Accidental injury represents the most common type of traumatic event to which a child or adolescent may be exposed, with a significant number of these children going on to experience posttraumatic stress disorder (PTSD). However, very little research has examined potential interventions for the treatment of PTSD in these children. The present trial aims to evaluate and compare child- and family-focused versions of a cognitive-behavioural early intervention for PTSD following accidental injury.The principal clinical question under investigation is the efficacy of an early, trauma-focused cognitive-behavioural intervention for the treatment of PTSD in children following accidental injury. Specifically, we compare the efficacy of two active treatments (child-focused and family-focused CBT) and a waitlist control (no therapy) to determine which is associated with greater reductions in psychological and health-related outcome measures over time. The primary outcome will be a reduction in trauma symptoms on a diagnostic interview in the active treatments compared to the waitlist control and greater reductions in the family-compared to the child-focused condition. In doing so, this project will also trial a method of stepped screening and assessment to determine those children requiring early intervention for PTSD following accidental injury.The present trial will be one of the first controlled trials to examine a trauma-focused CBT, early intervention for children experiencing PTSD following accidental injury (as opposed to other types of traumatic events) and the first within a stepped care approach. In addition, it will provide the first evidence comparing the efficacy of child and family-focused interventions for this target group. Given the significant number of children and adolescents exposed to accidental injury, the successful implementation of this protocol has considerable implications. If efficacious, this early intervention will assist in reducing symptoms of traumatic stress as well as preventing chronic disorder and disability in children experiencing acute PTSD following accidental injury.Controlled-trials.com: ISRCTN79049138 Posttraumatic Stress Disorder (PTSD) is an anxiety disorder that can develop following exposure to various traumatic events. It consists of three core symptom clusters; re-experiencing of a traumatic event, emotional numbing or avoidance of reminders of that event and physiological hyperarousal. Children and adolescents may experience PTSD following exposure to a wide variety of traumatic events, including sexual or physical abuse, war, natural disasters, accidents and medical-related traumas. The trauma that triggers the development of PTSD may be either of a recurring nature , or it may be a single incident trauma  or injury).Approximately 25% of children and adolescents report experiencing a significant traumatic event by the age of 16 years . PrevaleImportantly, recent research has indicated little difference in terms of distress and impairment between children meeting full and partial criteria for PTSD . SeveralIn terms of consequences, PTSD is a chronic and debilitating disorder that is associated with significant impairments in both social and academic functioning . When coTrauma-focused CBT (TF-CBT) has demonstrated the strongest level of empirical support as the treatment of choice for PTSD in adults. Specifically, models of the psychological impact of trauma suggest that the way in which people remember and recount threatening events significantly affects how well they manage and adjust to those experiences . SimilarIn light of these important differences, it has been suggested that the generalization or application of knowledge gained from treatment outcome studies with young people who have experienced child sexual abuse to young people who have experienced trauma other than abuse may be highly problematic . In moreChemtob, Nakashima and Hamada  and SteiOverall, research has demonstrated strong support for TF-CBT in the treatment of childhood PTSD, however, controlled investigations of youth exposed to single-incident traumas are lacking. On the limited evidence available, it appears that trauma-focused CBT may be an effective treatment for PTSD in children and adolescents exposed to accidental injuries such as MVAs. However, more controlled trials and empirical evidence is required and several important questions remain unanswered, including the importance of parental involvement in treatment and optimal timing of interventions.The role of parenting behaviours and parental anxiety have long been recognised as crucial factors in the development and maintenance of childhood anxiety disorders [e.g. ]. RecentTo date, very few treatment studies in this area have included a parental treatment component, with those that have concluding that a combined parent and child trauma-focused CBT condition results in the best outcomes for children. Although the recent pilot study conducted by Smith et al.  did inclAnother issue which requires attention concerns the optimal timing for delivery of CBT interventions for childhood PTSD. In terms of the course of PTSD in children and adolescents, the current adult literature and the few existing prospective studies of children presenting with PTSD suggest that a steep decline in PTSD rates may be expected within the first year following the traumatic event -28. HoweStrategies for identifying those most at risk for the development or continuation of poor psychological adjustment after trauma may represent the best way forward. Kenardy and colleagues  reportedThus, there is a pressing need for a controlled trial examining the efficacy of a trauma focused, CBT early intervention for the treatment of PTSD in children following exposure to accidental injury. Further, a controlled trial examining the impact of CBT on psychosocial as well as health related outcomes is necessary. A trial of this sort provides the opportunity to examine whether trauma-focused CBT interventions are suitable for PTSD resulting from accidental injury, as opposed to repetitive trauma such as sexual abuse or natural disasters. Given the importance of parental involvement in the treatment of other child anxiety disorders, yet the lack of examination of this issue in the treatment of youth PTSD , there is also a need to examine whether CBT interventions can be enhanced through the addition of a parent-based component.The study protocol presented here provides an overview of the present trial including a description of the methods, design, and current status of the trial as well as a discussion of the possible implications that may arise from the findings.child-focused' and 'family-focused') and one comparison ('waitlist') condition. The study will be conducted across 3 hospitals in Australia, The Royal Children's Hospital (RCH) in Brisbane, The Mater Children's Hospital (MCH) in Brisbane and the Women's and Children's Royal Hospital (WCH) Adelaide. There will be six measurement occasions: screening at 1-2 weeks post-hospital admission, baseline, 4-weeks post-baseline, post-intervention, and follow-ups at 6 and 12 months after the post-intervention assessment. A half crossover design will be used to allow baseline and post-test comparisons between the two active treatment conditions and waitlist control group on primary and secondary outcome measures, as well as increasing the sample size for the final evaluation at follow-up. Thus, all children in the waitlist condition will be randomly allocated to one of the two active treatment conditions following their initial waiting period.This study is designed as a three arm randomised controlled trial with two active interventions , the Royal Children's Hospital (Brisbane) Children's Health Service District Ethics Committee (protocol number HREC/09/QRCH/41), The Mater Health Services (Brisbane) Human Research Ethics Committee (protocol number 1305C) and the Children Youth and Women's Health Services (Adelaide) Human Research Ethics Committee (protocol number REC2149/2/2112). The study is funded by an NHMRC Project Grant (569660). The measures to be administered at each time point are listed in Table family-focused' intervention will receive both the child and parent program whereas participants in the 'child-focused' intervention condition will receive only the child program.The intervention to be evaluated consists of two integrated but distinct programs. The first program is for parents, "My child and the accident: A story with a good ending"  and the These programs were developed and piloted as part of a research project conducted by two of the authors (VC & JK). In both treatment conditions, participants will be seen by a psychologist individually. All sessions in both treatment programs are of approximately 1.5 hours in duration. In the family-focused intervention condition, the parent and child programs will be delivered consecutively, with parents completing parent sessions first. The parent program consists of four sessions and aims to: provide psychoeducation about PTSD, as well as a rationale for the child program; focus on danger perceptions and the way these change after a traumatic incident (with an emphasis on factors such as the possible communication of threat from parent to child and reinforcement of perceptions of danger); focus on changes in parenting practices after a traumatic incident affects a family member and encourage parents to think about their own parenting behaviours and whether these are likely to be helpful or not; and support parents to develop the skills to effectively parent a child experiencing PTSD.The child/adolescent program consists of six sessions. This program takes a strengths-based, resilience-building approach; is age appropriate, incorporates creative drawing and writing tasks and aims to: provide psychoeducation about the role of thoughts, behaviours (avoidance) & physical reactions in anxiety (and in PTSD in particular); emphasize the importance of the young person's story of his/her accident and their perceptions of current danger or threat; assist children/adolescents in identifying the 'hot spot' thoughts  in their story; provide young people with the skills to challenge their hot spot thoughts and to manage their "intruder thoughts" ; and plan for the future (relapse prevention).Participants will include children and adolescents aged between 7 and 16 years (and their parents). Children who have presented to either the Emergency Department of the hospital wards following an accidental injury  will be eligible for inclusion. A complete list of inclusion/exclusion criteria is given in Table Recruitment procedures will be identical across the three hospital sites and will involve several stages.Within each hospital's Emergency, Medical and Surgical wards, Research Nurses or Research Assistants will identify eligible participants according to inclusion/exclusion criteria. Eligible families will be contacted while still in the hospital and given an Information Sheet and a "Consent to Contact" form.Eligible families who have given their permission to be contacted will be contacted either by phone  1-2 weeks after their initial admission to the hospital. If the family remains interested in participating in the study, consent and assent forms will be completed and the Child Trauma Screening Questionnaire [CTSQ: ], a brieAt approximately 4-6 weeks following the young person's initial admission to the hospital, those participants judged to be at risk for PTSD will undergo a face-to-face diagnostic assessment. At this time, a diagnostic interview, the Clinician Administered PTSD Scale - Child and Adolescent Version  will beParticipants who fulfill either full or partial diagnostic criteria for PTSD and agree to participate in the study will be randomly assigned to either the waitlist condition (WLC) or one of the two active treatment conditions; child-focused cognitive-behavioural therapy (CF-CBT) or family-focused cognitive-behavioural therapy (FF-CBT). Participants in the WLC will be randomly assigned to one of the two active treatment conditions after 10 weeks of having been in the WLC (following a re-assessment at this time).Participants in the FF-CBT condition will complete the parent program in the first four weeks, and to evaluate changes in parent and child behaviours over this time, a brief assessment will be conducted following the completion of parent sessions, or at 4-weeks post-baseline assessment. To enable a comparison to participants in the CF-CBT and WLC groups, all participants will complete this assessment at 4-weeks post-baseline assessment. Measures to be delivered at this assessment point are outlined in Table All participants will be followed up at post-intervention and 6 and 12 months following the completion of treatment, using the assessment battery outlined in Table This study will aim to recruit a total of 140 participants (approximately 45 in each condition). Recruitment will be carried out over an 18-24 month period at three hospital sites. A comparison of the two active treatments to the waitlist control condition will establish the basic efficacy of both CF-CBT and FF-CBT. A comparison of the two active treatment conditions will test whether the efficacy of CBT for PTSD following accidental injury can be enhanced with parent involvement. Importantly, the battery of questionnaires used in this study will allow assessment of whether the active treatment conditions are associated with improvements in psychological symptoms as well as health-related quality of life. Further, the design of the study will allow for an examination of improvements and maintenance of gains (for the active treatments) over a 12-month follow-up period.It is hypothesized that:• Following treatment, compared to participants in the WLC, participants in both active treatment conditions will demonstrate significantly greater reductions in child trauma, anxiety and depressive symptoms, significantly higher ratings on a health-related quality of life measure and significantly reduced ratings of functional impairment.• Compared to participants in the CF-CBT treatment condition, participants in the FF-CBT treatment condition will demonstrate significantly greater reductions in child trauma, anxiety and depressive symptoms, significantly higher ratings on a health-related quality of life measure, significantly reduced ratings of functional impairment, significantly greater reductions in parental anxiety, trauma and depressive symptoms and significantly increased parental perception of their own ability to support their child.• These treatment gains (and differences) will be maintained over 6- and 12-month follow-up.The primary outcome measures will involve an assessment of posttraumatic stress symptoms. Diagnostic status, symptom severity and associated disability/impairment will be assessed using the Clinician Administered PTSD Scale for Children .Secondary outcome measures include a variety of measures administered to assess child and parent outcomes.Child secondary outcomes include: trauma symptoms/risk indicators, assessed using the Child Trauma Screening Questionnaire ; PTSD sParent secondary outcomes include: parent PTSD symptoms, assessed through the self-report, Posttraumatic Stress Diagnostic Scale ; parentIn addition, overall distress and improvement will also be assessed using a Global Assessment of Impairment/Improvement Scale developed by the authors.Subsidiary outcomes will be measured. Specifically, satisfaction with the treatment program will be measured through an author developed Program Satisfaction Questionnaire, completed by parents and children following the intervention. Severity of injury will be assessed using the Injury Severity Score  and AbbGiven the limited research examining trauma-focused CBT interventions for childhood PTSD following accidental injury, calculations of power was based on existing preliminary evidence and related trials. Although research has traditionally demonstrated large effect sizes for the comparison of CBT interventions to no therapy or control conditions in the treatment of child anxiety disorders, there is less reporting of the expected effect size between two active treatments. Previous research conducted by Cobham et al.  demonstrThe study was powered on the comparison between the family-focused versus the child-focused intervention, as this comparison would require the most power whilst also allowing sufficient power to detect differences between the waitlist and active treatments. For the evaluation of diagnostic change (e.g. whether children became free of their PTSD diagnosis), and accounting for an attrition rate of approximately 20%, a sample size of approximately 140 participants in total across the three groups, waitlist, FF-CBT and CF-CBT, will provide sufficient power to detect differences of a large magnitude between family treatment and child only treatment conditions. Greater power will be obtained by randomizing waitlist participants into the two active treatments following the completion of their waiting period.Randomisation of participants to treatment conditions will be performed by a staff member who is not involved in the recruitment, collection of data or interview procedures. Blocked randomisation is determined using a computer program which generates a list of random numbers and allocation to one of the three conditions. Randomisation will occur immediately following the completion of the baseline interview with the child, with participants informed at this time. Participants will commence their allocated condition one week later.Statistical analyses will be performed by a team member who has not been involved in assessing eligibility of participants, allocation of individuals to treatment conditions, administration of treatment, collection of follow-up assessment data or entering of data. Prior to investigating treatment outcome, the three groups will be compared for pre-treatment equivalence on demographic and baseline assessment measures. If significant differences are found, this will be taken into account by including these variables as covariates in outcome analyses. The outcome data will be analysed and reported in terms of statistical significance of differences between groups in change over time on outcome, clinical significance of the change, and effect sizes.Primary analyses will be undertaken on an intent-to-treat (ITT) basis, including all participants who have been randomized to a condition, regardless of withdrawal from the condition/study. Separate analyses will also be conducted for those who complete treatment. Differences in diagnostic status across groups will be examined using chi-square analyses for each assessment occasion. Continuous data will be analysed using the preferred approach of linear mixed effects modelling, which is able to include participants whose data may be missing at various time points. Other outcomes such as demographic characteristics and reasons for dropout will be described.This trial represents an opportunity to test the potential benefit of a trauma-focused CBT intervention for the treatment of childhood PTSD following accidental injury (as opposed to other forms of traumatic events). The proposed research is of enormous significance and provides innovations at many points along a clinical care continuum. For the first time, children and adolescents presenting to Australian hospitals following an accidental injury will receive a routine screen of their psychological functioning. Unfortunately, this does not currently occur, despite the well documented adverse psychological consequences associated with exposure to such traumatic events ,43. SuchThe proposed research aims to use a stepped screening and assessment approach to identify those young people most at risk of maintaining a diagnosis of PTSD over time. This represents a particularly important step forward in terms of indicated early intervention, whereby we are able to accurately identify and then treat those children/adolescents who are at greatest risk of the very significant repercussions of PTSD. At a broad level then, this research has the potential to promote a new model of service provision and improve the efficiency of care provided through the hospital systems to these young people.The current research also provides an opportunity to examine treatments that focus on both full and partial PTSD in children/adolescents following an accidental injury. To date, no treatment studies  have been conducted exclusively with this population . In addition, the present research provides an opportunity to examine the importance of incorporating a family component within such interventions. The design of the study allows for an examination of whether child or family-focused interventions are associated with greater improvements, which to date, has not been evaluated in a systematic way with children and adolescents experiencing PTSD following any sort of single-incident trauma. Conducting an RCT with this population represents a significant advancement of scientific knowledge in this field. In addition, in relation to the follow-up of treated participants, the proposed research will be the first RCT with children experiencing PTSD following an accidental injury to include a 12-month follow-up point.Finally, although the primary focus (in terms of outcomes) of this research is psychiatric , this study provides an opportunity to also examine outcomes relating to physical health. Specifically, it allows an examination of whether children's and parents' perceptions of children's health-related quality of life and physical functioning are improved following the two active interventions. If effective in improving children's psychological well-being, physical health, treatment adherence and quality of life, these interventions may have significant benefit for improving medical and psychological outcomes in the population as a whole.In summary, the major benefits of the proposed research will be to provide a more structured psychological care pathway for children/adolescents presenting with accidental injuries, and to create an evidence-base for easily disseminated psychological interventions designed to meet the needs of the significant number of Australian children and adolescents who experience adverse emotional effects following an accidental injury.The study commenced in February, 2010. Participants will be recruited during an 18-20 month period, from February, 2010 to September, 2011. The trial is expected to end in December, 2012.The authors declare that they have no competing interests.JK, VC, RN, and BM developed the trial protocol and wrote the applications for NHMRC Grant 569660. JK and SM further developed the details of the trial protocol. SM drafted the manuscript. All authors contributed to the editing of the manuscript. All authors have read and approved the final manuscript.JK. Professor Kenardy has specific expertise in the psychological impact of trauma in children and adolescents. He also has extensive experience in the design and execution of clinical trials of psychological interventions.VC. Dr Cobham has specific expertise in the psychological impact and treatment of anxiety disorders (including PTSD) in children and adolescents. As the primary author of the interventions, she will provide clinical expertise to the project in supervising therapists and overseeing the provision of the interventions. She also has specific expertise in the design and execution of clinical trials of psychological interventions.RN. Associate Professor Nixon's primary area of expertise is in the etiology, maintenance and treatment of child and adult posttraumatic stress disorders.BM. Associate Professor McDermott has investigated and published on the effects of single-event trauma following natural disasters and been involved in design of child and adolescent PTSD interventions.SM. Trial Manager for the trial and Postdoctoral Research Fellow with Professor Justin Kenardy. She also has expertise in the execution of clinical trials of psychological interventions for child anxiety.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/10/92/prepub


Mother-to-child transmission of human immune deficiency virus (HIV) is a multifactorial event highly associated with advanced maternal HIV disease and obstetric incidents taking place during parturition. Thus, various approaches to prevention may be beneficial. Although the time and the route of materno-fetal HIV transmission are still not sufficiently clear, much speaks in favor of a late HIV transmission, most probably taking place during parturition or the phase before the delivery. The fetus is remarkably protected by the placenta and the intact fetal membranes against many viral infections during gestation. These conditions change at parturition and the chance for a transition of HIV-infected carrier cells or virus into the fetal compartment increases. Proinflammatory cytokines secreted at the materno-fetal interface accumulate in amniotic fluid and may chemoattract and stimulate potentially HIV-infected immunocytes. After rupture of membranes, maternal cells of the decidua are directly exposed to the amniotic fluid. Aside from the contamination of the fetal skin at vaginal delivery as a debatable route of infection, blood-to-blood contacts and the fetal swallowing of contaminated amniotic fluid may be the major path of fetal HIV infection. For the fetal prophylaxis of an intrauterine infection, the application of zidovudine is recommended. However, cesarian section before the onset of labor leads also to a diminution of the transmission rate. As the transmission seems to have both systemic and local causes, it makes sense to combine different intervention strategies. Whether a combination of zidovudine and elective cesarean section can lower the transmission risk further has to be evaluated.


The most powerful study designs maycombine elements of both family and association studies applied to proteomic andtranscriptomic analyses. Such studies may provide appreciable advances in our understandingof the genetic aetiology of complex disorders.Historically genotypic variation has been detected at the phenotypic level, at the metaboliclevel, and at the protein chemistry level. Advances in technology have allowed its directvisualisation at the level of DNA variation. Nevertheless, there is still an enormous interestin phenotypic, metabolic and protein property variability, since such variation givesinsights into potential functionally important differences conferred by genetic variation.High-throughput transcriptomics and proteomics applied to different individuals drawnfrom a population has the potential to identify the functional consequences of geneticvariability, in terms of either differences in expression of mRNA or in terms of differencesin the quantities, pI(s) or molecular weight(s) of an expressed protein. Family studies candefine the genetic component of such variation  and with the genotypingof well-spaced markers can map the causative factors to broad chromosomal regions. Association studies in the variant proteins have the greatest power toconfirm the presence of


Sexual size dimorphism (SSD) is a key evolutionary feature that has been studied in many organisms. In a wide range of species, this pattern is more complex because of polymorphism within each sex. However, it is not known whether the magnitude and direction of SSD could be affected by alternative developmental trajectories within sexes. Our aim was to test whether an intrasexual polymorphism, facultative paedomorphosis , could affect SSD variation patterns in European newts.We report here the first evidence that SSD varies depending on the paedomorphic or metamorphic ontogenetic pathway. In species with a consistent female-biased SSD, paedomorphosis decreased the SSD level, but did not affect its direction. In species with moderate female-biased SSD or variable SSD patterns, paedomorphosis changed the magnitude, or both the magnitude and the direction, of SSD.Our study highlights the importance of developmental processes for shaping SSD patterns in populations in which contrasting life-history pathways evolved. European newts express different SSD patterns depending on their developmental pathway , as well as their species and population. These findings emphasize the importance of studying alternative morphotypes, which are found in a wide range of animal groups, to understand the evolution of SSD. The difference between the sexes in terms of body size is a fundamental characteristic that can lead to important biological insights. Selective forces have driven the size of males and females in opposite directions as a result of competitive advantages in ecology (food and habitat use), fecundity, or sexual selection  ,2. On thPaedomorphosis in tailed amphibians implies the existence of two morphs that differ by the retention of gills at the adult stage, i.e., a mixture of larval and mature characters ,13. The Triturus): the smooth newt , the palmate newt , and the alpine newt . The high incidence of both morphs in particular populations of these three species [European newts have served as important models for the study of SSD due in large part to their considerable variation in both extent and direction of intersexual body size differences -17. Neve species ,19 is a  species ,17.In the present study, our objective was to investigate patterns of SSD variation by studying populations of the three European species in which a large number of paedomorphs and metamorphs can be found syntopically (paedomorphosis is too rare in other European newt species to use them in studying SSD). More specifically, we aimed at determining (1) whether there is an effect of paedomorphosis on sexual size differences; and (2) whether this pattern is species-specific and/or population-specific. Because facultative paedomorphosis implies the retention of larval traits in the adults, we expect a change in the magnitude of SSD between morphs. Particularly, we hypothesised that paedomorphs with underdeveloped bodies would exhibit less pronounced SSD patterns than metamorphs.The alpine newt and the smooth newt were examined from the Herpetological Collection of the Institute for Biological Research "Siniša Stanković", Belgrade, Serbia. All these individuals were preserved and stored in the collection before 1994. Palmate newts were sampled directly in the field in 2008. Three populations of each species were examined , distance between legs , head width (HW), head length (HL), forelimb length (FLL), hind limb length (HLL) and tail length (TL). All measures of the alpine and smooth newts  were taken on alcohol-preserved specimens. The specimens of palmate newt were measured in a field laboratory on phenoxyethanol-anesthetised individuals (0.1%) and released in their study sites within one day of capture.PC1 = mean female PC1score - mean male PC1score) as a measure of sexual dimorphism. Also, to estimate differences in size between morphs, we used mean "size" PC1 scores of paedomorphs and metamorphs of the same sex and calculated a heterochronic size dimorphism index (HSD IPC1 = mean metamorph PC1score - mean paedomorph PC1score) as a measure of size dimorphism among morphs of the same sex. Differences in the level of SSD between morphs were calculated as the difference between the calculated SSD indices .In tailed amphibians, body size is most frequently assessed by snout-vent length, which is defined as the linear measure from the tip of the snout to the cloaca. However, this single linear measure might not fully describe size differences in overall body architecture . To explm specimens  was repeatedly drawn randomly, with replacement, from the original sample . PCA analyses were performed on each of 1000 generated datasets. We calculated the mean "size" PC1 scores for males and females in each population and morph. The SSD Index, HSD Index and PE Index were calculated for each of the bootstrapped datasets. To compare SSD between morphs, we used a bootstrap test against the null hypothesis that the morphs share the same SSD Index. To simulate this null hypothesis, the data for each pair of morphs were merged into a common pool of specimens. Bootstrap samples were then repeatedly drawn and SSD Index was computed. The magnitude of the difference in SSD was then compared to the value obtained from the original data. For each pair-wise combination, 1000 rounds of bootstrap re-sampling were used. If the difference between the means of pairs of bootstrap samples exceeded the observed differences in at least 5% of the total number of iterations, we could reject the null hypothesis that the means are equal [A possible source of error may come from discrepancies in the sample sizes. We used a bootstrap procedure to generate 1000 samples. A "bootstrap sample" of re equal .All statistical analyses were performed in SAS statistical software .The body size of paedomorphs and metamorphs was characterised by a set of seven linear measurements. To explore size dimorphism between sexes and morphs we performed a principal components analysis (PCA). The first principal component PC1) explained the largest proportion of overall variation (66%). All analysed morphometric traits, except head length (HL), had similar loadings on the first axis and evenly contribute to variation in body size. Therefore, the individual scores on PC1 were used to estimate the differences in overall body size. As our sample consists of sub-samples that largely differ in sample size  and in the species with slightly variable SSD , paedomorphosis was associated with reduced SSD and similar sizes of females and males. However, in the species with the most varied SSD , the effects of morph type on SSD are also more variable because paedomorphosis changes both the magnitude and the direction of SSD in a population-specific manner. These findings highlight the importance of studying alternative morphotypes in a wide range of animal groups, such as horned beetles , marine Variation of SSD according to alternative ontogenetic pathways  can originate partly from the magnitude of the size differences between the sexes. In this regard, Griffith  showed tDespite the trends for each species, large variation across populations was found within each species with respect to the effect of paedomorphosis on SSD. This finding is not very surprising because the growth and life-history of newts  rely on environmental factors that will vary across populations, such as temperature , drying Although further study is needed to elucidate the mechanisms that underlie the obtained patterns, our results offer two general lessons. First, the importance of studying alternative morphotypes, which are found in a wide range of animal groups, to understand the evolution of SDD. Second, understanding the link between body size variation and paedomorphosis is crucial to explain the ultimate advantages of alternative morphs.These authors contributed equally to this work. All authors read and approved the final manuscript.


The effects of photodynamic treatment (PDT) on venules include vascular leakage accompanied by oedema formation, vasoconstriction and blood flow stasis. The goal of this study was to gain insight into the mechanism underlying these vascular events by studying one of the earliest observations after PDT, granulocyte adhesion, in an in vitro model. For this purpose human umbilical vein endothelial cells (HUVECs) preincubated with Photofrin II (PII) were illuminated with red light and incubated with neutrophils. PDT led to a dramatic change in the morphology of the endothelial cells. Clearly, neutrophils adhered to the subendothelial matrix and their adherence coincided with an increase in the percentage of exposed subendothelial matrix by the gradual contraction of endothelial cells. Furthermore, the increase in adherence was dependent on drug dose, illumination time and the time delay after PDT. The neutrophil adherence could be inhibited by anti-beta2-integrin antibodies, which suggests that the alphaL-, alphaM- or alphaX-beta2 receptors of the neutrophil mediated this phenomenon. At 4 degrees C or by preincubation of the neutrophils with staurosporin, their adherence to the subendothelial matrix exposed by PDT of endothelial cells could be prevented. Apparently, activation of the beta2-integrin receptor by interaction with the subendothelial matrix is necessary for the increased binding of neutrophils. Taken together, these in vitro findings suggest that the PDT-induced contraction of the endothelial cells permits neutrophil adherence to the subendothelial matrix. It is conceivable that a similar mechanism contributes to the initial adherence of granulocytes to the vessel wall as observed after PDT in vivo.


A system's wiring constrains its dynamics, yet modelling of neural structures often overlooks the specific networks formed by their neurons. We developed an approach for constructing anatomically realistic networks and reconstructed the GABAergic microcircuit formed by the medium spiny neurons (MSNs) and fast-spiking interneurons (FSIs) of the adult rat striatum. We grew dendrite and axon models for these neurons and extracted probabilities for the presence of these neurites as a function of distance from the soma. From these, we found the probabilities of intersection between the neurites of two neurons given their inter-somatic distance, and used these to construct three-dimensional striatal networks. The MSN dendrite models predicted that half of all dendritic spines are within 100µm of the soma. The constructed networks predict distributions of gap junctions between FSI dendrites, synaptic contacts between MSNs, and synaptic inputs from FSIs to MSNs that are consistent with current estimates. The models predict that to achieve this, FSIs should be at most 1% of the striatal population. They also show that the striatum is sparsely connected: FSI-MSN and MSN-MSN contacts respectively form 7% and 1.7% of all possible connections. The models predict two striking network properties: the dominant GABAergic input to a MSN arises from neurons with somas at the edge of its dendritic field; and FSIs are inter-connected on two different spatial scales: locally by gap junctions and distally by synapses. We show that both properties influence striatal dynamics: the most potent inhibition of a MSN arises from a region of striatum at the edge of its dendritic field; and the combination of local gap junction and distal synaptic networks between FSIs sets a robust input-output regime for the MSN population. Our models thus intimately link striatal micro-anatomy to its dynamics, providing a biologically grounded platform for further study. The brain has an immensely complex wiring diagram, but few computational models of brain regions attempt accurate renditions of the wiring between neurons. Consequently, these models' dynamics may not accurately reflect those of the region. Key barriers here are the difficulty of reconstructing such networks and the paucity of critical data on neuron morphology. We demonstrate an approach that gets around these problems by using the available data to construct prototype neuron morphologies, and uses these to estimate how the probability of a connection between two neurons changes as we change the distance between them. With these in hand, we constructed artificial three-dimensional networks of the rat striatum and find that the connection distributions agree well with current estimates from anatomical studies. Our networks show features and dynamical implications of striatal wiring that would be difficult to intuit: the dominant input to the striatal projection neuron arises from other neurons just at the edge of its dendrites, and the main inhibitory interneurons are coupled locally by electrical connections and more distally by chemical synapses. Together, these properties set a unique state for the input-output computations of the striatum. The mammalian brain is a vastly complex structure at every level of description. Faced with the sheer breadth of neuron and receptor types, many researchers are abandoning attempts to intuit the ‘essential elements’ of a neural circuit, instead building large-scale models of neural circuits, modelling neuron-for-neuron Establishing the detailed network of the striatum is a particular priority, given the large number of experimental and theoretical studies seeking to understand its computations in vitro slice of striatum can induce the appearance of three or four cell assemblies – co-active groups of cells – within an One compelling reason for choosing to model at one-to-one scale is to explore a key question that can not be approached any other way: are there natural scales for the size of striatal regions involved in computing input-output functions? Much thought has been devoted to this question. The “domain” theory of striatum First though we had to build a model of the striatal network. Complete reconstructions of neural circuits are technically challenging, so quantitative data on the inputs and outputs of a single neuron are often incomplete or absent, while many published values are rough estimates. One way around this problem is to use reconstructions of stained dendrites and axons as guides To overcome these problems, we developed a stochastic approach based on the density of overlapping neurites, determining the densities from prototype dendrite and axon models. We applied this approach to reconstructing the three-dimensional GABAergic microcircuit of the adult rat striatum. Building prototype dendrite and axon models for MSNs and FSIs allowed us to determine any omissions or inconsistencies in existing quantitative data, and to establish constraints on the dendritic locations of afferent input. Using these models to reconstruct the three-dimensional network, we could address key questions about striatal micro-anatomy: how sparsely is the striatum connected? What are the comparative numbers of contacts for each type of connection? Are there natural spatial scales of the sub-networks within it? And do these scales correspond to previous electrophysiological The striatal GABAergic microcircuit, shown in Four connection types make up the microcircuit. First, MSNs extend local axon collaterals that synapse on other MSN dendrites. Long-established anatomically The connection statistics between MSN pairs are partially known. Conservative anatomical estimates place 600 synapses on one MSN from other MSNs There is less data on the statistics of FSI connectivity. Previous estimates of the number of FSIs afferent to a single MSN place bounds of 4–27 FSIs per MSN minimum probability of contact between a pair of neurons is proportional to the density of their overlapping neurites. This is a passive process: numbers of contacts exceeding this minimum probability thus imply active processes, especially axon guidance towards specific types of target neurons. We encapsulate the role of active processes as an increase in the effective density of the axon. As we will see, this relatively simple model is able to capture the known statistics of the microcircuit's connectivity.Our aim was to construct a stochastic model of the three-dimensional network of the adult rat striatum, and study the statistics of contacts between the striatal GABAergic neurons. By “contact” we mean whether or not one neuron connects to another: a contact is one or more synapses or gap junctions. Our starting hypothesis was that, in a three-dimensional, non-laminar structure like the striatum, the We could then compute the expected spherical volume that was occupied by dendrite (or axon) at a given distance from the neuron body . Then, in bodies . We treaWe chose the Burke algorithm The probability of a dendritic segment branching or terminating is a function of its diameter. To determine these probability functions, Burke et al As for most neuron types, detailed data on the diameters of dendrites at branch and termination points are not available for MSNs and FSIs, and so we could not define the probability functions and apply the Burke algorithm directly. Instead, we gathered morphological data on the known properties of their dendritic trees  to find the total number of spines at distance Putting this all together, the estimated total dendritic volume We already have a functional description of total axon diameter as a function of distance effective volume of the axon. Exact values for Finding the axon-density constant in the The axon model (equation 10) gives the straight-line change in axon diameter. Yet, both MSN and FSI axon collaterals wander extensively within their overall field voxels that were Both MSNs As we are assuming that the probability of finding a neurite is invariant for a given distance from the soma, we proceed by considering successive spherical shells of width If the number of Having obtained an estimate of the probability that a voxel contains an axon or dendrite, we could calculate the probability that a voxel contains an intersection between the neurites of two neurons. Let us denote the distance between the somas of the two neurons as  spheres . The cenThe total expected number of neurite intersections between two neurons at a given distance ocircuit , and theWe first define a volume of striatum we went want to model. The striatum contains 84900 MSNs per We explored the dynamical implications of some of our anatomical findings, using a computational model of the striatum drawn from our previous work We ran two sets of simulations: one set used networks constructed within The evolutionary algorithm searches successfully found usable Burke algorithm parameters for both MSN and FSI dendrograms. The resulting parameters are given in The resulting probability functions for branching and termination of the MSN and FSI dendrites are shown in ry small , only a The resulting MSN dendrogram models made some interesting predictions. Second, the model MSN dendrograms predicted that existing data on total dendrite length and estimates of spine counts are mutually inconsistent. The median total dendrite length, averaged over all instantiated dendrograms, was A third prediction is that the spines are in abundance in the proximal dendrites. We plot the histogram of the MSN dendrograms' mean spine counts in dendrite .We used the instantiated dendrograms to find the mean total volumes ribution . The traThe importance of close-fitting to the tails becomes clear when we consider the probabilities of finding a neurite-occupied voxel, and the subsequent intersection calculations. When we compute the probability of finding a dendrite-occupied voxel , we see Finally we turn to actually computing the expected number of intersections for each of the MSN and FSI connection types in the striatal GABAergic microcircuit : local aWe performed a simple procedure to find plausible values of the axon density constant for MSN  for each of the four connection types over a wide range of distances between the somas (We found that the distributions for the expected number of intersections were well-fit by functions of the formWe used the expected intersection functions (equation 20) to construct model striatal networks, which we could examine for their predictions of striatal connectivity. We built networks within a dded see . For eveThe constructed networks predict that, for all but the FSI gap junctions, the numbers of and distances between connected pairs of neurons have Gaussian distributions , despiteThe microcircuit and connection statistics). The number of MSNs contacted by one FSI , synapses  and gap junctions from our previous work in vivo. Increasing the FSI density increased the proportion of FSIs that did not fire, but also resulted in a broader and more heterogenous firing rate distribution. Despite this, the median firing rate of active FSIs was consistent across the changes in FSI density. The FSIs' firing rates of up to 80 spikes/s were also consistent with those observed in vivoWe first established the impact of the different FSI densities on the dynamics of the model. We examined the effects of the different spatial scales of the inter-FSI gap junction and synaptic networks by then building models with swapped FSI-FSI gap junction and synaptic interconnection functions; hence, in these models, a FSI was synaptically coupled to FSIs close by, but gap-junction coupled to FSIs further away, inverting the spatial scales predicted by our network model. The same mean distances of MSN and FSI afferents to one MSN imply that there will be a strong, non-monotonic, distance-dependent effect of those inputs on MSN activity. We examined this using a 1 mm-on-the-side cube of model striatum  – a size chosen so that we could stimulate the inputs to the MSN nearest the centre of the cube by up to twice the mean distance of the afferents. To test the impact of input from a range of distances on the central MSN, we synaptically stimulated all the neurons in successive tral MSN .distance . Across distance , but wasdistance . We alsodistance , but theWe have established a complete protocol for constructing a biologically-realistic network from first principles. The process described here is of general interest: in principle it could be used to model any region of the brain. It is particularly suited to the reconstruction of three-dimensional networks in non-layered structures, and we used it to reconstruct the GABAergic microcircuit of the adult rat striatum.Attempting to specify construction algorithms for the dendrites and axons showed specifically where quantitative morphology data were missing . The network model predicts each FSI contacts around 3000 MSNs, which may explain why the FSIs, despite being few in number, are able to potently suppress MSN activity across the striatum When we applied the intersection functions to construct the striatal network models, we found though that almost all distributions of numbers of contacts and their distances were Gaussian. The network models predicted that each MSN receives an average of 728 inputs from other MSNs, when considering the complete network. Confidence in this result stems not just from the tuning to match the data on probability of contact within The numbers of contacts for the other connection types were dependent on the percentage of FSIs in the network. Mean numbers of contacts in the 1% FSI network are consistent with existing estimates for the number of FSIs contacting one MSN The network models made two striking predictions about the spatial organisation of contacts in the striatum. First, that the networks of gap junctions and synapses inter-connecting FSIs were on different spatial-scales: the log-normal, left-skewed distribution of gap junction distances implies each FSI makes most of its electrical connections with immediately neighbouring FSIs; the Gaussian distribution of synaptic distances implies each FSI makes most of its synaptic connections with FSIs more distally. Second, that inputs to a MSN from either FSIs or other MSNs are on the same length scale of The anatomical model results point to some intriguing implications for the spatial scales of computation in the striatum. The “domain” theory se  away . Therefoin vitro induced the appearance of a few cell assemblies within an The spontaneous organisation of activity in striatum is consistent with such a widespread network of effective MSN-MSN connections. Carillo-Reid et al The network model also showed that the density of FSIs affects both the number and spatial-scales of connections. We showed that these anatomical effects are reflected in changing dynamical properties of the FSI network in the computational striatum model. Changing the FSI density altered the distribution of FSI firing rates, decreasing the proportion of active FSIs, but increasing the range of rates. However, irrespective of the FSI density, the FSI network remained in a globally-asynchronous state, with many FSIs completely or nearly silent. Though contrary to previous reports that networks of spiking neurons coupled by both gap-junctions and inhibitory synapses promote globally synchronised activity e.g. , our finThe local and distal networks formed respectively by the inter-FSI gap junctions and synapses produced characteristic properties of the MSN population dynamics too. The MSN population activity was remarkably consistent across changes in FSI density , despiteWe also saw that in the absence of active FSIs, the MSN population activity was globally reduced compared to all normal models with active FSIs. This result in a larger and anatomically more detailed model confirms our previous finding that removing all FSIs reduces model MSN activity The results of this work have further applications in the study of both single-neuron and network-level dynamics. By using our found parameters for the Burke algorithm, it is possible to generate many MSN and FSI dendritic morphologies, each consistent with current morphological data. Hence, instantiating the same multi-compartmental model e.g.  on multiWe have focussed on the principal GABAergic microcircuit of the striatum here, as this provides the basis for the most immediate, powerful control over the output of the striatum Our model is a stochastic realisation of the adult striatum; the modelling of developing striatal connectivity is a stern challenge given the current paucity of data Figure S1Implications of distance-dependent connections for MSN output. We stimulated all neurons within a 50 µm wide spherical shell at varying distances from the centre of a 1mm-on-the-side cube of striatum (84900 MSNs). Here we used a network with a FSI density of 3% (2547 FSIs) to check that the effects on the centre MSN were consistent even with increasing numbers of FSIs. A. The total number of neurons per shell increases exponentially with increasing distance from the centre; here and in all other panels we plot distances as the inner radius of the shell. B. The probability of any chosen neuron in that shell contacting the central MSN falls exponentially with increasing distance. C. All stimulated neurons received approximately 1250 spikes/s excitatory input for 4 seconds. The mean firing rate of MSNs in the shell fell slightly with increasing distance; the mean firing rate of FSIs in the shell was roughly constant (the first shell contained only one FSI). D. The number of neurons projecting to the centre MSN peaked at the same distance for both MSN and FSI afferents. E. In response to the same input as the stimulated neurons, the centre MSN's firing rate follows the inverse of the distribution of inputs across the shells. F. The centre MSN's inter-spike interval (ISI) coefficient of variation (CV), indicating the irregularity of the spike train, was more modulated by the distance of the afferent input than for the 1% FSI network.(0.19 MB PDF)Click here for additional data file.Text S1Specification of the Burke algorithm and the evolutionary algorithm used for searching the Burke algorithm parameter space; also includes a list of missing/desired morphological data.(0.12 MB PDF)Click here for additional data file.


FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology.Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence  The single cell gel electrophoresis, called shortly 'comet assay', as well as the micronucleus (MN) test are broadly applied test systems to check for genotoxic effects. In addition to classical cytogenetic methods for scoring chromosomal aberrations, fluorescence in situ hybridization (FISH) is used in genetic toxicology for analysis of chromosome damage with increased efficiency and specificity for identifying certain kinds of chromosomal aberrations. The comet assay, MN test and FISH presented in International Programme on Chemical Safety (IPCS) guidelines among the most often studied genotoxicity endpoints for the monitoring of genotoxic effects of carcinogens in humans [A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems n humans . RecentlThe comet assay is a rapid and very sensitive fluorescent microscopy-based method for measuring DNA damage, protection and repair at the level of individual cells -7. In thThe MN test is one of the preferred methods for assessing DNA damage at the chromosome level. It permits to measure both chromosome loss and chromosome breakage ,17. Metain situ by microscopy analysis. FISH technique protocols and wide variety of current applications of FISH technology are presented [in vitro and in vivo by chemical and physical agents [FISH is a powerful technique for localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. The detection of nucleotidic sequences on examined DNA molecule consists in hybridizing a DNA probe to its complementary sequence on chromosomal preparations. Probes are labeled either directly, by incorporation of fluorescent nucleotides, or indirectly, by incorporation of reporter molecules that are subsequently detected by fluorescent antibodies. Probes and targets are finally visualized resented -33. Strul agents -37.Compared with other assays, analysis of comets and MN bring along several advantages, including speed and ease of analysis and no requirement for metaphase cells in MN test and no need for dividing cells in the comet assay. However, results from the comet assay alone reflect only the level of overall DNA damage in single cells. The same is typical for MN test as it does not even permit to distinguish MN containing whole chromosomes from MN containing chromosome fragments. The introduction of FISH  in cometFISH permitted to supplement potential of the comet assay with an opportunity to recognize genome regions of interest on comet images. Thus, Comet-FISH is applied for the analysis of damage and repair of different genes, chromosomes and chromosome regions compared to whole genomic DNA within the comet, or visualization of genomic loci in three-dimensional organization of chromatin and elucidation of mechanism of comet formation and DNA organization in comets. By MN test combined with FISH the genetic contents of the MN can be characterized. The application of FISH probes allows to distinguish MN originating either from chromosome loss or breakage and to determine the involvement of specific chromosomes and chromosome fragments in MN formation. Using MN-FISH the clastogenic or aneugenic action of different factors, the chromosomal origin of spontaneous and mutagen-induced MN, and the relative contribution of all chromosomes in MN formation can be studied.Therefore, FISH was recognized as a valuable addition to comet and MN assays ,39. The Comet-FISH was first applied in human cells to compare the localization of specific chromosomal domains in native interphase nuclei with their distribution in comet-head and -tail after electrophoreses . As heatVarious DNA probes were successfully applied with the comet assay for analysis of damage and repair of specific genome loci . The size of the region of interest investigated by Comet-FISH varies from gene  to wholeMicroscopic evaluation of the Comet-FISH images includes record of the number of probe signals and their localization on comet. The position of the fluorescence signals indicates whether the sequence of interest lies within the undamaged (head) or within or in the vicinity of a damaged (tail) region of DNA. Repositioning of gene-specific signals from tail to head over the incubation period provides evidence for repair of all the lesions within and around the locus of interest . The levHowever, doing FISH on comet assay preparations is different from that of routine FISH mainly by the fact that it is performed not on flattened interphase nuclei fixed to a glass slide but on three-dimensional (3-D) preserved ones. This 3-D state reflects to some degree the real organization of chromatin in the living cell. On the other hand, the 3-D shape leads also to serious difficulties in the visualization and scoring of the signals . Thus, aAlthough the comet method is very popular, there is still no agreement on how the comet tail is formed. Understanding of comet formation and the factors influencing this process is necessary for the comet assay correct application in genetic toxicology. In the comet assay, cells are electrophoresed in a way that fragmented and relaxed DNA migrates towards anode further than intact DNA, producing a situation resembling a comet. Relaxation of DNA loops was proposed to be the primary basis for comet formation under neutral ,49 and ap53 gene was applied to cells that had been damaged by ionizing radiation. The results obtained favored the idea that both strands in a loop migrate into the tail, but separately, even in cases in which one strand is broken and the complementary strand is intact [The application of FISH has allowed to explain further aspects of comet formation. An important question is whether the complementary strands within a loop migrate into the tail independently or together upon alkaline denaturation and electrophoresis. Comet-FISH with a probe for the s intact .DHFR gene and one of ends of MGMT gene to leave the comet head even when the DNA is released from its supercoiling in CHO cells was explained by their attachment to 'matrix-associated region' [Before the era of Comet-FISH it was generally accepted that, when single cell gel electrophoreses is performed undamaged DNA remains in the comet head and the fraction of damaged DNA moves to the comet tail. FISH experiments indicated that besides the presence of breaks there are other factors determining the ability of particular DNA-sequences to migrate into the tail. These include the nature of the damage and organization of chromatin . DNA fro region' . Thus, tin vitro and in vivo genotoxic effects and understanding of their biological impact.Combined application of FISH with the comet assay offered the unique possibility to evaluate gene or chromosome damage and repair relative to the overall genome and compare repair rates of individual genes This methodology permits to detect mutagen-induced site-specific breaks in DNA regions that are relevant for development of various diseases or to recognize genome targets of action of environmental genotoxic agents. Recognition of sites of damage, promotes interpretation of induced TP53, KRAS, APC) in primary human colon and colon adenoma LT97 cells. Here this approach really facilitated studies on effects of nutrition-related carcinogens [Comet-FISH was found to be suitable for detection of DNA damage induced by genotoxic compounds e.g. in colon cancer relevant genes  [Comet-FISH revealed that strand breaks in the human tumor suppressor adiation  and in madiation . PrefereRL-2336)  and in nComet-FISH is also an effective alternative for measuring transcription-coupled repair (TCR), since the comet assay constitutes an extremely sensitive test for detection of DNA damage and repair by genotoxic agents at subtoxic, physiologically relevant exposures. Application of Comet-FISH for analysis of TCR was discussed elsewhere .DHFR and MGMT genes in CHO cells and p53 gene in human cells treated with H2O2 or photosensitizer plus light to induce oxidative damage were monitored using Comet-FISH with oligonucleotide probes for 5' and 3' regions of the genes investigated. CHO cells shown preferential repair of oxidative damage in the MGMT gene. Strand breaks in the human p53 gene were repaired more rapidly than total DNA. This approach can be applied to other genes treated with a range of damaging agents [Localization and repair rates of g agents .Ret, Ab1 and Trp53 genes fragmentation in Comet-FISH assay was proposed as in vivo biomarkers of X-radiation exposure in C57BL/6 and CBA/J mice. At the same time the comet assay alone, when applied to the same specimens, produced no significant results because of interindividual variability [It has been shown, that damage of specific genes can be applied as biomarkers of genotoxic exposure. iability .Comet-FISH in UV-A-irradiated human lymphocytes with whole chromosome painting (wcp), centromere-, telomere- and region-specific probes demonstrated comparably high sensitivity of chromosomes X and 8 towards UV-A-induced DNA damage . StudyinLeukaemia-specific chromosome damage (breakage at 5q31 and 11q23) in TK6 lymphoblastoid cells exposed to melphalan, etoposide or hydroquinone was studied using Comet-FISH . Comet-FIn our experiments Comet-FISH with telomere-specific peptide nucleic acid (PNA) probes was applied for measuring telomeric DNA sensitivity toward drugs used in cancer therapy in normal human leukocytes ,64 and iFISH analysis of MN is based on the achievements of interphase FISH . CommercMN test was successfully combined with different kinds of DNA probes which recognize centromeres, other chromosome-specific regions and whole chromosomes inside of micronuclei and main nuclei. The analysis of MN combined with centromeric DNA probes for all chromosomes allows discrimination between centromere negative MN or MN originating from chromosomal breakage (clastogenic effect) and centromere positive MN or MN containing whole chromosomes (aneugenic effect). Сentromere detection can be expected to be more accurate in distinguishing the two main types of MN than anti-kinetochore antibody staining  because Application of chromosome-specific centromeric probes permits evaluation of different chromosomes sensitivity toward genotoxic agents. This approach also allows detection of non-disjunctional events  in binucleated cells . The appHowever the identification of the chromosome-specific contents of MN is still very incomplete due to a lack of methods by which the DNA within the MN could be fully investigated. Moreover, the absolute number of fragments enclosed in a MN could not be quantified precisely. To our knowledge till now there are no successful attempts of application of interphase chromosome-specific multicolor banding (ICS-MCB) ,77,78 foMN can arise after mitosis from acentric chromosomal fragments or whole chromosomes that are not included in each daughter nucleus. Therefore, in MN test, chromosome aberrations are detected indirectly via DNA loss from the nucleus leading to MN in the cytoplasm of the cell. FISH technique permits to identify the chromosomal origin of MN and thus improve our understanding of mechanisms of MN formation.Anaphase aberrations and MN formation in woman lymphocytes were compared using pancentromeric and X-chromosome painting probes. It was shown, that micronucleation of the X chromosome in women's lymphocytes is probably the result of frequent lagging behind of the X chromosome during anaphase .XPD-defective human fibroblast cells demonstrated that the XPD gene product plays a role in the events which protect human cells from the aneugenic effects of chemicals [GSTM1 positive subjects showed an increased centromere negative MN frequency and GSTT1 null subjects showed an elevated centromere positive MN frequency [FISH with MN test permits to reveal involvement of different genes in induction of MN and aneuploidy. CBMN assay with centromere-specific probes in hemicals . The datrequency .Thus, MN-FISH combined with analysis of anaphase aberrations and genetic polymorphisms has contributed to the understanding of the processes that accompany the formation of MN.in vitro and in vivo. The characterization of MN contents is crucial for understanding of mechanisms of genotoxicity of chemicals and radiation.FISH analysis of MN with application of the different kinds of DNA-probes offered the possibility to precise the nature of genotoxic effects revealed in MN test. Application of MN-FISH in many researches has allowed to reveal the occurrence of different chromosomes in spontaneous and induced MN and to identify potential chromosomal targets of mutagenic substances. The clastogenic or aneugenic nature of action of many substances has been identified The distinction between the clastogenic and aneugenic effects , by identifying the origin of MN, is important for genotoxicity testing or for biomonitoring of genotoxic exposure and effect. This approach with application of centromeric and chromosome-specific centromeric probes has appeared useful and widely applied in various researches.in vitro genotoxicity testing in studies of toxins of phytoplankton domoic acid (DA) and okadaic acid (OA) in human intestinal cell line Caco-2 [Tripterygium hypoglaucum (level) hutch in mouse NIH 3T3 fibroblasts [Centromere-specific FISH analysis of the MN was applied for e Caco-2 , Al, Cd,e Caco-2 , industrroblasts , anti-turoblasts .in vivo of exposure to nitrous oxide in lymphocytes of operating-room nurses [in vivo. The results obtained are useful for understanding of possible by-effects of action of medicines.MN-FISH was applied for analysis of genotoxicity m nurses  and antim nurses . By usinm nurses , while am nurses  and antim nurses  are aneuEnvironmental lead exposure increases both centromere-positive and centromere-negative MN in blood lymphocytes of children, however, the contribution of centromere-positive MN was significantly higher than in the controls . The corApplication of FISH with MN test allows not only to distinguish between clastogenic and aneugenic effects, but also enables to discriminate between two mechanisms of aneuploidy induction: chromosome loss into MN or chromosome non-disjunction so that one daughter cell becomes trisomic and the other becomes monosomic ,72. It wStudies with chromosome-specific centromeric probes support the observation that chromosome non-disjunction is the major mechanism of spontaneous ,95 and iWell known fact of a non-random distribution of chromosome damages arise spontaneously ,100 and With application of chromosome-specific centromeric probes in was shown that both the X and the Y chromosomes are overrepresented in lymphocyte MN of men but that the Y chromosome is overrepresented only in older subjects . Occurrein vitro increased frequency of micronucleation and malsegregation of chromosomes X and 8 in different age groups of women [in vitro probably as only cells with non-lethal chromosome aberration could survive to be detected [in vitro in human lymphocytes treated with chemotherapeutic agents melphalan, chlorambucil and p-N,N-bis(2-chloroethyl) aminophenylacetic acid [Treatment of lymphocytes with aneuploidogens vincristine and demecolcine of women . Aneuplodetected . Non-distic acid .Reasons of preliminary inclusion of some chromosomes in spontaneous and induced MN require further investigation. But it is known, that chromosome-specific aneuploidy play key roles in the development and progression of cancers. Thus, precise identification of the specific chromosomes and chromosome regions involved in the observed alternations should be continuously important areas for future research.Analysis of chromosomal contents of spontaneous MN in normal woman lymphocytes using SKY  and FISHIn spontaneously arising MN of blood cells of patients with immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF) chromosome 1 appeared to be present in a higher proportion compared to chromosomes 9 and 16. Chromosome 18, not associated with the ICF syndrome, showed no signal in any of the MN observed . FISH anWe studied the involvement of chromosomes 7, 18 and X in mitomycin C (MMC)-induced MN using wcp and chromosome-specific centromere probes. It was shown that X-chromosomal material was over represented in female- and under represented in male-derived MN. MN with centromeric and wcp signals from chromosome X in MMC-treated human leukocytes is presented in Figure Until now, FISH has not been widely applied in plant mutagenesis because DNA probes required for chromosomes of particular plant species are very limited. The study  is a rarIn summary, combined application of the FISH technique with comet and MN assays permits to improve the ability of these widely used genotoxicity tests.Tests for estimation of genotoxicity belong to the express methods and should be easy and rapid in application. These advantages determine some limitations of methods, namely inability to recognize damage of certain loci of genome. MN and comet assays with application of different kinds of FISH probes offer unique possibility to detect on the same specimen the total DNA and chromosome damage and evaluate damage of specific regions of genome as well.MN and comets appear by loss of DNA material from the nucleus in micronuclei and in comet tail, respectively. Therefore, both methods reflect secondary rather than primary effects of DNA damage. FISH analysis of origin and contents of MN and comets promotes the better understanding of mechanisms of their formation necessary for correct application of methods.Special modifications for concurrent application of FISH with comet and MN assays were elaborated. It was confirmed that data obtained with FISH on MN and comets are comparable with results of metaphase and interphase FISH.Comet-FISH technique permits to gain valuable and reliable information, particularly about DNA damage and repair in general and also in relation to the organization of the nucleus. Some questions relating to the behaviour and organization of DNA within the comet were clarified using FISH technique. Comet-FISH was applied for detection of DNA damage and repair of cancer relevant genes, for measuring transcription-coupled repair, identification of genome targets of action of various genotoxic agents, including anti-tumour preparations.MN-FISH permits to discriminate aneugenic and clastogenic effect in MN and to recognize contents of MN. MN-FISH was applied in various researches for elucidation of mechanisms of genomic instability, distribution of chromosome breaks in genome and, to some extent, the etiology of certain human maladies.Indeed, the available data demonstrate that FISH technique is able to develop the genotoxicity assessment using the comet assay and MN test.The author declares that they have no competing interests.GGH wrote the manuscript. Author read and approved the final manuscript.


Self-inflicted foreign bodies in the male urethra and urinary bladder are an emergency that urologists may rarely have to face. A case of an electrical wire inserted in the male urethra and coiled in the bladder is presented.A 53-year-old male presented with the inability to void and bloody urethral discharge after having introduced an electrical wire in his urethra for masturbation 3 hours earlier. He had made several unsuccessful attempts to remove it.The variety of these objects may be impressive and removal of the foreign body may be quite challenging requiring imagination and high-level surgical skills., In this case an electrical wire was used and the diagnostic as well as the therapeutic steps for its removal are presented. Self-insertion of foreign bodies into the male urethra and urinary bladder for autoerotic stimulation is a rather rare emergency condition that an urologist may encounter. A case of an electrical wire inserted in the male urethra and coiled in the bladder is presented.A 53 year old male presented with the inability to void and bloody urethral discharge after having introduced an electrical wire into his urethra for masturbation 3 hours earlier. He had made several unsuccessful attempts to remove it.During the physical examination, the two ends of the wire were observed in the urethral meatus Figure . An x-raA large number of self-inflicted foreign bodies have been reported in the male urethra and urinary bladder -5. The vThe most common reason for self-insertion of a foreign body into the male urethra is of erotic or sexual nature, especially masturbation or sexual gratification -4. A menClinical presentation may vary from asymptomatic to swelling of external genitalia, dysuria, poor urinary stream or retention, bloody or purulent urethral discharge and ascending urinary tract infection ,2.Depending on the type of foreign body and its location, various methods of removal have been described, including meatotomy, cystoscopy, internal or external urethrotomy, suprapubic cystostomy, Fogarty catheterization, and injection of solvents. Removal of the foreign body may be quite challenging requiring imagination and high-level surgical skills. Endoscopic therapy is the standard. The most suitable method is relevant to the size and mobility of the object. In the majority of mobile objects inside the urethra, the mobility is towards the bladder where, after having been pushed, the foreign body can be grasped by forceps or retrieval baskets. Nephroscopes have been used for the retrieval of screws as well as magnetic retrievers for galvanic objects . The YAGA self-inflicted foreign body in the urethra and bladder is a rare situation. Endoscopic manipulation is the preferred first-line treatment and if unsuccessful, open procedures may be necessary.The authors declare that they have no competing interests.KS was the patient's surgeon and has been involved in drafting the manuscript and revising it critically for important intellectual content. GKoritsiadis has made contributions to conception and design. GKoutalellis contributed to the analysis and interpretation of data and was also the doctor who examined the patient in the emergency room. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


We report a case of vascularised fibula osteocutaneous flap used for composite cervical spinal and posterior pharyngeal wall reconstruction, in a patient with recurrent skull base chordoma, resected by an anterior approach via median labio-mandibular glossotomy approach. Bone stability and pharyngeal wall integrity were simultaneously restored Microvascular transfer of fibular bone has been used in many ways in reconstructive surgery. A case of skull base chordoma, which recurred multiple times, was resected by an anterior approach via median labio-mandibular glossotomy approach. Vascularised fibula osteocutaneous flap was used for cervical spinal and posterior pharyngeal wall reconstruction. The case demonstrates the complexity in the management of recurrent skull base chordomas, and the aptness of the flap in simultaneously restoring the bone stability and pharyngeal wall integrity.A 41-year-old male with a history of chordoma of the cranio-vertebral junction operated upon multiple times earlier, presented with complaints of pain, limitation of neck movements, and difficulty in swallowing. The first surgery was done eight years back through a posterior approach, at which time the tumour was decompressed and an occipito-cervical stabilization was done. This was followed by transoral decompressions done on two occasions. Neurological examination revealed exaggerated deep tendon reflexes and extensor plantar reflex on the right side with intact cranial nerve functions, normal muscle bulk and power in all four limbs. A computed tomography (CT) scan showed a large recurrence Figures  involvinAfter necessary preoperative evaluation and counselling he underwent a sub total excision of the tumour by median labio-mandibular glossotomy approach. Reconstruction was carried out using a vascularised fibula osteocutaneous flap. The fibular strut was used to reconstruct the vertebral column. It was wedged between healthy bone of the lower clivus and the C3 vertebral body, and held in place with a small mini-plate at the lower end of the graft. The skin paddle was used to reconstruct the posterior pharyngeal wall which was largely scarred from the previous surgeries and was excised with the tumour. The artery was anastomosed to the lingual artery and the vein to one of the tributaries of internal jugular vein in the neck. The recipient vessels in the neck were in close proximity to the donor vessels and so the length of the vascular pedicle was adequate. Chordomas are comparatively slow growing malignant neoplasms derived from notochord. In the cranial region the tumours usually arise from the clivus. Clival chordomas usually present in the third and fourth decades of life and there is slight male preponderance. Surgical resection with or without adjuvant radiotherapy is the treatment of choice. Because of their locally invasive nature into the cervical spine, parasellar structures, and intradural extensions, true microscopic total removal of clival chordomas is typically not possible. The endoNon vascularised bone grafts have been used successfully for the reconstruction of cervical spine. Iliac crest bones, autogenous and allogeneic segments of fibula have been reported.5 The suc6et al.[et al.[The advantage of vascularised bone is the superior healing in compromised settings. Pedicle flaps from the ribs have been used for thoracolumbar spinal reconstruction.10 Fibula11et al. have desl.[et al. have repThough there are a few earlier reports of this reconstructive technique, our case stands out as it demonstrates the complexity in the management of recurrent skull base chordomas, and the aptness of vascularised fibula osteocutaneous flap in the composite reconstruction of pharyngeal wall and cervical spine.


A Baltic population of Atlantic sturgeon was founded ~1,200 years ago by migrants from North America, but after centuries of persistence, the population was extirpated in the 1960s, mainly as a result of over-harvest and habitat alterations. As there are four genetically distinct groups of Atlantic sturgeon inhabiting North American rivers today, we investigated the genetic provenance of the historic Baltic population by ancient DNA analyses using mitochondrial and nuclear markers.A. oxyrinchus descended from a relatively small number of founders originating from the northern extent of the species' range in North America.The phylogeographic signal obtained from multilocus microsatellite DNA genotypes and mitochondrial DNA control region haplotypes, when compared to existing baseline datasets from extant populations, allowed for the identification of the region-of-origin of the North American Atlantic sturgeon founders. Moreover, statistical and simulation analyses of the multilocus genotypes allowed for the calculation of the effective number of individuals that originally founded the European population of Atlantic sturgeon. Our findings suggest that the Baltic population of A. oxyrinchus colonized the Baltic Sea ~1,200 years ago, suggesting that Canadian specimens should be the primary source of broodstock used for restoration in Baltic rivers. This study illustrates the great potential of patterns obtained from ancient DNA to identify population-of-origin to investigate historic genotype structure of extinct populations.These results demonstrate that the most northerly distributed North American  Acipenser sturio, found in France (Gironde basin), and the Atlantic sturgeon A. oxyrinchus inhabiting the rivers and coastal waters from the Gulf of Mexico to the Canadian Maritime Provinces. Although classified as sister species and showing some phenotypic similarities, approximately 60 million years of isolation  was invns – see  choosingA. sturio and A. oxyrinchus have several diagnostic substitutions in the flanking region of the microsatellite locus Aox-23  and A. The size of the founding population in the Baltic Sea in the Early Middle Ages was inferred from seven microsatellites and mtDNA control region sequences. The following population history was assumed in our simulations: a small part of the source (Canadian and Mid-Atlantic) populations colonized the Baltic Sea at 1,200 years before present (ybp), then the populations of both sides of the Atlantic kept a constant size  until the Baltic population became extinct. The Baltic founder population was assumed to experience a single-generation bottleneck, because the population size of species having a potential to produce a huge number of offspring is expected to show a dramatic increase after they settle themselves in a suitable environment. However, we also tested bottleneck periods of different lengths, as well as a gradual increase of the population size after the colonization, to check the sensitivity of the results to this assumption. Coalescent simulations were iterated 1,000,000 times, varying the effective population size of the founders as well as the source. Uniform prior distributions are assumed for both founder  and source  females as well as mutation rates . In general, fishes are characterized by very low mutation rates and sturgeons have one of the lowest mutation rates within all vertebrates . As we apδ = 0.001) with the smallest distances were selected and used in the final analyses. Local weighing and calculations of the posterior density functions were carried out for each locus using the R functions.The approximate Bayesian computation (ABC) method  was applmt: mitochondrial; a: ancient; NA: North America.AL initiated the study, did statistical analysis and wrote the paper. UA did experimental work. SL did statistical analysis (hybrid detection). NB provided archaeological samples and background information. LD did morphological analyses. TLK did analysis of NA populations and revised the language. SM did demographic modelling. All authors read and approved the final manuscript.A. sturio (As) and A. oxyrinchus (Ao) :464–9; no accession numbers are archived in Genbank).Alignment of partial d-loop sequences of Click here for file


Abnormal bleeding may result from deficiency of one of the clotting factors.A 13-year-old Indian girl, presented with a history of bleeding following minor injury, epistaxis, and hematuria, bleeding from gums, subcutaneous bleeding and gastro intestinal tract bleeding. She underwent a complete coagulation profile test, CT scan and ultrasound. Platelet Factor 3 dysfunction was diagnosed to be the cause of bleeding. Patient was transfused fresh frozen plasma and platelet as part of treatment. Condition of the patient after four months of diagnosis deteriorated and she started bleeding in tears and sweat.A thorough examination and proper workup are necessary to determine the exact cause and rule out serious conditions. Depletion of any of the clotting factor leads to bleeding from different sites. Platelet factor 3 availability (PF3-A) being an insensitive to plasma coagulation factors are highly specific and reproducible . ClottinA 13-year-old school-going Indian girl child complained of bleeding from gums, hematuria, sub cutaneous bleeding, gastrointestinal bleeding which she regurgitates and collects in her oral cavity, prolonged bleeding after minor trauma and history of epistaxis. Patient later developed conjunctival hemorrhage leading to bloody tears and hematohidrosis. There was no family history of bleeding. The complete coagulation profile  is normaSubconjunctival hemorrhage and hematohidrosis observed as the secondary presenting clinical feature of PF3 dysfunction for the first time in the present study. Sodhi and Jose  earlier A case of PF3 dysfunction shows an unusual clinical secondary entity of hematohidrosis and bloody tears for the first time. It can be very complicating for the clinician. A thorough examination and proper workup are necessary to determine the exact cause and rule out serious conditions.Parents of the patient gave the written informed for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The author declares that there are no competing interests with any author.KLM was solely responsible for conducting, writing and editing this manuscript.


Posterior shoulder fracture-dislocation is a rare emergency condition with poor prognosis when there is a delay in diagnosis and presence of associated injuries.We present a case of a neglected four-part fracture-dislocation of the proximal humerus in a 34-year-old Greek woman. Except from the substantially displaced and comminuted tuberosity fractures, an anterolateral defect of approximately 50% of the articular surface was apparent. Open reduction of the humeral head was followed by reconstruction of the proximal humerus with allograft impaction, transfer of lesser tuberosity to the humeral defect and anatomic fixation of the greater tuberosity and humeral neck fractures. At two and a half years postoperatively, the humeral head was revascularised and properly articulated with the glenoid fossa.The presented case underlines the variability of injury pattern, the potential of missed diagnosis and the need for preserving the humeral head in young patients regardless of the amount of articular surface defect and disruption of soft tissue attachments. Posterior locked shoulder dislocation is an uncommon injury  which may be misdiagnosed and overlooked in up to 60% of cases . The speWe present a case of a neglected four-part posterior fracture-dislocation of the proximal humerus in a young woman. The vascularity and integrity of the humeral head were at high risk due to a large reverse Hill-Sachs lesion (50% of the articular surface) and severely displaced tuberosities fractures. Open reduction and internal fixation of the humeral neck and greater tuberosity fractures in combination with grafting and transfer of the lesser tuberosity to the humeral defect led to joint stability, viability of the humeral head and favourable functional outcome.A 34-year-old right-hand dominant Greek woman, presented at the Upper Limb Clinic of the Hospital complaining of persisting pain and stiffness in her right shoulder. The symptoms began 3 months earlier after a fall on her outstretched hand from a height of approximately 3 metres. The patient reported that the initial clinical assessment in the local emergency department and the anteroposterior radiograph of the right shoulder did not reveal any significant abnormality and a diagnosis of shoulder sprain and contusion was established. Pain medication was prescribed and a sling was applied for 10 days. After that time, the patient was re-examined and physical therapy with active and passive shoulder and upper limb exercises was commenced. As there was no improvement in pain and shoulder mobility, she was finally referred to our clinic for a second opinion and further evaluation.On physical examination, her shoulder looked flattened anteriorly and both acromion and coracoid processes appeared to be prominent at the anterior part of the shoulder. There was an internal rotation deformity of 30° and any effort to passively or actively move the glenohumeral joint was extremely painful. Forward elevation of 40°, no external rotation and inability to completely supinate the forearm were also identified. The patient did not have any neuromuscular deficit and her medical history was unremarkable in terms of previous injuries in the shoulder region or other medical comorbidities. The anteroposterior radiograph of the right shoulder illustrated the marked internal rotation of the proximal humerus and the typical "lightbulb sign". The greater and lesser tuberosities were fractured and displaced from each other and from the humeral head. A further undisplaced fracture line at the anatomic neck of the proximal humerus was also evident Figure . Becauseet al. [® DBM Putty, Osteotech, Eatontown, NJ) and stable fixation of the lesser tuberosity was achieved with two partially threaded 4.0 mm titanium screws (Figure According to these findings, open reduction and reconstruction of the proximal humerus was considered necessary. Under general anaesthesia, the patient was placed in a beach chair position and the glenohumeral joint was assessed via a deltopectoral approach. The axillary nerve was palpated to ascertain its position but it was not mobilised. The long head of the biceps was still intact and both tuberosities were localised and circumferentially released from the newly formed granulation tissue and immature callus. As the capsule was torn and detached along with the lesser tuberosity, mobilisation of the bone fragment in a "trap-door" manner allowed easy access and visualisation of the glenohumeral joint. The humeral head was found to be dislocated posteriorly, the posterior labrum was pulled out from the glenoid and a layer of fibrous tissue covered the glenoid cavity Figure . After met al. . Aiming s Figure . The grePostoperatively, the extremity was placed in a sling with the shoulder in neutral rotation and slight abduction. At 4 weeks, passive shoulder and pendulum exercises were initiated and the patient was advised to use the sling for another 4 weeks. At 8 weeks, a more aggressive physical therapy with active assisted range-of-motion and strengthening exercises was instituted as plane X-rays showed maintenance of joint congruency and early signs of bone healing. Despite the instructions for examination at regular intervals, the patient did not return for follow-up until two and a half years postoperatively. She reported that her shoulder was totally painless without any limitations during daily activities. She could actively elevate and abduct her arm 150° and 120°, respectively. In internal rotation, she reached the L2 vertebra and external rotation was 40°. Plane radiographs Figure  and CT set al. [et al. [The rarity of incidence of posterior-fracture dislocation, the potential for delay in diagnosis and the lack of evidence-based management strategies make this specific injury type challenging to treat. Recently, Robinson et al.  divided  [et al. . Even ifApart from the severity of injury and fracture deformity, the final prognosis is further affected by the extent of the underlying glenoid or reverse Hill-Sachs lesion ,6. As exet al. [et al. [et al. [et al. [The transfer of lesser tuberosity instead of subscapularis alone was first introduced by Hawkins et al. . The ostet al. . Finkels [et al.  reported [et al.  noted si [et al.  found raAlthough avascular necrosis of the humeral head is unpredictable and may occur in any posterior fracture-dislocation pattern, neglected injuries and fracture of the anatomic neck substantially increase the above incidence . AccuratPosterior shoulder fracture-dislocation continues to be a "diagnostic trap" for the unaware physician despite the advances in imaging techniques and the continuous flow of information about the risk of missed diagnosis. In neglected injuries, open reduction of the humeral head, stable fixation of all of the associated fractures and filling of the anterolateral defect with graft and/or transfer of lesser tuberosity may lead to optimum result and good functional recovery.The authors declare that they have no competing interests.BEC prepared and submitted the article. PP collected and analysed the data while CGD critically revised the manuscript. Each author read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The sensitivity of eight cell lines established from treated and untreated patients with small cell carcinoma of the lung (SCCL) was tested in the clonogenic assay with 1 h and continuous exposure to aclarubicin (ACLA), adriamycin (ADR), daunorubicin (DAU) and mitoxantrone (MITO). The sensitivity to ADR, DAU and MITO covariated, and varied with a factor of five. The sensitivity to ACLA was independent of the sensitivity to ADR and varied only within a factor of two. Only ACLA showed pronounced increased potency with continuous incubation, and ACLA was the most potent drug in the three cell lines least sensitive to ADR. Two resistant cell lines were selected by treating NCI-H69 in vitro with DAU. One cell line (9-fold resistant to DAU) expressed large amounts of P-glycoprotein, the other cell line (4-fold resistant to DAU) had barely detectable glycoprotein. Both lines acquired resistance to ADR, ACLA and MITO. The cross-resistance to ACLA and MITO was only partial and ACLA was still the most potent drug on these lines. The sensitivity to ACLA of the cell lines least sensitive to ADR suggest that ACLA partially circumvents mechanisms of multidrug resistance. Together with the pronounced increase in potency with prolonged exposure, these results suggest that ACLA has a mechanism of action different from the 'classical' anthracyclines. In this context mitoxantrone is more similar to the classical anthracyclines although its structure is more dissimilar.