding that health professionals (physicians or nurses) are more likely to report drinking and driving than other university graduates even after controlling for possible confounders. The role of these health professionals in educating the population regarding the health consequences of drinking and of drinking and driving has been long advocated for ,26,27. YAlso never reported for Europe is our finding that the prevalence of self-reported drinking and driving seems to be diminishing in more recent years, at least among women. Whether this is a true trend or simply a reflection of an increase in socially desirable answers to the question related to drinking and driving remains to be investigated. It could also be the case that subjects enrolling in the cohort in more recent years are substantially different from the earlier participants. However, since our sampling framework has not changed and neither has changed our invitation protocol, we do not believe this to be an explanation for this finding. Analyses of US data from 1983 to 2003 suggest that there has been a decrease in the overall percent of drinking and driving among motor vehicle drivers and motorcyclist . This reAs with other public health problems, an array of interventions to reduce the prevalence of alcohol amongst road users has been set in practice around the world, including legislation prohibiting driving at or over certain levels of alcohol in blood, or the recommendation that advice on this matter is provided in the context of routine general medical visits . Most ofOur findings are amongst the first on the high prevalence of drinking and driving among Spanish. Particularly worrisome is the fact that health professionals reported this habit even at higher rates. Multidisciplinary interventions  are needed to reduce this serious health risk.The author(s) declare that they have no competing interests.MSG conducted some of the analysis, drafted the first manuscript and incorporated all comments and suggestions. SP collaborated in the literature review and the interpretation of results, FG and JI made substantial comments to the earlier analyses findings and manuscript drafts, and so did MMG, who also was involved in the statistical analysis.All authors read and approved the final manuscript.not been published elsewhere and does not overlap or duplicate their published work.All authors confirm that the content has The pre-publication history for this paper can be accessed here:


Deregulated cell cycle progression and loss of proliferation control are key properties of malignant cells. In previous studies, an interactive transcript abundance index (ITAI) comprising three cell cycle control genes, [MYC × E2F1]/p21 accurately distinguished normal from malignant bronchial epithelial cells (BEC), using a cut-off threshold of 7,000. This cut-off is represented by a line with a slope of 7,000 on a bivariate plot of p21 versus [MYC × E2F1], with malignant BEC above the line and normal BEC below the line. This study was an effort to better quantify, at the transcript abundance level, the difference between normal and malignant BEC. The hypothesis was tested that experimental elevation of p21 in a malignant BEC line would decrease the value of the [MYC × E2F1]/p21 ITAI to a level below this line, resulting in loss of immortality and limited cell population doubling capacity. In order to test the hypothesis, a p21 expression vector was transfected into the A549 human bronchogenic carcinoma cell line, which has low constitutive p21 TA expression relative to normal BEC.Following transfection of p21, four A549/p21 clones with stable two-fold up-regulated p21 expression were isolated and expanded. For each clone, the increase in p21 transcript abundance (TA) was associated with increased total p21 protein level, more than 5-fold reduction in E2F1 TA, and 10-fold reduction in the [MYC × E2F1]/p21 ITAI to a value below the cut-off threshold. These changes in regulation of cell cycle control genes were associated with restoration of cell proliferation control. Specifically, each transfectant was capable of only 15 population doublings compared with unlimited population doublings for parental A549. This change was associated with an approximate 2-fold increase in population doubling time to 38.4 hours (from 22.3 hrs), resumption of contact-inhibition, and reduced dividing cell fraction as measured by flow cytometric DNA analysis.These results, likely due to increased p21-mediated down-regulation of E2F1 TA at the G1/S phase transition, are consistent with our hypothesis. Specifically, they provide experimental confirmation that a line with slope of 7,000 on the p21 versus [MYC × E2F1] bivariate plot quantifies the difference between normal and malignant BEC at the level of transcript abundance. Bronchogenic carcinoma (BC) is the leading cause of cancer-related death in the United States and most industrialized nations . It is rIn an effort to identify biomarkers that diagnose BC with more accuracy than cytomorphologic criteria, this laboratory developed and has employed a highly quantitative, quality controlled RT-PCR method, Standardized RT (StaRT)-PCR -4. With In previous studies of the association between the [MYC × E2F1]/p21 ITAI and malignancy, it was observed that there is substantial variation in transcriptional regulation of p21 among BC tissues . In someWAF1/CIP1 gene is a key regulator of cell cycling /p21 ITAI accurately distinguishes normal from malignant lung samples. The hypothesis was tested that this empirically derived cut-off is mechanistically determined and is a mathematical representation of the difference between normal BEC and malignant BEC. First, the malignancy ITAI genes MYC, E2F1, and p21 were measured in an additional set of primary normal and malignant lung samples to more firmly establish the threshold separating them. Then, experimental studies were conducted on the human BC line, A549. This line was chosen because it has down-regulated p21 expression, in part due to p14ARF deletions , resultiPreviously, we reported that a cut-off threshold of 7,000 for the MYC × E2F1]/p21 ITAI accurately distinguishes normal from malignant BEC . An addi/p21 ITAIBecause the MYC × E2F1]/p21 ITAI is a ratio between two values, the cut-off is best represented as a line on a bivariate plot. On a plot of all normal or malignant samples the ITAI threshold is represented as a line with a slope of 7,000 that passes through the origin  was significantly (p < 0.05) increased two-fold in each of the A549/p21 clones compared to the parental A549 Figure . StaRT-PExpression of Hexa-His-tagged p21 was observed in each clone (lanes 2–5) but not in the parental A549 (lane 1). A higher molecular weight protein(s) was observed in the lysates from the clones suggesting a non-specific antigen/antibody interaction. This increase in total p21 expression was associated with a greater than 5-fold significant (p < 0.05) decrease in E2F1 TA in each clone Figure . Further5, 5 × 105, and 1 × 106 cells) of the A549/p21 clonal populations, the cells ceased to proliferate and viability decreased at 80% confluency further suggesting p21-mediated contact inhibition at high saturation densities (data not shown). While the differences in proliferation rates were significant between parental A549 and the A549/p21 clones, there were no significant differences among any of the individual clones. Each of the A549/p21 clones was capable of approximately 15 total doubling times based on the estimated number of cells in the established initial foci (≈1 × 102 cells) compared to the estimated number of cells in the seventh passage, which was the last passage that they were capable of (data not shown). These passage limitations are similar to the passage limitations of cultured normal BEC  enables quantitative experimental investigation of the relationship between MYC, E2F1, and p21 individual transcript abundance values and the ITAI cut-off value that distinguishes normal from malignant BEC. For example, a malignant BEC line with a low [MYC × E2F1] value relative to most BC samples may be malignant if the p21 value also is relatively low. This was the case with parental A549. Although the A549 [MYC × E2F1] value is lower than in most malignant tissues  supports these two selection processes. Each successfully isolated A549/p21 clone had total p21 expression that was elevated relative to parental A549, but was low enough to allow 15 doublings, which was sufficient for the studies reported here.The absence of inducible clones could have been due to a number of factors, including promoter degradation, deleterious vector integration, and/or sub-optimal selection conditions affecting either the tetracycline repressor vector or the p21 expression vector.In each A549/p21 clone we observed 1) a two-fold increase in p21, accompanied by 2) a 5-fold decrease in E2F1 TA and 3) that a 10-fold reduction in the ITAI was sufficient for each A549/p21 clone to cross the cut-off threshold of 7,000. This was associated with marked reduction in cell cycle progression and cell proliferation in the A549/p21 clones.p21 exerts its inhibitory function in either late G1 or late G2 of the cell cycle ,27. ConsMYC TA level was not decreased considerably in the A549/p21 stable clones compared to the parental A549 in the context of a five-fold decrease in E2F1 TA level. This is in contrast to reports that in some contexts E2F1 is a transcription factor for MYC  and thatin vitro and in vivo /p21 ITAI distinguishes between malignant and non-malignant phenotypes. This will guide development of more accurate diagnostic tests and more effective gene-targeted therapeutics.The parental A549 cell line was cultured in RPMI 1640 with 10% fetal bovine serum . The A549/p21 clones 1, 29, 30, and 32 were continuously maintained in RPMI and 10% Tet-system approved FBS  with 450 μg/mL Geneticin (G418 sulfate)  unless otherwise noted.Culture conditions for normal and malignant BEC were described previously . Normal Sixty-five primary normal BEC samples and thirty-one primary bronchogenic carcinoma samples  were obt5'CACCATGTCAGAACCGGCTGG3' and reverse primer 5'TCAATGGTGATGGTGATGAT3') were suitable for directional Topoisomerase (TOPO) cloning  into pENTR/D-TOPO. To amplify the p21 complete cDNA from the pcDNA3.1/GS, the PCR conditions were 94°C/15 sec., 58°C/30 sec., and 72°C/60 sec. for 35 cycles (slope = 9.9) in an air thermocycler . Each of five 10 μL PCR reaction mixtures contained 2 mM dNTPs, 10×, 30 mM MgCl2, template (pcDNA3.1/GSp21), forward and reverse primers (0.1 μg/μL), Platinum Pfx DNA Polymerase , and RNase-free water. After amplification, the five reactions were combined and electrophoresed in a 2% (NuSieve/SeaKem) agarose gel containing ethidium bromide. The PCR product was visualized under UV and extracted following the Qiagen gel extraction protocol . The TOPO reaction was performed according to manufacturer's instructions. The pENTR/D-TOPO-p21 vector was isolated, quantified, and sequenced to confirm integrity and orientation. Next, the Gateway™  LR site-specific recombination system was used according to manufacturer's instructions to recombine the p21 ORF from the entry vector (pENTR/D-TOPO-p21) with the destination vector (pT-REx-DEST30), which contains two tetracycline operon (TetO2) sites within the human CMV promoter for tetracycline-regulated expression of the inserted gene.The GeneStorm hORF Expression Vector (pcDNA3.1/GS), containing the complete cDNA sequence of the cyclin-dependent kinase inhibitor 1A  (Accession L25610) was obtained from Invitrogen, Inc., ; The Gateway cloning system  was used to create the p21 expression vector. Primers were designed to amplify the full-length p21 cDNA insert  in 500 μL of RPMI and 10% Tet-system approved FBS  without antibiotics. Twenty-four hours later, A549 cells were transfected with the pcDNA/TR vector  that expresses the Tet-repressor. For each well of cells transfected, 1 μg of vector DNA (pcDNA/TR) was diluted into 50 μL of Opti-MEM I Reduced Serum Medium . Two microliters of Lipofectamine 2000 (LF2000)  was diluted into 50 μL of Opti-MEM I Medium  and subsequent transfection was performed according to LF2000 manufacturer's instructions. At 24 hours post-transfection, the cells from each well were trypsin-dissociated and inoculated into corresponding 10 cm plates containing fresh growth medium, RPMI and 10% Tet-system approved FBS  without antibiotics. The next day, selective medium was added containing blasticidin S-HCl  at the A549 minimum 100% lethal dose (LD100) of 7 μg/mL. Within one week, individual foci developed, and each of these were individually dissociated, transferred to a single well in a 24-well dish and cultured in selection medium. The pcDNA/TR-A549 clonal populations were expanded and then sequentially transfected with the pT-REx-DEST30-p21 response vector by means described above but using the A549 minimal LD100 for Geneticin (G418 sulfate)  at 450 μg/mL as the selection antibiotic pressure. The pcDNA/TR/p21-A549 transfectants were maintained under dual blasticidin S-HCl and Geneticin selection. Lowest basal and the highest tetracycline-induced p21 TA level after addition of 1 μg/mL of doxycycline was assessed in passage 4 cells from each clone, with passage 1 being the passage of the initial double antibiotic resistant clones.A549 cells were trypsin-dissociated, and each well of a 24-well dish was inoculated with 10The initial screen for inducible exogenous p21 expression was done on cells from passage 4 of the A549/p21 clones. All mRNA, protein, flow cytometric, and growth kinetic assessments were performed at passage 5 and compared to parental A549 with an equivalent number of passages post-transfection.For cultured or primary cells, pelleted cells were dissolved in TriReagent  and total RNA was extracted according to manufacturer's instructions and previously reported methods . Then, a5'GTGATGGTGATGATGACCG3' while the forward was 5'ACCCTTGTGCCTCGCTCAG3'). In order to measure total p21 exogenous plus endogenous TA level, forward and reverse primers 5'GCCTGCCCAAGCTCTACCT3' and 5'GAGAAGATCAGCCGGCGTT3', respectively, were designed that hybridize in the coding region of p21 and not the 3' Hexa-His-tag sequence. Previously reported data for TA measurement of MYC, E2F1, and p21 in cultured normal BEC, all malignant BEC except H146  and total protein concentration was determined colorimetrically by the bicinchoninic acid (BCA) assay . Samples were solubilized in NuPAGE LDS Sample buffer  and 20 μg was loaded on a denaturing 7% NuPAGE Tris-Acetate gel . Protein bands were transferred to Invitrolon PVDF membranes  as described by the manufacturer. Then, the membranes were blocked with 5% non-fat dry milk in 1× TBST (blocking buffer) for 1 hr at room temperature. For determination of exogenous p21 protein expression, blots were incubated overnight with Anti-His Tag Mab  (1:2000) recognizing the Hexa-Histidine fusion tag of p21 transactivated from the expression vector contained in the stable A549/p21 clones. Blots were then washed with 1× TBST and incubated with the secondary antibody, HRP-conjugated anti-mouse IgG  (1:5000) for 1 hr at 37°C. Blots were washed three times with 1× TBST and once with 1× TBS. Detection was performed by the commercially available ECL detection system . For determination of total p21 protein expression (exogenous plus endogenous), blots were incubated with Anti-Waf1 (Ab-1) Mab  (1:500). HRP-conjugated goat anti-mouse IgG  (1:2000) was used as the secondary antibody. Methodology was the same as described above. The Hexa-Histidine tag that flanks the ORF of p21 was necessary for determining expression of p21, both mRNA and protein, that was exogenously synthesized. To date, there is no evidence suggesting deleterious effects of Hexa-Histidine tags on the function of p21. Further, because the relative expression of His-tagged p21 was low in the A549/p21 clones, unexpected alterations in expression profiles and phenotype are less likely.6 cells/T75 flask). To calculate the number of population doublings at each passage, the log2 of the number of cells inoculated was subtracted from the log2 of the cell count for the cells harvested. This information was used to determine the number of population doublings for each clone.Sub-cultures of the clonal populations were maintained and periodically sub-cultured for determination of maximum number of passages at optimal confluency . The stable clones were maintained with RPMI and 10% FBS containing 450 μg/mL G418. In order to rule out potential effects of the antibiotic on cell proliferation, the experiment was repeated in antibiotic-free medium. Both experiments were repeated in triplicate and the doubling time was calculated by plotting the graph of cell count versus time using the third degree polynomial.Cell doubling time was determined by counting cells from trypsinized monolayers. Viability was assessed based on trypan-blue  exclusion. 30 mm Petri dishes containing 2 × 105, 5 × 105, and 1 × 106 cells) were inoculated per T75 flask for determination of percent confluency at which cells ceased dividing.For further demonstration of growth characteristics of the A549/p21 clonal populations, variable numbers of cells  using SPSS 11.5.1 for Windows . Determination of mean TA and cell number, standard deviation of TA and cell number, cell doubling time, and creation of graphs were accomplished using Excel 2000 .ELC and JCW each have significant equity interest in Gene Express, Inc., which produces and markets StaRT-PCR reagents used in these studies.TGG conceived and designed experiments, and was responsible for TA measurement in cultured samples, construction and cloning of p21 expression vector, stable transfection of vector and construction of A549/p21 clones, preparation of the exogenous and endogenous and exogenous p21 SMIS, TA measurement in A549 and A549/p21 clones, Western blot analysis, cell proliferation characteristic analyses, prepared cells for flow cytometric DNA analysis, statistical analysis, and was the primary author of this manuscript. MWH was responsible for TA measurement in cultured samples and critical review of the manuscript. ELC was responsible for TA measurement in primary samples and critical review of the manuscript. JCW coordinated and obtained funding for the study and revised the manuscript.TA, ITAI values and descriptions of all samplesClick here for file


Restoration of p21 expression was accomplished using adenoviral infection. As anticipated from the loss of a cell cycle inhibitor, p21(-/-) FLS grow more rapidly than WT FLS. In examining migration towards biologically relevant RA synovial fluid, p21(-/-) FLS display a marked increase  in migration compared to WT cells. Moreover, this effect is independent of the cell cycle since chemical inhibitors that block the cell cycle have no effect on migration. In contrast, p21 is required to repress migration as restoration of p21 expression in p21(-/-) FLS reverses this effect. Taken together, these data suggest that p21 plays a novel role in normal FLS, namely to repress migration. Loss of p21 expression that occurs in RA FLS may contribute to excessive invasion and subsequent joint destruction.Rheumatoid arthritis (RA) is characterized by synovial hyperplasia and destruction of cartilage and bone. The fibroblast-like synoviocyte (FLS) population is central to the development of pannus by migrating into cartilage and bone. We demonstrated previously that expression of the cell cycle inhibitor p21 is significantly reduced in RA synovial lining, particularly in the FLS. The aim of this study was to determine whether reduced expression of p21 in FLS could alter the migratory behavior of these cells. FLS were isolated from mice deficient in p21 (p21 Proper regulation of the mammalian cell cycle is vital for cellular homeostasis. Alterations in the cell cycle components have been associated with several disease states. Progression through the different phases of the cell cycle is dependent on the activities of cyclin dependent kinases (cdks) bound to their cognate cyclins ,2. AnothRheumatoid arthritis (RA) is a chronic inflammatory and destructive disease . The fib(-/-)  and wild-type  mice were purchased from the Jackson Laboratory . Mouse knees were excised from WT or p21(-/-) mice and pooled. Isolated mouse synovial tissues were digested with collagenase, dispase, and DNAse I, and single cell suspensions were obtained [p21obtained ,25,26. ASynovial fluid (SF) specimens were collected during arthrocentesis from patients who met the American College of Rheumatology criteria for a diagnosis of RA. All specimens were obtained with approval of Midwestern University's Institutional Review Board.(-/-) fibroblasts were plated at 1.8 × 104 cells/well in 1 ml DMEM + 10% FBS in a standard 24-well tissue culture plate and incubated at 37°C. At different time points cells were washed with PBS, trypsinized and their number quantified by hemocytometer counts with trypan blue. Analysis was performed in triplicate wells and results are expressed as the mean ± standard error (SE).WT and p21(-/-) FLS were cultured for 18 h in DMEM containing 1% FBS. One hour prior to the assay, this media was removed, cells were rinsed twice with PBS, and media was replaced with DMEM containing 0.1% FBS. FLS (2.6 × 104 cells/well in DMEM + 0.1% FBS) with or without mitomycin C  were added to the bottom wells of a 48-well microchemotaxis chamber . The chambers were inverted and incubated for 2 h (3 h when MMC was present) in a 5% CO2 atmosphere at 37°C, allowing for cell attachment to the membrane. Upon righting the chambers, dilutions of synovial fluids collected from patients with RA (or control agents) were added to the top wells and the chambers were incubated overnight at 37°C. PBS served as a negative control, whereas recombinant human basic fibroblast growth factor  served as a positive control. The next morning, non-migrated cells were detached with a cotton swab, membranes were removed, fixed in methanol, and stained with Diff-Quik . Checkerboard analysis was performed in a similar manner, except that the concentrations of RA SF were varied in the upper and lower chambers. Dilutions of RA SFs  were added to the cell suspension in the bottom wells as well as on the opposite side of the membrane, when appropriate. Each condition was analyzed in quadruplicate and migrated cells from membranes mounted on glass slides were quantified in three representative high power fields. Quantification of high powered fields was accomplished by analyzing photographs of chemotaxis spots taken with a Nikon Coolpix E5000 (5.0 megapixel) camera mounted on a Nikon Eclipse TS100 inverted microscope. Chemotaxis data appeared normally distributed based on examination of histogram plots, and statistical analysis was performed using a Student's t-test.Chemotaxis was performed with minor modifications to a protocol previously optimized for RA synoviocyte chemotaxis . WT and (-/-) FLS were treated with identical conditions as used for chemotaxis assays described above. Cells were trypsinized, counted using trypan blue, plated at 2.6 × 104 cells/well into two separate chemotaxis chambers, and inverted to allow cell adherence to the membrane. FLS from one chamber were fixed with MeOH exactly 2 h after plating, allowing sufficient time for cell attachment but not enough time for proliferation. Counts from this chamber allowed an exact determination of the number of cells plated for each group. WT and p21(-/-) FLS in the second chamber were plated with or without 10 μg/ml MMC. After 2 h, the second chamber was righted and PBS was added to the top side of all wells. After an 18 h time period, the time allowed for migration in all chemotaxis experiments, the membrane was removed and the WT and p21(-/-) FLS were fixed with MeOH and counted.To assure that local proliferation on the membrane was not occurring, WT and p21(-/-) FLS were infected with an adenovirus overexpressing p21 (Adp21) and compared to mock infected WT FLS, mock infected p21(-/-) FLS or p21(-/-) FLS infected with an adenovirus that produces an irrelevant bacterial protein . Initially, FLS infected with Adp21 and AdlacZ were subjected to β-galactosidase staining to estimate a concentration that resulted in infection of nearly 100% of cells . All infections were completed by incubation with virus for 4 h in DMEM + 5% FBS. Mock infected WT and p21(-/-) fibroblasts were incubated with the same media in the absence of virus. After incubation, cells were washed three times with PBS and full growth media was replaced for 4 h. Cells were incubated overnight in a 1:10 dilution of the full growth media. Chemotaxis assays were performed as described above the following day.To restore p21 expression, p21(-/-) mice. We first assessed whether FLS from p21(-/-) mice possessed proliferative characteristics, as was expected from the loss of a cell cycle inhibitor. Figure (-/-) FLS were plated into 24-well plates. At various time points, cell number was determined and by 44 h after plating the cells, more p21(-/-) FLS were consistently present relative to WT FLS  (data not shown) FLS migrate to RA SF in a dose-dependent manner. Even RA SF dilutions of 1:1000 from some patients could significantly increase FLS migration when compared to background migration represented by PBS (p < 0.05).Little is known about whether the reduced p21 expression exhibited in RA FLS may be related to alterations in FLS migration. To address this issue, we isolated FLS from knee synovium of WT and p21S Figure  and p21((-/-) FLS in response to RA SF, we employed 48-well microchemotaxis chambers and determined cell motility towards dilute RA SFs. Cell counts were normalized to their background migration by representing the data as fold-increase over PBS. Figure (-/-) FLS when compared with WT FLS (p < 0.05). A representative chemotaxis experiment is shown in Figure (-/-) FLS. The data shown are representative of three independent experiments, which consistently suggest that p21(-/-) FLS exhibit significantly enhanced migration towards RA SFs (p < 0.05).To directly compare migration of WT and p21(-/-) FLS may simply be the result of a non-specific enhancement of chemokinesis, we performed several checkerboard assays. A representative assay (Table To examine whether the increased migration of p21(-/-) FLS in the presence and absence of MMC at the end of the assay and compared it with the number of cells plated at the start of the assay. Figure (-/-) FLS treated with MMC was significantly below the number of p21(-/-) FLS that were not MMC treated (p < 0.05). In contrast, there were no significant differences between WT and p21(-/-) FLS that were or were not MMC treated. This suggests that MMC completely halted proliferation and that any demonstrable changes in chemotaxis in the presence of MMC are not due to proliferation on the membrane.To examine whether the enhanced migration could be explained by differences in proliferation, we performed chemotaxis assays using cells that were pretreated with MMC to stop cell division. To assure that local proliferation on the membrane was not occurring in the presence of MMC, we determined the number of WT and p21(-/-) and WT FLS chemotaxis assays using seven separate RA SFs in nine independent experiments, there is significantly more migration by the p21(-/-) FLS compared with WT FLS (p < 0.05). Figure (-/-) FLS. It should be noted that the counts in chemotaxis assays using mouse FLS isolated on different dates occasionally varied greatly between experiments (background migration as well as induced migration), although the trend was always identical, such that the p21(-/-) FLS migrated significantly more than the WT FLS. Therefore, in the presence of MMC, a cell division inhibitor, we still demonstrate that loss of p21 confers an increased migratory behavior on FLS when compared with WT cells (p < 0.05). Of note in Figure (-/-) FLS.Figure (-/-) and WT fibroblasts. Therefore, the enhanced migration may be specific for another chemoattractant, or a combination of chemoattractants found in the RA SF.To begin to assess whether the enhanced migration may exhibit specificity with regards to the chemoattractant used, we performed several chemotaxis assays using bFGF as the chemoattractant. Figure (-/-) FLS with p21 would reduce their excessive migration. First, to demonstrate that our adenoviral construct produced functional p21, we infected p21(-/-) FLS and compared the growth rate with that of p21(-/-) FLS infected with an adenovirus producing green fluorescent protein. Growth of FLS expressing p21 was significantly inhibited compared with FLS expressing green fluorescent protein . Next, to establish whether p21 is solely responsible for the enhanced migratory characteristics, we reconstituted p21(-/-) FLS with p21 and compared chemotaxis to cells infected with AdlacZ. Figure (-/-) FLS infected with Adp21 was comparable to WT FLS that were mock-infected. This migration was significantly below mock-infected p21(-/-) FLS or those infected with an adenovirus producing an irrelevant bacterial protein, β-galactosidase.We next aimed to use an adenovirus to increase p21 and establish whether reconstituting p21(-/-) mice. We first assessed whether FLS from p21(-/-) mice possessed proliferative characteristics, as was expected from the loss of a cell cycle inhibitor. Figure Expression of p21 in RA synovial tissue is significantly decreased when compared with the same tissue from osteoarthritis patients. Expression of p21 inversely correlates with thickness of the RA synovial lining . Similar(-/-) FLS migration to RA SF suggests that p21(-/-) FLS exhibit significantly enhanced migration (p < 0.05). Our checkerboard assays (Table (-/-) FLS proliferate quicker than WT FLS, the non-MMC treated FLS in Figure (-/-) mice migrated more than those from WT mice. Overall, chemotaxis assays performed in the presence of a cell cycle inhibitor suggest that the changes noted are not the result of enhanced proliferation by migrating cells. Moreover, reconstitution of p21 into p21(-/-) FLS is sufficient to significantly reduce migration of these cells, suggesting that the loss of p21 in FLS may enhance their ability to migrate towards the proinflammatory constituents of the RA joint.A direct comparison of WT and p21(-/-) embryonic fibroblasts were also examined in this study, but were determined to not exhibit differences in migration when compared with WT cells [(-/-) fibroblasts that were isolated from synovium obtained from 5–8 week old mice, whereas the previous study employed fibroblasts derived from an embryo. The previous study examined cell migration by wounding of a confluent monolayer of cells, whereas our study examined specific directed chemotaxis and chemokinesis. An additional key difference is that our study used RA SF as the chemoattractant to assess whether the multiple biological constituents of these disease-related fluids may display differences in chemoattraction between p21(-/-) and WT fibroblasts. In fact, when we utilized bFGF as a sole chemoattractant in seven independent chemotaxis assays, we do not see consistent differences between migration of p21(-/-) and WT fibroblasts, which is in-line with the previously published findings where p21(-/-) assays were performed in the presence of growth serum [Recently, Besson and coworkers  demonstrWT cells . There ath serum .(-/-) VSMCs [(-/-) cells with exogenous p21, transduction of Gax cDNA once again inhibits VSMC migration. Thus, increasing Gax upregulates p21 and inhibits VSMC migration. This appears consistent with our studies, in which a loss of p21 in FLS results in excessive migration and reconstituting p21 is capable of reducing the exuberant migration to RA SF. Overexpressing p21 alone in WT VSMCs did not influence cell migration. In our hands, similarly, infecting WT FLS with a high titer of Adp21 did not inhibit cell migration to RA SF (data not shown).Previous studies in vascular smooth muscle cells (VSMCs) involving the homeobox transcription factor growth-arrest specific homeobox (Gax) have also established a tie between p21 and cell migration . Overexp-) VSMCs . Upon rein vitro has recently been associated with the rate of joint destruction in vivo [FLS locomotion can be regarded as an important pathogenic mechanism contributing to the invasion of cartilage and bone in RA. Grafting of RA FLS alone to SCID mice, in the absence of a functional immune system, results in chronic arthritis, underscoring the potential key role of these cells in driving disease ,33. This in vivo . Invasiv in vivo , and the in vivo . We demoWe previously demonstrated that RA FLS exhibit decreased expression of p21, and now observe that a lack of p21 may contribute to excessive migration of FLS. These data suggest that p21 plays a novel role in normal FLS in repression of migration. Further, a lack of p21 expression, as occurs in RA FLS, may contribute to excessive invasion towards the biological chemoattractants found in RA SF.AdlacZ = adenovirus expressing β-galactosidase; Adp21 = adenovirus expressing p21; bFGF = basic fibroblast growth factor; cdk = cyclin-dependent kinase; DMEM = Dulbecco's modified Eagle's medium; FBS = fetal bovine serum; FLS = fibroblast-like synoviocytes; Gax = growth-arrest specific homeobox; MMC = mitomycin C; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; SF = synovial fluid; VSMC = vascular smooth muscle cell; WT = wild-type.The authors declare that they have no competing interests.JMW designed and developed all aspects of the study, performed chemotaxis experiments, drafted the manuscript, and addressed reviewers concerns. KK, DJS, NGS, and MSR each contributed a significant number of chemotaxis assays, worked with adenoviruses, and participated in growth and maintenance of FLS. DJP performed the majority of chemotaxis counting, while MVV participated in chemotaxis counting as well as in the design of experiments and interpretation of data. JCS performed all animal work and isolation of FLS cultures from mice. HP conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


In recent years, online services for problem drinkers have been developed. This paper describes ongoing efforts to improve one of these services, the Alcohol Help Center.This report summarizes new modules added to the Check Your Drinking (CYD) screener, a component of the Alcohol Help Center, to make the CYD screener more useful to periodic heavy drinkers, as well as to regular alcohol consumers. Participants’ initial reactions to the CYD screener and the changes in their drinking habits at a three-month follow-up are presented. The CYD screener provides a free personalized Final Report that compares the user’s drinking to that of others in the general population of the same age, gender, and country of origin. Current alcohol consumption and demographic characteristics are collected as part of the CYD screening process. After users were presented with a customized Final Report, they were hot-linked to a volunteer feedback survey. The voluntary feedback survey asked about impressions of the CYD Final Report. Respondents agreeing to participate were sent a follow-up survey after three months.21 = 41.5, P < .001) and accurate . Respondents who participated in the three-month follow-up displayed reductions in drinking compared to baseline . We recruited 388 volunteers  who were registered users of another free-to-consumer online eHealth service. Of the 343 respondents agreeing to participate in the three-month follow-up, 138 accessed the survey, and 97 provided complete data . Compared to moderate drinkers, current problem drinkers judged the Final Report to be more useful (34% vs. 69%, χ Improvements can still be made to make the CYD screener more relevant to specific populations, particularly periodic heavy drinkers. There is a need to further tailor algorithms that can present questions only relevant to specific populations. There also appears to be a need to further customize the Final Report for respondents who identify themselves as infrequent heavy drinkers. These improvements will be made, and a randomized controlled trial is planned to conduct a rigorous evaluation of the CYD screener as an intervention to help problem drinkers. Hazardous alcohol consumption has been identified as one of the five leading contributors to the global burden of disease, and it results in enormous economic costs -7. Yet vThere have been a number of reports of online services for problem drinkers . ManIn order to provide a preliminary outcome evaluation of the CYD screener, a three-month follow-up survey was also conducted. Two hypotheses were tested in this outcome evaluation. Hypothesis one predicted that respondents would be drinking less at three months’ follow-up as compared to baseline. Second, previous research has indicated that respondents’ perceived risk might be an important incentive to adopt health protective behaviors . Thus, Recruitment for this pilot study was conducted by an email invitation sent to registered users of a separate free-to-consumer website program, the Stop Smoking Center . A standThe study was approved by the standing ethics committee of the Centre for Addiction and Mental Health. The email invitation described the purpose of the survey, how long it would take (about 10 minutes), and that the use and storage of the data would ensure anonymity. Responding to the email invitation was taken as informed consent. The design of the survey followed international guidelines set forth to protect privacy ,26. The The same survey methods were employed for the three-month follow-up survey as for the baseline survey. A full copy of the follow-up survey is available in Appendix 2. First, respondents were asked about their current smoking status, whether they currently drank alcohol, and to what extent they currently perceived themselves to be at risk of getting hurt or sick because of their drinking . Respondents who were current drinkers were then asked the same items from the CYD screener (see below), this time with respect to their drinking in the last three months. Results from the follow-up survey were linked to the baseline survey using respondents’ unique user ID number.Univariate comparisons were made of the baseline survey results, comparing respondents who did or did not complete the voluntary feedback survey at baseline and also comparing problem and nonproblem drinkers. A repeated-measures multivariate analysis of variance was employed to test hypotheses one and two. Differences in drinking from baseline to follow-up were compared for respondents who did or did not report reductions in their perceived risk associated with drinking between the baseline and follow-up time points.The CYD screener is available for public access . The surThe Final Report begins with a summary pie chart that compares the respondent’s drinking in a typical week to that of others of the same age group (six different age groups), gender, and country of origin. Recent population comparison data are currently available for Canada and the United States; UK data have been added since this study and data from other countries will be added at a later date -36. The The Final Report then continues with two drinking feedback graphs—a bar chart comparing the respondent’s drinking on each day of the week to that of others of the same age group and gender (data only available from Canada), and a pie chart comparing the frequency of heavy-drinking days (five or more drinks on one occasion) to that of others of the same age, gender, and country of origin. This last graph, in particular, was added with the specific intent to provide useful feedback to infrequent drinkers. Respondents who drink five or more drinks on one occasion once per month or more are alerted to the increased risks associated with this type of consumption . A list Email invitations were sent out to 7741 registered users of the Stop Smoking Center who registered between October 27, 2004 and July 27, 2005 and had active email accounts. Of these potential participants, 1085 recipients hot-linked to the survey using the unique link provided in each email (participation rate = 14%). Of these, 973 started the baseline survey; 9 respondents were removed because they said they were taking the test for someone else; 231 were removed because they identified themselves as nondrinkers, and 1 respondent did not complete the CYD survey, resulting in a final sample size of 732. Of these 732 respondents, 388 (53%) completed the voluntary feedback survey to give their impressions of the Final Report (completion rate = 40%). t = 3.2, P = .001) and more likely to be male  compared to current moderate drinkers. Problem drinkers were also more likely than moderate drinkers to live outside the United States or Canada . Further inspection of the country of origin revealed that 48% of the respondents who lived outside of the United States or Canada lived in the United Kingdom. As expected, problem drinkers consumed more alcohol in a typical week  and experienced more drinking consequences  compared to moderate drinkers. Problem drinkers also rated themselves as significantly more likely to get hurt or sick because of their drinking compared to moderate drinkers .21 = 41.5, < .001), to be surprised by how much more they drank than others , and to feel that the summary accurately outlined and captured their drinking . Because one of the main intents of updating the screener was to provide useful information for infrequent drinkers, two further analyses were conducted comparing problem drinkers who were frequent or infrequent drinkers. Compared to problem drinkers who drank more than once a week (n = 103), those who drank weekly or less (n = 27) appeared just as likely to find the Final Report somewhat or extremely useful . In addition, problem drinkers who consumed five or more drinks (on one occasion) once a month or more (n = 111) were just as likely to find the Final Report somewhat or extremely useful as those problem drinkers who consumed five or more drinks less than once a month . There was some difference in the proportion of frequent (more than weekly) and infrequent (weekly or less) problem drinkers who thought the feedback accurately depicted their drinking . However, there was no difference between frequent heavy drinkers (five or more drinks monthly or more) and infrequent heavy drinkers on how accurate they felt the Final Report to be .There were also a number of significant differences regarding impressions of the Final Report between problem and moderate drinkers see . Problem21 = 6.6, P = .01) and the days of the week drinking bar chart useful . Few problem or moderate drinkers found the frequency of heavy-drinking days pie chart useful . One potential difficulty in interpreting respondents’ ratings was that the feedback charts were generated with population data from Canada or the United States, so they would be less relevant to respondents from other countries. Analyses were conducted to explore the proportions of Canadians and Americans who endorsed each chart and were marginally higher than those reported by the full sample (not shown).Respondents were asked if they found the three drinking feedback charts useful see . Compare4,76 = 12.2, P = .001) and of reduction in perceived risk . In addition, there was a significant interaction between time and perceived risk . Subsequent univariate analyses exploring this interaction revealed significant interactions for the variables: number of drinks in a typical week , greatest amount drunk on one occasion , number of drinking-related consequences , and AUDIT scores . Inspection of the observed means for these variables revealed that respondents who had a reduction in their perceived risk from baseline to follow-up also had reductions in their drinking from baseline to follow-up. Respondents with no reduction or an increase in their perceived risk displayed little or no reductions in their drinking from baseline to follow-up.Of the 343 respondents who agreed to participate in the three-month follow-up survey, 138 accessed the survey and attempted to provide responses (participation rate = 40%). Responses from 41 participants could not be used because the unique respondent ID number was not associated with the participants’ data.  This left 97 participants who provided complete follow-up data (completion rate = 70%). Finally, 16 of these respondents did not complete the baseline voluntary feedback survey and, as such, had not provided an assessment of perceived risk at baseline, leaving 81 respondents with complete data to test hypothesis two. A repeated-measures multivariate analysis of variance (MANOVA) was conducted to test both hypotheses one and two simultaneously. The two independent variables were time (baseline versus three-month follow-up) and change in perception of risk (reduction in perception of risk from baseline to follow-up versus no reduction or increase in perceived risk). Four drinking variables were included as dependent variables: number of drinks in a typical week, greatest amount drunk on one occasion, number of drinking related consequences, and AUDIT score. Baseline and follow-up values for these dependent variables are displayed in Problem drinkers were more likely to find the Final Report useful, surprising, and accurate than moderate drinkers. As the primary target of this website is current problem drinkers, it was intended that the Final Report should be found most useful to this group . Of the feedback elements in the Final Report, both the original typical week pie chart and the days of the week bar chart were endorsed by about a third of respondents as being useful. The frequency of five or more drinking days pie chart was not often endorsed as being useful. It was discouraging to see how few respondents found the frequency of five or more drinks pie chart useful. This element of the CYD screener was added specifically to make the Final Report more relevant to infrequent heavy drinkers. Some qualitative responses from participants also highlighted that the Final Report was considered inaccurate by irregular drinkers.As with an evaluation of the earlier version of the CYD , a signiThere was a significant reduction in drinking measures from baseline to follow-up. While this finding supports hypothesis one, it should be stressed that this finding does not confirm that the reduction in drinking was due to use of the CYD screener because there was no control group in this study. In addition, reductions in estimates of perceived risk from baseline to follow-up were associated with reduction in drinking. This finding provides support for the importance of perceived risk as a potential mediator of the impact of self-help interventions such as the CYD screener. However, as with the preliminary support that the CYD may lead to reductions in drinking, a proper randomized controlled trial is needed in order to confirm this hypothesis .Not all respondents filled out the voluntary feedback survey, suggesting that caution should be taken regarding the generalizability of the results. It should, however, be noted that there were no systematic differences between completers and noncompleters on the variables we measured. In addition, there was a substantial attrition of respondents from baseline to follow-up, again leading to cautions regarding the validity of the results ,43. FinaAn upgraded version of the CYD screener will include a modified assessment algorithm and Final Report for participants with irregular drinking patterns rather than using the same assessment and Final Report for all users. Finally, a randomized controlled trial is underway to establish whether participation in the CYD screener will result in sustained reductions in alcohol consumption.


Excessive use of the Internet has been associated with attention deficit hyperactivity disorder (ADHD), but the relationship between video games and ADHD symptoms in adolescents is unknown.A survey of adolescents and parents  was performed assessing daily time spent on the Internet, television, console video games, and Internet video games, and their association with academic and social functioning. Subjects were high school students in the ninth and tenth grade. Students were administered a modified Young's Internet Addiction Scale (YIAS) and asked questions about exercise, grades, work, and school detentions. Parents were asked to complete the Conners' Parent Rating Scale (CPRS) and answer questions regarding medical/psychiatric conditions in their child.There was a significant association between time spent playing games for more than one hour a day and YIAS (p < 0.001), overall grade point average (p ≤ 0.019), and the "Inattention" and "ADHD" components of the CPRS . No significant association was found between body mass index (BMI), exercise, number of detentions, or the "Oppositional" and "Hyperactivity" components of CPRS and video game use.Adolescents who play more than one hour of console or Internet video games may have more or more intense symptoms of ADHD or inattention than those who do not. Given the possible negative effects these conditions may have on scholastic performance, the added consequences of more time spent on video games may also place these individuals at increased risk for problems in school. The introduction of the telegraph in the nineteenth century ushered in a new era of communication and social development. Further advances in technology led to the creation of the telephone, radio, and television. Recently, the Internet has become the pinnacle of interchange in the modern world and facilitates many different modes of communication. Each generation has raised concerns regarding the negative impact of media on social skills and personal relationships. The Internet appeals to adolescents for many reasons and has become a social connection for many with uses including messaging, e-mail, gaming, education, and music.The Internet and other media types are reported to have important social and mental health effects in adolescents. The association between television viewing and obesity, attention disorders, school performance, and violence has been reported -6. LikewThe effect of video games on adolescents is not well characterized despite a growing body of evidence demonstrating their addictive nature and popularity -15. IndeDespite recent negative attention, some studies have shown possible positive effects of video games on development. One study by Li et al. found a positive association between motor development and cognitive behavior in preschool age children . Other sThe term "video games" does not always differentiate between console and Internet/computer video games but instead, suggests a loose clustering. Console video games include Nintendo, Sony Playstation, Microsoft Xbox, and others. Internet video games refer to computer games played online in a community setting with other players. Although similar in nature, several important differences exist. Console games can be played with other people, but most games are "single player" and are meant to be played alone. Internet games however, are designed for "multi-player" use and are played with others online, usually at distant sites. Console games are less expensive than Internet games, and do not require a computer. The genre of video games played on the Internet versus console games also differs in content. Console game themes include sports, action, strategy, family, puzzle, role-playing games, and simulation, while video game themes designed for Internet use are more specific and are mainly action and strategy. The video game market, regardless of type, is a multi-billion dollar industry that generally targets children and adolescents.The relationship between video games and ADHD is unknown. The incidence of ADHD continues to rise and is a significant challenge on medical, financial, and educational resources ,33. ADHDThis study examined the relationship between video game use and symptoms of ADHD. Other parameters studied included body mass index (BMI), school grades, work, detentions, and family situation.th and 10th grade students at the school (n = 221). The survey included sections for students (five pages) and parents (two pages) to complete independently, as well as a consent form which needed to be signed by both the student and parent for participation in the study. All survey data was anonymous. Surveys were collected (n = 162) through the school Guidance Office. Eighteen surveys were omitted due to incomplete responses. The final subject pool comprised 144; 72 each from parents and students. Original power calculations were based on a reported 10% prevalence of psychiatric disorders in the adolescent population and called for a total of 200 students for a power of 0.80. However, statistically significant results were reached after analysis of 144 completed questionnaires and led us to conclude that the study could be terminated at that point.After receiving IRB approval, subjects were recruited from a local high school in Vermont. Permission from school officials was obtained and contact was made with the guidance office and school teachers. Surveys were distributed to all 9Time spent playing videos games, watching television, or using the Internet was assessed using a time scale of less than one hour, one to two hours, three to four hours, or greater than four hours. The student survey material included Young's Internet Addiction Scale, modified for video game use  . This scThe dependent variables reported in numerical format  were analyzed using the student's t-test and the Mann-Whitney test. The latter method is based on median values and is the preferred method when testing small sample sizes. Data reported as "yes/no"  was analyzed using the chi-square test. Results were considered significant if p ≤ 0.05. Time spent playing video games, watching television, and using the internet was the independent variable. The time intervals compared were for student who spent less than one hour or greater than one hour on a particular activity. The one hour cutoff was used because it gave a more even distribution of sample sizes between the two groups although other time intervals were also compared.The study cohort comprised 72 students; 31 males and 41 females in the ninth and tenth grade. Average age was 15.3 ± 0.7 years. Subject demographics are shown in Table The mean BMI for adolescents who watched less than one hour of television per day was 20.28 ± 2.33 and 22.11 ± 4.01 for those who watched more than one hour of television  and ADHD  behavior in those who played video games for more than one hour Table . No signThere was a trend toward lower grades in students who surf the Internet and play video games for more than one hour, but these results were not significant Table . HoweverMales were significantly more likely than females to spend greater than one hour a day playing console or internet video games . Twenty males reported playing video games for more than one hour a day versus only one female adolescent reported playing internet video games for more than one hour. There was no significant relationship between gender and time spent watching television or on the Internet. We also found no significant association between time spent on any media form and students who worked, had married parents, received more detentions per month, or exercised more frequently.ADHD among children and adolescents has been attributed to both genetic and environmental factors. Of the The prevalence of ADHD in adolescents is reported to be 4–7% ,38. ThisThis study found no association between video games use and oppositional or aggressive behavior. Previous research has shown a positive correlation between violence in video games and aggressive behavior ,14,20,21The effect of television viewing on BMI has been reported in several studies ,2,5,6. WTime on the Internet was not associated with increased BMI; a trend towards decreasing BMI was found in adolescents who use the Internet for more than one hour. Our findings suggest that current recommendations to limit television and video game times for children should be followed .Both console and Internet video games were associated with an increase in addiction scores as measured by YIAS-VG. The YIAS-VG assesses the degree to which video games negatively impact different social factors including daily activities, relationships, sleep, and daily thoughts. The increase in YIAS-VG scores imply that playing video games for more than one hour a day does have a negative impact on relationships and daily activity. We did not define a cutoff on the YIAS-VG to identify "excessive" video game use but the scores in our cohort were not high enough to be considered as evidence of "Internet Addiction" ,36.GPA was lower in those who played video games for more than one hour. Even though this study cohort had a relatively high overall GPA, the difference between an "A" (less than one hour of video games) versus a "B" (more than one hour of video games) is a significant change in grade. For students who are less academically proficient, this may be especially important. There was also a trend towards a lower GPA in students who watch television for more than one hour. Excessive television has been reported to be associated with poor school performance .This investigation found that playing console and Internet video games for more than one hour a day has negative social and academic effects in adolescents. This association does not depend on being "addicted" to video games or playing for excessive time periods. Furthermore, there was no difference between playing video games on the Internet or on a console system. The intensive nature of video games is likely to cause this time dependent relationship between video games and behavior disorders, regardless of whether it is over the Internet or on a console system.Several limitations of the study exist. This cross-sectional comparison of video games and ADHD does not allow for cause-effect relationships to be established. Therefore, it is impossible to say whether playing video games leads to an increase in ADHD symptoms, or if adolescents with more ADHD symptoms tend to spend longer times playing video games. Prospective studies to examine this relationship more closely are certainly justified. The subject cohort was also not representative of all groups. The large majority of students who responded to the survey were Caucasian, not involved with drugs or alcohol, had married parents, and did well in school. Thus, the association between video games and ADHD in other cohorts cannot be inferred. This study was designed to analyze adolescents who spent more than one hour of time playing video games. It would be interesting to examine the latter cohort in more detail to determine if there is a linear relationship between time spent playing video games and ADHD symptoms or academic performance, or if some other relationship exists among those who spend excessive time on these activities.To our knowledge, this is the first study to find an association between video game use and ADHD symptoms in adolescents. Assessment of ADHD risk factors often involves identification of home and academic environmental factors. Parental relationships, early childhood development factors (i.e. preterm delivery), and excessive Internet use are associated with ADHD later in life. Identification of these and other risk factors that contribute to ADHD will lead to prevention and earlier treatment strategies.This article was amended post-publication. The Beck Depression Inventory was originally listed in Appendix A Table , but has


Anopheles gambiae s. l. is generally thought to utilize puddles over bare soil as its prime larval habitat and to avoid standing water populated with vegetation. In Kisian, Kenya near Kisumu, water often pools in grassy drainage areas both during and after periods of infrequent rains, when typical puddle habitats become scarce because of drying. This raised the question of whether An. gambiae has the behavioural flexibility to switch ovipositional sites when puddles over bare soil are unavailable.Physical objects like vegetation can influence oviposition by mosquitoes on soil or water substrates. An. gambiae s. l. were offered paired choices between wet, bare soil and wet soil populated with mixed grasses or grasses of differing height. No-choice tests were also conducted by giving females either grassy soil or bare soil.To test whether presence and height of grasses influenced oviposition, wild-caught gravid In choice tests, females laid four times more eggs on bare, wet soil than soil populated with grasses. However in no-choice tests, egg output was not significantly different whether grasses were present or not. Females laid significantly more eggs on soil populated with short grass than with medium, or tall grass.An. gambiae s. l. has the capacity to oviposit into grassy aquatic habitats when typical puddles over bare soil are unavailable. This knowledge will need to be considered in the design and implementation of programmes aimed at reducing malaria transmission by suppression of An. gambiae s. l. immatures.This work shows  Anopheles gambiae oviposition, while visual contrast strongly influences finding of prospective ovipositional sites [Physical and chemical cues influence acceptance of ovipositional sites by mosquitoes -3. Darknal sites ,5. Odoural sites  and decral sites .Anopheles culicifacies oviposition [Anopheles hermsi increased in accordance with the density of aquatic macrophytes (Myriophylluym aquaticum) up to 1,000 stems m-2 [Anopheles funestus breeds mainly in marshes and swamps that contain tall grasses and other plants [An. gambiae is generally thought to utilize puddles over bare soil as its prime larval habitat [An. gambiae deposited eggs in rice fields at all stages of vegetative maturity. More recently, Fillinger et al. [An. gambiae avoids habitats with emergent vegetation like grasses. They and Minakawa et al. [An. gambiae can be commonly found in association with grasses. However, it is unclear whether presence of larvae in grassy habitats results from hatch of eggs placed on and around wet grasses, or whether larvae were carried there by flowing water. The question addressed in the current laboratory study was whether An. gambiae has the behavioral flexibility to oviposit on and around wet grasses both when bare soil is and is not available.Physical objects like vegetation can influence oviposition on soil or water substrates -10. Riceposition . Howeverr plants . An. gam habitat -15 and t habitat  reportedr et al.  have stra et al.  provide An. gambiae s. s. Thus, results reported here represent An. gambiae s. l..Experiments were performed in the Entomology laboratory of the Kenya Medical Research Institute (KEMRI) in Kisian, Kenya using feral females aspirated from houses near Kisian from 28 April to May 28, 2004, and from 15 April and 15 May 2005. These blood-fed females were kept for two days in cardboard cups  covered with wet cloth towels to provide moisture. Ten percent honey solution was also provided as an energy source. Gravid females were transferred into 60 × 60 × 60 cm white BugDorm-2 cages  held on a lab bench near a bank of windows providing ambient nightlight. The response variable reported is total egg numbers. No detectable differences emerged in proportion of females retaining eggs. Although polymerase chain reaction was not performed on the wild-caught mosquitoes, previous samples from this area  containeAn. gambiae larvae. These plugs were inserted into white cups of similar dimensions; they contained a mixture of Bermuda grass (Cynodon dactylon), sedge grass (Scirpus steudneri), and Rhode grass (Chloris gayana). Grass coverage of the soil ranged from 50 to 70%. Comparison cups were filled with soil taken from the same location but with no grass parts included. All the cups were saturated with tap water from a deep bore hole. A pair of cups with grass-covered vs. bare soil was offered to ca. 60 gravid females in a BugDorm-2 cage. Eleven replicates, each using a different batch of mosquitoes, were performed through time for choice tests.Plugs of soil (8 cm height × 8 cm diam.) with associated grasses were collected outside the KEMRI compound near areas supporting The no-choice test was conducted (seven replicates) by providing 40 gravid females with a single ovipositional cup containing either grass-covered or corresponding bare soil. The following morning, grass stems were cut at their base and plants as well as soil were examined very thoroughly for the presence of eggs with the aid of a dissecting microscope.Soil plugs with grasses of differing height and corresponding bare soil were collected near the KEMRI compound. Over 90% of the soil was covered by grasses as compared to 50–70% in 2004. Grasses were divided into three height categories: short (1–2 cm), medium (13–16 cm), and tall (27–39 cm). It is acknowledged that species of grasses co-varied with the height categories such that effects cannot be attributed to height alone. Each grass category, paired with its control bare soil, was offered to 20 wild-caught gravid females.Trifolium burchellianum (80%) and Cynodon dactylon (20%); medium grasses were Cynodon dactylon (50%) and Scirpus steudneri (50%); and tall grasses were Chloris gayana, the most dominant grass around Kisian.Thirty gravid females were offered: short, medium, and tall grasses for oviposition. This test was replicated six times using different batches of females. Samples of the grasses used were dried and stored in layers of dry paper for subsequent species confirmation. Short grasses consisted of cape clover, t-test in two-choice tests and no-choice tests. Differences in egg outputs in three-choice tests were calculated as a proportion of the total number of eggs laid in each cage and compared using 1-way analysis of variance (ANOVA) [Differences in egg output were analysed by paired  (ANOVA) . Means wAn. gambiae females deposited four times more eggs on bare wet soil than on soil covered by grasses . Bare soil received 1,495 ± 224 eggs (mean ± SEM) while grass-covered soil received 353 ± 115 eggs.Wild-caught t = 1.0, df = 6, P = 0.35). Both in choice- and no-choice tests, some eggs were laid on the leaves or stems of grasses   . Short grasses were more acceptable than medium and tall grasses when no bare soil was present. Overall, egg abundance on grass-covered soil relative to bare soil decreased from 20 to less than 8% as the density of the grasses increased from 50–70% (in 2004) to 90% (in 2005). The density and height of grasses might act as a barrier precluding females from approaching sufficiently close to touch the wet substrate or to land. The speculation is offered that, if no bare soil is available, females might persist in exploring a wet grassy site and eventually alight and oviposit. In some cases, eggs appeared to have rained down upon grass leaves and axils (Figure When offered a choice, An. gambiae should be expected in rice fields [An. gambiae is an ovipositional generalist [An. gambiae s. l. immatures.This work confirms and extends previous claims that e fields  and othee fields ,18. Femae fields , as wellneralist  rather tJH designed and carried out all the experiments, analysed and interpreted data, as well as drafted and revised the manuscript. EDW was P. I. of the grant supporting this work; he participated in the conception of the study, data collection and interpretation, and revision of the manuscript. PEO helped collect grass samples and data. FA identified grass species. JV provided institutional support for this study. JRM participated in the study design, data collection and interpretation, as well as drafting and revising the manuscript, and submitting the manuscript. All authors read and approved the final manuscript.


Other yeast mitochondrial studies have led to the characterization of an equilibrative fatty acid-stimulated Ca2+ transport activity. To gain a deeper understanding of the regulation of mitochondrial divalent cation levels we further characterized the efflux of Ca2+ and Mg2+ from yeast mitochondria.Divalent cations are required for many essential functions of mitochondrial metabolism. Yet the transporters that mediate the flux of these molecules into and out of the mitochondrion remain largely unknown. Previous studies in yeast have led to the molecular identification of a component of the major mitochondrial electrophoretic MgSaccharomyces cerevisiae were suspended in a salt-based suspension medium, Ca2+ and Mg2+ were released from the matrix space. Release did not spontaneously occur in a non-ionic mannitol media. When energized mitochondria were suspended in a mannitol medium in the presence of Ca2+ they were able to accumulate Ca2+ by the addition of the electrogenic Ca2+ ionophore ETH-129. However, in a KCl or choline Cl medium under the same conditions, they were unable to retain the Ca2+ that was taken up due to the activation of the Ca2+ efflux pathway, although a substantial membrane potential driving Ca2+ uptake was maintained. This Ca2+ efflux was independent of fatty acids, which have previously been shown to activate Ca2+ transport. Endogenous mitochondrial Mg2+ was also released when mitochondria were suspended in an ionic medium, but was retained in mitochondria upon fatty acid addition. When suspended in a mannitol medium, metal chelators released mitochondrial Mg2+, supporting the existence of an external divalent cation-binding site regulating release. Matrix space Mg2+ was also slowly released from mitochondria by the addition of Ca2+, respiratory substrates, increasing pH, or the nucleotides ATP, ADP, GTP, and ATP-gamma-S.When isolated mitochondria from the yeast 2+ and Mg2+ release was activated by increased ionic strength. Free nucleotides, metal ion chelators, and increased pH also stimulated release. In yeast cells this release is likely an important mechanism in the regulation of mitochondrial matrix space divalent cation concentrations.In isolated yeast mitochondria Ca Free Mg2+ in cells is quite low (0.3 – 3%) as compared to the total amount of Mg2+ due to buffering by polyphosphates, nucleic acids, and free nucleotides [2+ level in the cell varies during changing physiological conditions. The free Mg2+ level increases slightly as ATP levels decrease. The total cellular Mg2+ level changes upon the addition of several hormones to cells [2+ homeostasis in several tissues [2+ transport exist to maintain proper intracellular levels.Mgleotides ,2. In mate low 0. – 3% as  tissues . This su2+ flux are largely unknown. The human homolog of a component of the main electrophoretic mitochondrial Mg2+ transporter in yeast has been cloned [The pathways involved in mammalian mitochondrial Mgn cloned . The pro2+ in a respiration-dependent manner [2+ into the cytoplasm [2+ levels [2+ transport [2+ at alkaline pH [2+ has been well characterized at the isolated organelle level, the cellular signals regulating mitochondrial Mg2+ transport have not been clearly elucidated.Heart mitochondria take up and release Mgt manner -8. Uncouytoplasm . There i+ levels . Hormoneransport . Fatty aaline pH . Even th2+ is plentiful (> 1 mM), yeast cells contain around 400 nmole Mg2+/mg protein [2+ decreased (0.02 mM), cellular Mg2+ decreased to levels near 80 nmole/mg protein. In times of ample Mg2+, the Mg2+ is taken up and stored in the vacuole. When these cells were then placed in a Mg2+ free growth medium, they survived by utilizing their vacuole stores. But once the cellular Mg2+ level became too low, the yeast cells died, in contrast to limitations in many other nutrients in which the cells survive in the G0 phase of the cell cycle [In yeast cells when medium Mgntiful >  mM, yeasntiful >  mM, yeas2+ uptake system in the plasma membrane have been identified [2+ transport is inhibited by aluminum [2+ transport proteins confers resistance to aluminum toxicity, the inhibition of Mg2+ uptake may be the primary cause for aluminum toxicity in yeast [2+ may also provide benefits to cells confronted with heavy metals. Increased cellular Mg2+ levels protected cells from toxic concentrations of Mn2+ by down-regulating Mn2+ transport [Two genes comprising the yeast Mgentified . Yeast paluminum  and polyaluminum . Since oin yeast . Mg2+ maransport .2+ is essential for many mitochondrial reactions including those of DNA and RNA metabolism [2+ blocks the yeast mitochondrial ATP-induced unspecific channel (YMUC) [2+ also inhibits mitochondrial anion uniport [2+ could be important for the mitochondrial distribution of many other ions. In this report we discovered that Mg2+ and Ca2+ are released from yeast mitochondria under conditions of increased ionic strength. In yeast cells this release pathway is likely an important regulator of mitochondrial divalent cation levels.Mgtabolism ,17 as wetabolism . Mg2+ bll (YMUC) . Mg2+ al uniport . Therefo2+ or undergo an increase in non-specific inner membrane permeability in the presence of Ca2+ [2+ by the addition of the electrogenic Ca2+ ionophore ETH-129 to intact cells [2+ after ETH-129 addition as measured by mitochondrial-targeted aequorin [2+-indicating dye antipyrylazo III , no mitochondrial swelling occurred  was maintained in a KCl medium after ethanol, Ca2+ and ETH-129 addition  to give the baseline level.In the KCl medium in the absence of Ca2+ influx, activation of a fatty acid stimulated Ca2+/H+ exchange led to the decreased ΔΨ. Because the ΔΨ was not dissipated to as great of an extent when mitochondria were suspended in a KCl medium, the Ca2+ efflux pathway described here appears to be less active than the one activated by fatty acids [In a previous report a large decrease in the ΔΨ occured after ETH-129 addition to mitochondria suspended in a mannitol medium in the absence of BSA  . Togethety acids .2+/mg protein. This is fairly consistent with a previously measured value of between 20 and 30 nmole Mg2+/mg protein [2+ was lost from the matrix space in a time-dependent manner  media containing a varied KCl concentration osmotically balanced with mannitol  Increasing the KCl concentration increased the rate of Mg2+ release from the matrix space. When no KCl was present virtually all of the Mg2+ remained in the mitochondria.Using atomic absorption spectrophotometry, we determined that isolated yeast mitochondria contain between 25 and 35 nmole Mg protein . When nor Figure . A slighl Figure inset. In2+ was also released from the matrix space when mitochondria were suspended in all other ionic media tested including K+ TES, NaCl, and tetramethylammonium (TMA) Cl (data not shown). Endogenous Mg2+ was released from the matrix space at slightly different rates in the different ionic media in increasing order TMA Cl < choline Cl < KCl < Tris Cl  where no swelling occurred , decavanadate, a YMUC inhibitor [2+ is fairly rapid even in the absence of ADP, EDTA, or ethanol, the addition of these compounds only slightly stimulated the rate of efflux.When non-energized yeast mitochondria were suspended in a choline Cl medium, the addition of either EDTA (ethylenediaminetetraacetic acid) or ADP slightly increased the Mge Figure . The comnhibitor ,28, was 2+ release occured. As shown in Figure 2+. Adding 2 mM EDTA resulted in a quick initial and then subsequently slower release of Mg2+. The addition of EDTA and ethanol together led to a very quick release of almost all of the endogenous mitochondrial Mg2+. The addition of ATP caused a slightly greater rate of Mg2+ release than ADP. The presence of carboxyatractyloside, an inhibitor of the adenine nucleotide translocator, did not alter the rate of Mg2+ efflux when ATP or ADP was added. Therefore ADP and ATP likely bind to a site on the outer surface of the inner mitochondrial membrane to release Mg2+. ADP and ATP were half maximally effective at concentrations of 0.43 mM and 0.22 mM respectively -N, N, N', N'-tetracetic acid) was not effective. EDTA and EGTA are metal ion chelators. These results suggest that an inhibitory metal ion blocks the Mg2+ release pathway at a site on the inner membrane facing the cytoplasm. EDTA likely binds the inhibitory metal ion to stimulate efflux. GTP and ATP-γS were nearly as effective as ATP in activating the Mg2+ efflux pathway. The nucleotide analog 5'-fluorosufonylbenzoyladenosine (FSBA) partially blocked the ADP-induced Mg2+ release suggesting that nucleotides do not activate release solely by chelating an inhibitory metal ion. The addition of 0.1 mM Ca2+ also led to a slight increase in the Mg2+ release rate. Unlike yeast plasma membrane Mg2+ transporters [3+ did not block mitochondrial Mg2+ efflux (data not shown). La3+ and ruthenium red, which block the Ca2+ uniporter in mammalian mitochondria [2+ release in yeast mitochondria.As described in Table sporters , Al3+ dichondria  were als2+ release . This data suggest that the mitochondrial Mg2+ efflux pathway studied in this report and the fatty acid-induced Ca2+/H+ exchange are distinct activities.Since fatty acids stimulate Cal medium , we soug2+ release from the mitochondrial matrix space in a KCl medium  while Mg2+ release does not occur spontaneously in a mannitol medium.The yeast mitochondrial Mgeviously ,34. The eviously  hexaammine [2+ levels changed with the levels of Mrs2p and Lpe10p. Mitochondrial electrophoretic Mg2+ uptake was absent in an MRS2 deletion strain. Mrs2p and Lpe10p are essential for yeast growth on nonfermentable carbon sources [2+ release described in this report. However, in the previous experiments Mg2+ was taken up by energized mitochondria in an Mrs2p-dependent manner where we found that Mg2+ was released from energized mitochondria [2+ in ionic or non-ionic media, while greatly differing Mg2+ efflux results occurred in this report under these conditions. In addition cobalt(III)hexaammine, an inhibitor of Mrs2p, blocked Mg2+ transport while having no effect on Ca2+ transport [2+ and Ca2+.Mitochondrial matrix space Mgtabolism ,38. Two tabolism ,39. MRS2xaammine . Mitochochondria . In the ransport , while tYOL027 and YPR125 genes are multicopy suppressors of the respiratory-deficient phenotype caused by deletion of MRS2 [2+ transporters. YOL027p has been demonstrated to mediate K+/H+ exchange activity in yeast mitochondria [2+ and K+ transport properties mentioned above either YOL027p or YPR125p may also function as a Mg2+/H+ exchanger. Mg2+ inhibited K+/H+ exchange activity in YOL027p-contaning submitochondrial particles. Deletion of YOL027 resulted in slightly altered mitochondrial transport rates of both Mg2+ and Ca2+ [YPR125 gene (40 % sequence identity) could be responsible for the remaining Mg2+ and Ca2+ transport activity because overexpression of YPR125 rescued growth of the YOL027 deletion strain and YPR125p also localized to mitochondria.The  of MRS2  and are chondria . Becauseand Ca2+ . It is p+/H+ exchange activity. A screen for nigericin-resistant mutants revealed that the Mdm31 and Mdm32 genes may be involved in mitochondrial cation homeostasis [Mdm31 and Mdm32 deletion mutants contained a two to three-fold higher level of mitochondrial Mg2+ than the wild type. Since yeast mitochondrial K+/H+ exchange is inhibited by Mg2+ [2+ [2+ levels to inhibit the endogenous mitochondrial K+/H+ exchanger to rescue the cells from nigericin-mediated death.When added to yeast cells certain ionophores such as nigericin selectively target mitochondria . Nigericeostasis . Mdm31 a by Mg2+ ,43, it wMg2+ [2+ . Knockin2+ release activity, while a similar activity has been demonstrated in mammalian mitochondria [2+ through a uniport mechanism or by respiration-dependent 'unspecific leak' and release Mg2+ by a Mg2+/H+ exchange [2+ uptake system, while the efflux described in this report is likely a Mg2+/H+ exchange. In this regard the respiratory substrate-induced release that occurs in yeast mitochondria in the absence of external Mg2+ resembles the respiration-induced release in rat heart attributed to Mg2+/H+ exchange [2+ in yeast mitochondria resembles a Mg2+ release activity in rat liver mitochondria that is stimulated by alkaline pH in the presence of fatty acids [2+ release also only occurred in an ionic medium. This suggests that yeast and mammalian mitochondrial Mg2+ transport may occur through similar mechanisms.We have shown that yeast mitochondria contain a Mgchondria . Mammaliexchange ,44. Our exchange  indicateexchange . The ionty acids . This ra2+-indicating dye and measuring mitochondrial Mg2+ levels we have identified Ca2+ and Mg2+ efflux activities stimulated by increased ionic strength in yeast mitochondria. Mitochondrial efflux did not appear to be spontaneously active when isolated mitochondria were suspended in a sugar-containing medium. Nucleotide di- and tri- phosphates, increasing pH, and divalent cation chelators also activated mitochondrial Mg2+ release. The function of this mitochondrial efflux pathway is likely important for regulating the matrix space concentrations of Mg2+, Ca2+, and perhaps other divalent cations.By using a Ca600 = 1.8–2.2). Mitochondria were isolated from spheroplasts as previously described [+), and 0.1 mM EGTA, pH 6.8. Protein concentration was determined using a mini-Biuret method using BSA as a standard. Samples were solubilized in deoxycholate (Na+), which was present at a final concentration of 1% by weight.The yeast strain W303-1A was grown aerobically at 30°C in a media containing 2% lactate, 1% yeast extract, 2% peptone, 0.05% dextrose, and 0.01% adenine at pH 5.0. Yeast cells were harvested during logarithmic growth phase  as an indicator. Safranine accumulation was followed spectrophotometrically, as a change in ΔA511-A533. Initial absorbances without mitochondria in the media were always set to identical values. After addition of mitochondria to the medium absorbances in different ionic strength media were shifted up or down compared to runs in the same media. However, the magnitude of the dye response was similar in the different ionic strength media. Therefore, the different runs were comparable after aligning their baseline membrane potentials after FCCP addition.For ion concentration determination atomic absorption spectrophotometry was performed using an AA-575 spectrophotometer (Varian). 2 mg of mitochondrial protein was incubated in the indicated media, spun down for 2 minutes in a microcentrifuge and the supernatant poured off. The mitochondrial pellet was solubilized with 0.6 mL 2 N perchlorate overnight. 0.5 mL was removed and diluted to 2 mL with deionized water prior to sample reading. For swelling experiments, solute permeability was monitored by light scattering using an SLM- Aminco DW-2C spectrophotometer in split beam mode at Antration . ChangesMedium free ion concentrations in the presence of chelators were determined using a computer program .PB designed and performed the experiments and wrote the manuscript. DP was involved in the conception of the experiments and supervised the work.