Medical students preparing for final exams need practical and theoretical knowledge. We evaluated a junior doctor led integrated programme delivering theoretical and practical teaching.An 8-week junior-doctor led teaching programme was set up for final year medical students. Theory, OSCE and bedside teaching on selected weekly clinical themes were run. Satisfaction was evaluated using a questionnaire survey.Almost all agreed that the programme was useful and that an integrated approach to teaching was more beneficial than separate and unrelated lectures and practical teaching. The majority agreed that theory sessions and practical sessions had improved their confidence for finals and agreed they felt more prepared for work as a doctor. Most agreed that the Facebook® group provided an easily accessible platform for communication and sharing learning resources. Some comments, however, highlighted limitations particularly in the ability to answer difficult questions.Integrated teaching by junior doctors in small groups appeared to be an efficient teaching method  for medical students, improving their confidence for finals and life as a doctor and provided useful opportunities for junior doctors to develop as clinical teachers. This can be a useful blueprint for other hospitals. Final year medical students need to acquire practical and theoretical knowledge to sit finals and start work. Teaching programmes addressing this must consider the complex context of adult learning that requires a variety of teaching methods , 2. FactFinal year medical students were assigned to Bournemouth and Poole Hospitals for 8-week attachments. The programme had eight themes on key medical emergencies and subjects often encountered in finals. These were taught in three styles of sessions Fig. : The levClassroom teaching focused on key facts, ways of remembering them, passing finals and being a safe doctor. Each hour session was split into 20–30 min blocks run by different tutors to provide variety and help maintain focus.Objective structured clinical examination (OSCE): In each hour session, two common stations were taught in small groups. Stations addressed physical examination skills and communication scenarios. Students learned by observing, practising and receiving feedback in a supportive environment.Buddy scheme: Each student was paired with a doctor for one-to-one bedside teaching aimed to recognize clinical signs, develop practical skills and provide pastoral support.Junior doctors signed up to specific sessions. They were expected to have passed OSCE stations themselves before being allowed to teach and to individually prepare for the sessions. Clear learning objectives and session resources were provided to enhance preparation and performance. All trainees had to undertake a ‘Developing the Clinical Teacher’ supervised learning event as a curriculum competency with a supervisor of their choice . This toThe project was reviewed by the local Research & Development department. We obtained confirmation that the project and data collection met criteria for a service evaluation rather than research as it involved evaluation of existing teaching sessions with questionnaires completed voluntarily and anonymously. This work did not require submission for review by ethics nor National Health Service permission. The work was carried out in accordance with the Declaration of Helsinki in particular anonymity was guaranteed and consent for evaluation was obtained.Thirty-four from a total of 38 (89 %) participated. All rated the programme as useful and 34/34(100 %) felt it was pitched at the right level. 23/38(60 %) participated in a more detailed questionnaire and provided comments. 22/23(96 %) attended theory and 21/23(91 %) attended OSCE sessions. All agreed that a programme of this style should be available to all final medical students and all agreed that an integrated approach is more beneficial at their stage than separate unconnected lectures and practical teaching. The majority felt that theory 20/23(87 %) and practical sessions 21/23(91 %) improved their confidence for the written and OSCE element of finals. 19/23(83 %) agreed that near peer teaching was more relevant and focused than sessions provided by more senior clinicians and 21/23(91 %) agreed that they felt more prepared for work as a doctor. 18/23(78 %) used the Facebook® group but 20/23(87 %) agreed that this was an easily accessible platform for communication and sharing resources. Feedback for the buddy programme was mixed with only 11/23(48 %) responses. Only 2/11(22 %) agreed that the buddy improved confidence for final year examinations and 5/11(45 %) were undecided. Pass rates for final year students attending the hospitals before the teaching programme were above 95 % and remained so for students undergoing the teaching.Free comments were thankful and complimentary of the programme and the doctors who taught them. They reported it to be ‘concise’, ‘well-organized’ and ‘relevant’. Students found teachers ‘approachable’ and ‘relatable’ and mentioned that OSCE practice with senior doctors can be ‘more intimidating’. Some comments, however, highlighted the limitations of near peer teachers stating, ‘when asked difficult questions they are less able to answer’. Teachers themselves found involvement beneficial to their development, improving teaching, organizational and leadership skills. The majority volunteered to teach in subsequent groups.Integrated learning for finals was well received. All students agreed this style of teaching on a chosen weekly subject in a variety of contexts  was an effective way of teaching, increasing confidence and should be available to all final medical students. Arguably an important factor contributing to the success was the attention to set up the programme bearing in mind factors that facilitate adult learning. It is well established that the focused learning time for an adult is 20 min. Without a suitable break, attention fluctuates down to three-four minute spurts. Altering lecture content in short classroom sessions and offering a variety of teaching environments may have contributed to maintaining attention in participants, maximizing retention of information and improving satisfaction , 2. The A strength of the programme was the peer and near peer teaching. Such teaching has been proven to be effective and an enjoyable way of providing information, and trainers can develop as role models , 7 In ouFeedback from the junior doctor teachers confirmed that this programme was a good opportunity to develop competences as clinical teachers and pass on their recent experiences of final year examinations. It also offered experience in educational leadership and management. Overall the programme was a positive experience for both students and junior doctor teachers and may serve as a model for other organizations hosting students and junior doctors.


We demonstrate that abundance, reproductive output, and growth rate of Common Gartersnakes living in field and forest habitats are good metrics of habitat suitability, whereas stress hormone levels show no difference in suitability between habitats. Thamnophis sirtalis). We conducted a validation study and confirmed that fCM concentrations indeed reflect circulating corticosterone concentrations. We estimated abundance, reproductive output and growth rate of gartersnakes in field and in forest habitat and we also measured fCM concentrations of gartersnakes from these same habitats. Common gartersnakes were more abundant and had higher reproductive outputs and higher growth rates in field habitat than in forest habitat, but fCM concentrations did not differ between the same two habitats. Our results suggest either that fCM concentrations are not a useful metric of habitat suitability in common gartersnakes or that the difference in suitability between the two habitats was too small to induce changes in fCM concentrations. Incorporating fitness metrics in estimates of habitat suitability is important, but these metrics of fitness have to be sensitive enough to vary between habitats.Measuring habitat suitability is important in conservation and in wildlife management. Measuring the abundance or presence–absence of a species in various habitats is not sufficient to measure habitat suitability because these metrics can be poor predictors of population success. Therefore, having some measure of population success is essential in assessing habitat suitability, but estimating population success is difficult. Identifying suitable proxies for population success could thus be beneficial. We examined whether faecal corticosterone metabolite (fCM) concentrations could be used as a proxy for habitat suitability in common gartersnakes ( Habitat loss is one of the greatest threats to global biodiversity . TraditiIdeal despotic distributions  are a clHabitat occupancy also can be a misleading metric of habitat quality because metapopulations often have source–sink dynamics . In a soIdeal despotic distributions, source–sink systems and ecological traps exemplify why abundance and occupancy are not necessarily useful measures of habitat suitability. Ecologists have been advocating the use of measures other than abundance to estimate habitat suitability for >30 years . AlthougCorticosterone concentrations are sometimes too variable to be a good indicator of habitat suitability. For example, different levels of human disturbance had no effect on corticosterone concentrations in eastern hellbenders . CorticoThamnophis sirtalis) living in field vs. forest habitat. We conducted a validation study and confirmed that fCM concentrations indeed reflect circulating corticosterone concentrations. We compared baseline fCM concentrations with abundance, individual growth rate and reproductive output of gartersnakes living in each habitat. Field and forest have very different thermal regimens; field is thermally superior to forest for snakes in our study area  concentrations as a proximate measure of habitat suitability for common gartersnakes (udy area . For thi2 plots in adjacent fields and forests and placed eight plywood coverboards (360 cm2) on each plot . We set up five pairs of 2500 mach plot . We systn = 3) and close to our enclosures in Québec (n = 17). In the laboratory, before putting females in the enclosures, we gave each female the opportunity to mate with three or four males collected from the same area. We observed every female mate during these encounters. We fed half of the snakes in each habitat two large earthworms per week and we fed the other half of the snakes four large earthworms per week to determine whether fCM concentrations could be an indicator of habitat quality only with certain caloric intake levels. We systematically placed females in each food and habitat treatment combination based on their body size to ensure that each combination had similar distributions of body sizes, because body size may influence growth, reproductive frequency and reproductive output. We measured the body temperature of each snake with an infrared thermometer immediately before feeding it. We measured the mass and snout–vent length (SVL) of each snake once per week and also attempted to collect a faecal sample from each snake once per week. Faecal samples were stored in individual sealed tubes and were kept on ice until they were returned to the laboratory, where they were frozen at −80°C until they were processed. Snakes started giving birth in late August and finished by early September. We removed all snakes from the enclosures after the snakes gave birth and returned all mothers and their offspring to their point of capture.We examined the growth rate, reproductive output and fCM concentrations of 20 female common gartersnakes in enclosures in Pontiac County, Québec, Canada . We built 2.67 m × 2.67 m × 1.33 m frames out of lumber and created walls for the enclosure by attaching polyethylene vapour barrier to the frames. We buried the bottom 10 cm of the walls of the enclosures in the ground to prevent snakes from escaping. We built six of these enclosures in field habitat and an additional six in forest habitat. Each enclosure contained a 60 cm × 60 cm plywood coverboard for shelter. In late May 2014, we placed 10 females in forest enclosures and the other 10 females in the field enclosures; snakes were housed either in pairs or on their own. We captured all female snakes for this experiment in fields and wetlands near Ottawa, Ontario  into a new tube and stored the extract at −80°C until we measured fCM content by commercial radioimmunoassay (RIA). The extraction efficiency for this procedure was estimated by splitting a sample in two, spiking one half of the sample with a known amount of corticosterone, measuring corticosterone/fCM content in each half and comparing recovered corticosterone with the amount added, for six independent samples; using this method, extraction efficiency was 50%. This value is relatively low compared with ∼90% extraction efficiency for the faeces of ground squirrels , which was previously used by rd curve .We calculated the change in mass of snakes as the difference in the mass of a snake between the beginning and the end of the experiment. Likewise, we calculated the change in SVL of snakes from the beginning to the end of the experiment. We compared changes in mass and SVL of snakes in field vs. forest habitat using general linear models in R . Snakes in the field grew more in terms of both mass  or on SVL increase .Common gartersnakes demonstrated a strong preference for field over forest habitat , nor were fCM concentrations affected by body temperature .Snakes in field and in forest did not differ in fCM concentrations . Yet fCM concentrations in our study did not differ between snakes living in field and in forest habitats, and also did not vary with body temperature. Stress hormones exhibit variable relationships with temperature across different taxa. Fish have demonstrated a consistently positive relationship between cortisol and temperature e.g.  between In animals, important differences between habitats include energetic  and structural differences (e.g. presence of cover or nesting sites), which can have different physiological consequences. Structural differences between habitats could lead to differences in predation risk, and animals could then experience increased stress hormones in the riskier habitat  of Canada Post-Graduate Scholarship to W.D.H., and NSERC Discovery Grants to K.M.G. and G.B.-D.Supplementary DataClick here for additional data file.


IRI). Following an acute episode of sustained myocardial ischaemia, the opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion, mediates cell death. Preventing MPTP opening at the onset of reperfusion using either pharmacological inhibitors [such as cyclosporin A (CsA) ] or genetic ablation has been reported to reduce myocardial infarct (MI) size in animal models of acute IRI. Interestingly, the endogenous cardioprotective intervention of ischaemic conditioning, in which the heart is protected against MI by applying cycles of brief ischaemia and reperfusion to either the heart itself or a remote organ or tissue, appears to be mediated through the inhibition of MPTP opening at reperfusion. Small proof‐of‐concept clinical studies have demonstrated the translatability of this therapeutic approach to target MPTP opening using CsA in clinical settings of acute myocardial IRI. However, given that CsA is a not a specific MPTP inhibitor, more novel and specific inhibitors of the MPTP need to be discovered – the molecular identification of the MPTP should facilitate this. In this paper, we review the role of the MPTP as a target for cardioprotection, the potential mechanisms underlying MPTP inhibition in the setting of ischaemic conditioning, and the translatability of MPTP inhibition as a therapeutic approach in the clinical setting.Mitochondria have long been known to be the gatekeepers of cell fate. This is particularly so in the response to acute ischaemia‐reperfusion injury (http://dx.doi.org/10.1111/bph.2015.172.issue‐8This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit  ANTadenine nucleotide translocaseCABGcoronary artery bypass graftCsAcyclosporin ACypDcyclophilin DDrp1dynamin‐related protein 1GSKglycogen synthase kinaseIPCischaemic preconditioningIPostischaemic postconditioningIRIischaemia‐reperfusion injuryLVleft ventricularMImyocardial infarctATPMitoKmitochondrial ATP‐sensitive potassium channelMPTPmitochondrial permeability transition poreOMMouter mitochondrial membraneOPA1optic atrophy 1PMIperioperative myocardial injuryPPCIprimary percutaneous coronary interventionRICremote ischaemic conditioningRISKreperfusion injury salvage kinaseROSreactive oxygen speciesSAFEsurvivor activating factor enhancementSTEMIST segment elevation myocardial infarctionVDACvoltage‐dependent anion channelTables of Linkset al., et al., et al., et al., et al., The mitochondria are crucial determinants of cardiomyocyte fate following an episode of acute myocardial ischaemia‐reperfusion injury (IRI). In particular, reperfusing acutely ischaemic myocardium induces the opening of the mitochondrial permeability transition pore (MPTP), an event that mediates cardiomyocyte death by uncoupling oxidative phosphorylation and causing mitochondrial swelling. Preclinical animal studies  channel, inhibits calcium‐induced MPTP opening in adult rat cardiac mitochondria  integrity and maintained mitochondrial function. However, no studies have investigated directly whether the MPTP is a target for cardioprotection in the setting of RIC.The mechanism through which the cardioprotective signal applied to an organ or tissue remote from the heart activates pro‐survival signalling pathways in the heart is not clear. Wang et al.  have sho(1) Passive pathway – ischaemic conditioning modulates factors such as cellular energy status, mitochondrial calcium and phosphate accumulation, oxidative stress, and intracellular pH changes, which are known to affect MPTP opening susceptibility  Active pathway – ischaemic conditioning activates a signalling pathway, which then modifies MPTP opening susceptibility by either interacting with putative components of the MPTP, or by modulating the same factors alluded to in the ‘passive pathway’.The actual mechanism through which the cardioprotective signal elicited by ischaemic conditioning mediates its inhibitory effect on MPTP opening at the time of myocardial reperfusion is not clear. A number of potential mechanisms have been proposed, and these can be broadly divided into two different categories  , IPC may reduce mitochondrial calcium overload during ischaemia and inhibit MPTP opening at reperfusion  at the onset of myocardial reperfusion, can limit MI size  have been linked to the activation of the RISK pathway and in the cases of IPC and IPost the activation of this salvage kinase cascade has been shown to inhibit MPTP opening .The activation of the Akt component of the RISK pathway is known to recruit the eNOS‐NO‐sGC‐cGMP‐PKG pathway and through this cascade , attenuating ROS production, improving glucose‐induced insulin release, preventing acidosis through enhanced coupling of glycolysis and glucose oxidation, and inhibition of fatty acid oxidation  ] or fission to form fragmented mitochondria [regulated by the mitochondrial‐fission proteins  ]  has also been identified as a key enzyme involved in cytoprotection in the heart  hypertension, on cardioprotection  and Cyclosporine and Prognosis in Acute Myocardial Infarction Patients (CIRCUS)  multi‐centre randomized clinical trials are currently investigating CsA therapy in PPCI‐treated STEMI patients.The ongoing CYCLosporinE A in Reperfused Acute Myocardial Infarction (CYCLE)  surgery, subjects the heart to acute global IRI as the heart is put onto and taken off cardiopulmonary bypass. The extent of perioperative myocardial injury (PMI) can be quantified by measuring the release into blood of cardiac enzymes, such as creatine kinase and Troponin T or I, the release of which has been linked to worse clinical outcomes. We have recently found that administering a single dose of CsA prior to CABG surgery reduced the extent of PMI in CABG surgery patients  underlying cardioprotection by ischaemic conditioning remains unclear, evidence suggests that it prevents MPTP opening at the reperfusion by modulating factors known to MPTP opening at this time, such as cellular energetic status, mitochondrial CaNone.


Food allergy is a major public health problem in children, impacting upon the affected individual, their families and others charged with their care, for example educational establishments, and the food industry. In contrast to most other paediatric diseases, there is no established cure: current management is based upon dietary avoidance and the provision of rescue medication in the event of accidental reactions, which are common. This strategy has significant limitations and impacts adversely on health‐related quality of life. In the last decade, research into disease‐modifying treatments for food allergy has emerged, predominantly for peanut, egg and cow's milk. Most studies have used the oral route , in which increasing amounts of allergen are given over weeks–months. OIT has proven effective to induce immune modulation and ‘desensitization’ – that is, an increase in the amount of food allergen that can be consumed, so long as regular  doses are continued. However, its ability to induce permanent tolerance once ongoing exposure has stopped seems limited. Additionally, the short‐ and long‐term safety of OIT is often poorly reported, raising concerns about its implementation in routine practice. Most patients experience allergic reactions and, although generally mild, severe reactions have occurred. Long‐term adherence is unclear, which rises concerns given the low rates of long‐term tolerance induction. Current research focuses on improving current limitations, especially safety. Strategies include alternative routes , modified hypoallergenic products and adjuvants . Biomarkers of safe/successful OIT are also under investigation. Food allergy is a major public health issue throughout the world, particularly in children. There is no established treatment for use in routine clinical practice: management involves avoidance of the culprit food(s) and rescue medication in the event of accidental reactions Food allergy is estimated to affect up to 6% of children in Europe Cow's milk, egg, peanut and tree nuts are the most common food allergens in children The spectrum of severity for symptoms during food‐allergic reactions is variable and includes life‐threatening anaphylaxis and even death Even with appropriate dietary avoidance, accidental allergic reactions are common: up to 40% of children allergic to cow's milk had at least one reaction every year according to one report Many factors contribute to rescue medication often not being used appropriately in the community when needed. Many parents/patients – especially teenagers – do not carry their medication at all times, find it difficult to identify anaphylaxis symptoms or to use auto‐injector effectively in these stressful situations Given the above, there is demand for a disease‐modifying treatment for food allergy, particularly for the most common, ubiquitous and dangerous allergens in terms of fatal anaphylaxis: cow's milk and peanut. Different strategies are under investigation, the most common approach being oral immunotherapy (OIT) with over 60 studies published in peer‐review journals. Results have been promising in terms of effectiveness and positive impact on parent‐assessed HRQoL. However, significant concerns remain regarding the safety of OIT, and its ability to induce permanent tolerance. Current research focuses in improving these two key issues. The benefits and pitfalls of OIT are summarized in Table OIT consists of giving increasing amounts of food allergen orally over weeks or months (‘updosing phase’). Once the target dose is reached, this amount is given on a regular basis, usually daily (‘maintenance phase’). This results in an allergen‐specific immunomodulatory effect. A recent meta‐analysis showed a reduction in skin prick test wheal size and an increase in specific IgG4‐blocking antibodies following OIT to cow's milk, egg and peanut OIT is effective in increasing the amount (or threshold) of allergen food‐allergic individuals are able to eat without experiencing an allergic reaction, an effect termed desensitization. A meta‐analysis by Nurmatov et al. not transient desensitization) after 3–5 years of active treatment – yet the same criteria are not applied to OIT. Further research is needed to clarify whether a longer course of OIT would increase rates of tolerance, and whether OIT only accelerates allergy resolution in those who would have developed natural tolerance without any intervention. As it is difficult to determine for how long OIT maintenance should be stopped to prove permanent tolerance (most studies utilize a ‘short’ off‐OIT phase of 2–8 weeks), the term ‘sustained unresponsiveness’ is preferred instead of tolerance It seems a little incongruous that for other forms of immunotherapy – such as subcutaneous immunotherapy (SCIT) for aeroallergens and venom, success is generally defined as tolerance  experience anaphylaxis to an OIT home dose, this at could be reported as a rate of 10% (patients) or 0.05% of doses (9125 doses administered over the 6 months). Furthermore, different classification systems are used to describe severity across studies, and the indications to administer adrenaline can vary considerably. In this context, there are major limitations to compare safety outcomes across studies and identify optimal OIT regimens.On the one hand, most studies report that OIT‐related reactions are generally mild and usually tend to decrease or resolve overtime. Probably many patients may undergo successful OIT without major safety concerns A few open studies of OIT have shown a positive impact on children's quality of life following successful OIT using validated questionnaires to assess HRQoL No studies have, as yet, assessed whether successful OIT improves nutrition by allowing the child to re‐introduce foods (such as cow's milk) into their diet, although the impact on nutrition may be limited given that OIT is not generally performed in very young children. Studies on cost‐effectiveness of OIT are also lacking at this time. OIT studies to date have focused on efficacy as the primary outcome. Whether this is the most important outcome for patients has not been evaluated. There is a need to involve patients and caregivers in defining relevant outcomes for studies on treatments for food allergy.Several approaches have been proposed to improve the safety of OIT. These include research on using alternative routes of exposure, modified hypoallergenic products and adjuvants for immunotherapy as well as on biomarkers of safe and successful OIT to facilitate patient selection Fig. .The identification of biomarkers associated with safe (and successful) OIT might help select suitable candidates more likely to respond safely to OIT, and screen out those candidates in whom OIT may lead to unnecessary risks. Although the outcome of OIT probably depends on multiple factors, some individual characteristics might have a predictive value. In a recent study of cow's milk OIT, patients who were withdrawn due to significant reactions had IgE binding to a broader diversity of peptides  and at higher intensity than those children who completed OIT successfully Several studies have used the sublingual route for food immunotherapy . This approach builds on existing evidence on the efficacy and good safety profile of sublingual immunotherapy (SLIT) for inhalant allergens Epicutaneous immunotherapy (EPIT) involves the application of a patch containing the food allergen to the skin. The epidermis is poorly vascularized, which might limit the potential for systemic reactions. In contrast, it contains high numbers of potent antigen‐presenting cells, which may allow immune modulation and enhanced efficacy The first trial on immunotherapy for food allergy was a RCT performed in the 1990s in 12 peanut‐allergic adults using SCIT (aqueous peanut extract) for 1 year The underlying rationale is to use products that have reduced or no ability to bind specific IgE and activate mast cells and basophils through modification of allergenic sites or epitopes. However, their ability to interact with T cells is preserved to allow immune modulation without triggering allergic reactions. Such products can be found ‘naturally’ in our diets or produced in the laboratory.independent of allergen consumption. Indeed, having IgE against specific linear peptides/epitopes is associated with clinical reactivity to baked foods, and a more persistent course of cow's milk and egg allergy There is now strong evidence that baked foods containing extensively heated cow's milk or egg in baked foods  are tolerated by approximately 70% of children with milk or egg allergy A novel approach to peanut desensitization is currently being tested in a Phase 2/3 study (NCT02149719) using boiled peanut. This has reduced allergenicity compared to raw or roasted peanut . Boiling appears to result in the loss of key allergenic components  into the cooking water E. coli (EMP‐123). Unfortunately, 5 of 10 peanut‐allergic adults required discontinuation due to significant allergic reactions The introduction of point mutations into known IgE‐binding epitopes of food allergens, either through chemical modification or site‐directed mutagenesis, can be used to develop recombinant proteins with reduced allergenicity. Hypoallergenic mutants of peanut, fish and apple allergens have been generated using the latter technique This approach uses overlapping peptides (protein fragments 10–20 amino acids long) which represent the entire sequence of the allergenic protein. These short peptides cannot cross‐link two IgE molecules, but can interact with antigen‐presenting cells. Such approaches have been successfully used for immunotherapy to aeroallergens Anti‐IgE appears to be is a very promising therapy for IgE‐mediated allergic diseases, and probably acts by two mechanisms: preventing free IgE molecule from binding to its receptors on effector cells, and through effects on effector cells including by downregulating the expression of the high‐affinity IgE receptor on mast cells, and decreasing basophil histamine release in vitro studies suggest that gut microbiota modulate immune programming, promote oral tolerance and are important inhibiting the development of the allergic phenotype. The utility of pre‐biotics , probiotics (live microorganisms that benefit the host) and/or symbiotics (a combination of both) in the prevention and/or treatment of allergic diseases has been addressed in different studies Evidence from animal models and E. coli was associated to the above‐mentioned rectal EMP‐123 vaccine for peanut allergy As bacteria are potent stimulants of Th1 immune responses, modified bacterial products are under investigation as adjuvants for allergen‐specific immunotherapy. In a dog model of food allergy, a single subcutaneous injection with a mixture of heat‐killed Listeria monocytogenes (HKLM) and either peanut, or milk and wheat, significantly reduced anaphylactic symptoms on oral food challenge Daily antihistamines have been used during OIT updosing in several studies There is little data relating to the longer‐term safety of OIT. Clearly, a major issue is that of sustained unresponsiveness vs. transient desensitization, the latter requiring ongoing regular exposure to maintain desensitization. One concern remains issues of compliance, particularly in teenagers where this can be a problem during OIT Finally, there is also a concern that OIT can result in the development of eosinophilic oesophagitis (EoE) and other food‐induced enteropathies. A systematic review reported EoE in up to 2.7% of patients undergoing OIT for IgE‐mediated food allergy  Disease‐modifying treatments for food allergy are under development and have shown promising results to date, in terms of efficacy. OIT does induce desensitization, but its ability to induce permanent tolerance once ongoing exposure has stopped is limited. More research is needed into strategies – such as combining routes of exposure or identifying biomarkers of safer and successful OIT – to improve both efficacy and safety of immunotherapy treatments for food allergy Fig. . Safety


Recent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53 000 clones tested using naïve screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high‐throughput expression for subsequent production and characterization. The pre‐screening of clone libraries by naïve screens followed by the pyrosequencing of the inserts allowed for a 106‐fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time‐consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine‐tuned naïve‐ and sequence‐based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market. Accordingly, the demand for biocatalysts in the form of free or immobilized enzymes, whole cell catalysts or cell‐free systems, with a high applicability potential in industry is increasing . As an example, microbial communities from approximately 2192 different sites distributed across the planet have been examined for their metagenomic content. They include habitats such as terrestrial , marine  and freshwater  habitats; other types of habitats included non‐marine saline and alkaline lakes, acid mine drainage systems, wastewater treatment sludges, compost (consortia bred on plant biomass) and eukaryotic‐associated microbiomes  . This suggests that we have apparently undersampled all representative types of habitats. Within the investigated sites, clones containing new enzyme activities or purified enzymes  were isolated and  characterized   the selective focusing on activity‐based enzyme mining, and the establishment of larger and diverse clone libraries , depends on the enzyme activity under screening and the substrates used in the search, among other potential factors. Of note, the abundance level of the corresponding genes encoding the enzyme activities of interest in microbial genomes (see comments below) and the activity level of the enzymes are important factors affecting the efficiency of the screening programmes. Having said that, other key potential driving factors, such as the metagenome source, the DNA extraction method, the cloning vector, the expression system or host cells, the screening technique and screening conditions, to cite some, are additional factors influencing the success of the enzyme identification process. As example, enhanced expression systems based on viral components that prevent transcription termination at metagenomic terminators resulted in a sixfold increase in the frequency of carbenicillin resistant clones , such as acylases, phosphatases, proteases, oxidoreductases, glycosyl hydrolases and lipases/esterases  . Note that actually at least 200 distinct substrate molecules have been successfully applied in assays for esterases/lipases biocatalysts at high throughput scale for selection in metagenomic clone libraries.Concerning the substrate‐dependent efficiency of screening programmes, a number of interesting patterns could be observed. Thus, it was demonstrated that the incidence rate decreased from 1:188  to 1:3937 (127 clones) and 1:15 625 (32 clones) when the library was screened for esterase and lipase activity, respectively, using 1% (v/v) tributyrin, tricaprylin and triolein as the indicator substrates . At least 15 distinct chromogenic and fluorimetric substrates, for which extensive frequency data are available, have been commonly and successfully employed for the screening of clones with glycosidase activity (from references given in Table S1). Cross‐linked azurine hydroxyethyl cellulose, a unique substrate for the measurement of endo‐cellulase, provided a major incidence rate (1:108)  , whereas p‐nitrosoaniline complemented with D‐glucose and used in combination for screening of α‐glucose dehydrogenase activity has been shown to exhibit the lowest hit rate (1:20 000) ‐ketoprofen‐specific hydrolytic activities  such as Lake Arreo (1:1152) or deep‐sea Matapan–Vavilov basin (1:667)  for each type of sequences, and the protein sequences can be analysed in detail to identify the domains or motifs that are specific for the desired activity or structurally classified by the active site modelling and clustering method  in bacterial genomes  and the very expensive and time‐consuming biocatalysts optimization phase while increasing the efficiency of the processes. However, there are very few cases in which a new environmental biocatalyst has been translated to a process in recent times  years from the time when a gene is identified until the industrial process is established Fig.   location of sites together with appropriated references and site characteristics are specifically described. Whether the habitats have been subjected to direct sequencing [for community structure analysis and gene content by high throughput (HTP) sequencing] or enzyme screening  is also cited.Table S2. Results of single references for the incidence rates of positive clones or enzymes. Examples are provided for the screening of oxidoreductase and glycosidase activity by using multiple substrates after naïve screens. The number of clones tested, the number of positive clones, the incidence rate, the substrate used and the reference are given. For abbreviations, see Fig. 4 legend.Click here for additional data file.


Plants have a long evolutionary history, during which mass extinction events dramatically affected Earth's ecosystems and its biodiversity. The fossil record can shed light on the diversification dynamics of plant life and reveal how changes in the origination–extinction balance have contributed to shaping the current flora.We use a novel Bayesian approach to estimate origination and extinction rates in plants throughout their history. We focus on the effect of the ‘Big Five’ mass extinctions and on estimating the timing of origin of vascular plants, seed plants and angiosperms.Our analyses show that plant diversification is characterized by several shifts in origination and extinction rates, often matching the most important geological boundaries. The estimated origin of major plant clades predates the oldest macrofossils when considering the uncertainties associated with the fossil record and the preservation process.Our findings show that the commonly recognized mass extinctions have affected each plant group differently and that phases of high extinction often coincided with major floral turnovers. For instance, after the Cretaceous–Paleogene boundary we infer negligible shifts in diversification of nonflowering seed plants, but find significantly decreased extinction in spore‐bearing plants and increased origination rates in angiosperms, contributing to their current ecological and evolutionary dominance. See also the Commentary by Niklas We performed multiple search queries in order to obtain the most comprehensive dataset of the plant fossils, on a global level. The approach that captured the maximum number of records involved querying the database for all described plant classes and orders individually. With this approach we obtained 35 666 entries, which spanned most of the Phanerozoic (the oldest occurrences dating back to c. 424 million yr ago (Ma)). We included all occurrences that were identified to the species level as well as those identified only at the genus level but without specific assignation (e.g. ‘Psilophyton sp.’). We did not revise fossil identifications provided by the PBDB. Several culling procedures are normally used to avoid including incompletely identified taxa, such as searching and excluding entries identified with aff., cf., sp. or other modifications to binomial species names  are commonly assumed to be the most reliable source of data to study plant evolution, because they usually contain many taxonomically useful characters and can therefore be more easily and reliably assigned to different plant groups ; and (3) flowering seed plants (i.e. angiosperms). Fossil occurrences were assigned to the different categories based on the supra‐generic taxonomic information provided by the PBDB and, when available, using the Index Nominum Genericorum . We matched the names of fossil‐genera in our dataset with those listed at The Plant List (http://www.theplantlist.org/) to identify which taxa are extant.Analyses of diversification should ideally be performed on monophyletic groups, that is, clades in a phylogenetic sense. However, although monophyly assessment is straightforward in phylogenetic analyses based on molecular data, many questions remain on the phylogenetic placement of several fossil taxa. In addition, restricting the analyses to clades would require splitting the already fragmentary fossil data into smaller subsets, reducing analytical power and precision. We therefore categorized vascular plant fossil‐genera into three major groups, distinguished by their functional ecology, morphology and relationships , nonflowering seed plants (34%) and angiosperms (32%). Angiosperms encompassed 58% of the diversity (with 257 genera) whereas the remaining genera were evenly distributed among the other two plant groups. The large majority of fossil occurrences included temporal ranges (minimum and maximum ages), which typically derive from the upper and lower temporal boundaries of the stratigraphic units to which fossils were assigned. In our dataset, temporal ranges spanned on average 12.7 Myr with a standard deviation of 12.6 Myr. In order to account for these dating uncertainties in the analyses of origination and extinction rates we considered the temporal ranges as uniform distributions and, from these, we randomly resampled the ages of each fossil occurrence following Silvestro et al. . We geneet al., http://sourceforge.net/projects/pyrate/; Silvestro et al., et al., We carried out the analyses of plant diversification using a hierarchical Bayesian model to infer the temporal dynamics of origination and extinction  in the extinction rate of spore‐bearing plants following the Frasnian–Famennian boundary, which did not take place in similar magnitude among seed plants  had less detectable effects on plant diversity. However, although Cascales‐Minana & Cleal  found noCooksonia; Wellman, et al., et al., The origin of land plants can be established from the Dapingian (middle Ordovician) based on the earliest known cryptospores, whereas the first sporangia is dated from the Katian (late Ordovician) and first robust evidence of macroflora is found at the Wenlock Series .We placed the origin of seed plants between the Givetian (late Middle Devonian), and the Famennian (Late Devonian). Our estimation slightly predates that derived by Anderson et al. , which cy Cleal, . Similaret al., th Century, there is still considerable controversy about the timing of angiosperm origin. The root of this debate is derived from different scenarios obtained from using macro vs microfossil evidence or paleontological vs molecular data. From the fossil record, Friis et al. , whereas Smith et al. , which represented key points of floral turnover and contributed to major diversity changes of plant life. However, there was a differential effect of those events on each plant group analyzed, and substantial differences in the impact of each event on diversification dynamics. Although many uncertainties remain regarding the age of origin of major plant clades, our analyses have provided new estimates that help in reconciling current fossil evidence with a robust probabilistic approach that takes into account a complex and variable preservation process.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries  should be directed to the Table S1 Raw data downloaded from Paleobiology DatabaseClick here for additional data file.Table S2 Complete reference list of the original downloadClick here for additional data file.Table S3 Fit of different distributions to the posterior estimates of the origination times of three plant groups calculated by Akaike Information CriterionClick here for additional data file.


Biology is an analytical and informational science that is becoming increasingly dependent on chemical synthesis. One example is the high‐throughput and low‐cost synthesis of DNA, which is a foundation for the research field of synthetic biology (SB). The aim of SB is to provide biotechnological solutions to health, energy and environmental issues as well as unsustainable manufacturing processes in the frame of naturally existing chemical building blocks. Xenobiology (XB) goes a step further by implementing non‐natural building blocks in living cells. In this context, genetic code engineering respectively enables the re‐design of genes/genomes and proteins/proteomes with non‐canonical nucleic (XNAs) and amino (ncAAs) acids. Besides studying information flow and evolutionary innovation in living systems, XB allows the development of new‐to‐nature therapeutic proteins/peptides, new biocatalysts for potential applications in synthetic organic chemistry and biocontainment strategies for enhanced biosafety. In this perspective, we provide a brief history and evolution of the genetic code in the context of XB. We then discuss the latest efforts and challenges ahead for engineering the genetic code with focus on substitutions and additions of ncAAs as well as standard amino acid reductions. Finally, we present a roadmap for the directed evolution of artificial microbes for emancipating rare sense codons that could be used to introduce novel building blocks. The development of such xenomicroorganisms endowed with a ‘genetic firewall’ will also allow to study and understand the relation between code evolution and horizontal gene transfer. The high‐throughput and low‐cost synthesis of DNA, for example, is the foundation of the field of synthetic biology (SB). The aim of SB is to provide urgent biotechnological‐based sustainable solutions to problems in the health, energy and environmental sectors  aims to endow biological systems with artificial chemistry absent in natural organisms Schmidt, . XB is aIntroducing new functional members of XNA into either DNA or RNA and ncAAs into proteins breaks the universality of the genetic code of living organisms, alienating these from other natural life forms Budisa, . This alet al., et al., Notably, the difference between SB and XB is that the former generates genetically modified organisms (GMOs) based on natural chemical building blocks [e.g. canonical amino acids (cAAs)], whereas the latter aims at creating chemically modified organisms (CMOs) in which the chemical building blocks are artificial , is only encoded by the single UGG codon, whereas arginine (around 5.5% distribution in the E. coli proteome) is decoded by six triplets  and there are 4E. coli, out of the 61 sense codons, two are unique (methionine and tryptophan) while the rest are degenerate, with amino acids being encoded by as little as two and as much as six triplets  analogues, homologues and surrogates into heterologous‐expressed proteins  and tRNAs Fig. . While mCorynebacterium glutamicum in the Met‐auxotroph E. coli strain B834 while supplying the strain with O‐acetyl‐homoserine and inorganic sodium azide (NaN3) as precursors  Budisa,  Fig. 4AA4A have Methanocaldococcus jannaschii TyrRS:tRNATyrCUA‐pair, and PylRS:tRNAPylCUA‐pairs from Methanosarcina barkeri and Methanosarcina mazei. This stop codon suppression (SCS) approach allows the site‐specific incorporation of ncAAs into a target protein, which is usually overexpressed  by re‐introducing amber stop codons into essential genes . Because the UAG codon lacks any meaning in that distinct strain, these otherwise lethal stop codons have to be rescued with M. jannaschii TyrRS:tRNATyrCUA for p‐azido‐l‐phenylalanine (pAzF) or biphenyl‐l‐alanine (bipA)  or simply reverting the amber sites back to their original meaning. Thus, long‐term evolution experiments of strain C321.ΔA in more diverse and rich media are needed to ensure that it can be biocontained without reversing its genotype.Codon reassignment mainly includes the removal of the endogenous translation components like tRNA and aaRS, synonymous codon swaps throughout the chromosome or essential genes (Lajoie IleUAU gene in E. coli because the third base of the tRNAMetUAC is modified by tRNAIle‐lysidine synthetase (TilS) to lysidine, yielding tRNAIleLAU that is read by wobbling at the third base position  by using a rescue plasmid expressing the Mycoplasma mobile IleRS:tRNAIleUAU pair to sustain cell viability. The rescue plasmid can be easily removed in E. coli ΔtilS and can be exploited to apply evolutionary pressure to reassign the Ile codon AUA to another cAA  and l‐N6‐(1‐iminoethyl)lysine (l‐NIL) by using PylRS:tRNAPylCUA‐pair. Cells can survive the deletion of Arg rare codons (argW and argU) only if the evolved PylRS‐system is co‐expressed and AGG positions in most essential genes are exchanged by synonymous codons . Although analytical data were not presented , the exchange of Arg at remaining AGG positions most probably took place given that both ncAAs used  are similar to the cAA Arg (‘similar replaces similar’)  and phenylalanine (Phe) are withdrawn. This synthetic auxotrophy can be exploited to screen a library of artificial chorismate mutase analogues, in which aroQ is only composed from a nine‐amino acid alphabet. It was found that even this reduced set of amino acids can lead to a functional, metabolically competent and near to wild‐type growing strain. However, the reduction led to a restricted diversity ending up with a destabilization of overall protein structure. These results indicate the need of more amino acids for robust proteins and explain the evolutionary force towards the increased diversity. A more general approach towards this issue was reported by Marlière and co‐workers: they showed the possibility of removing Trp from the genetic code  as an analogue for leucine (Leu) can be isolated from a suitably designed cultivation experiment  as Trp source. Using the mutagenic agent it was possible to obtain a derivative exhibiting preference towards the ncAA. Nevertheless, those old experiments were not accompanied by precise analytic measurements to quantify the extent of ncAA incorporation . Moreover, those experiments were conducted in ‘rich’ medium in which standard amino acids, vitamins and nucleobases were supplemented. Therefore, only a small portion of the cellular machinery had to adapt to the incorporation of the ncAA. Bacher and Ellington . Yet they found a strain tolerating high levels of 4F‐Trp to a level at which it was toxic for the parental strain. Due to the lack of next generation sequencing, only a few relevant genes were sequenced but these were unchanged , while others like trpS, aroP and tyrR exhibited single nucleotide polymorphisms. Expression of trpS and aroP mutant proteins in trans in the ancestral strain led to a growth advantage in the presence of 4F‐Trp. However, those mutations are not sufficient to let the C600 strain incorporate the ncAA at high levels . Nevertheless, this experiment improved our understanding of serial evolution experiments to change the chemical composition of the proteome.Already in the late 1950s, Cowie and Cohen  demonstrllington  adapted E. coli cultivation experiments by the groups of Lenski, Ellington, Bacher and Wong, we also set out to adapt this bacterium towards an ncAA. In particular, our group completely substituted Trp by the analogue Thienopyrrole‐alanine  in a Trp auxotrophic strain . However, supplementing these cells with Trp reverses them to ‘natural’ ones given that they still favour the incorporation of the canonical building block. To achieve a Trp‐independent reassignment  at all UGG codons across E. coli's genome – an experimental strategy for biocontainment still needs to be developed and executed  the need for specific biological functions; (ii) the abundance of elements and precursors in past habitats on earth and (iii) the nature of existing solvent(s) and available energy sources in the prebiotic environment Budisa, . Thus faNone declared.


Identifying subpopulations within a study and inferring intercontinental ancestry of the samples are important steps in genome wide association studies. Two software packages are widely used in analysis of substructure: Structure and Eigenstrat. Structure assigns each individual to a population by using a Bayesian method with multiple tuning parameters. It requires considerable computational time when dealing with thousands of samples and lacks the ability to create scores that could be used as covariates. Eigenstrat uses a principal component analysis method to model all sources of sampling variation. However, it does not readily provide information directly relevant to ancestral origin; the eigenvectors generated by Eigenstrat are sample specific and thus cannot be generalized to other individuals.We developed FastPop, an efficient R package that fills the gap between Structure and Eigenstrat. It can: 1, generate PCA scores that identify ancestral origins and can be used for multiple studies; 2, infer ancestry information for data arising from two or more intercontinental origins. We demonstrate the use of FastPop using 2318 SNP markers selected from the genome based on high variability among European, Asian and West African (African) populations. We conducted an analysis of 505 Hapmap samples with European, African or Asian ancestry along with 19661 additional samples of unknown ancestry. The results from FastPop are highly consistent with those obtained by Structure across the 19661 samples we studied. The correlations of the results between FastPop and Structure are 0.99, 0.97 and 0.99 for European, African and Asian ancestry scores, respectively. Compared with Structure, FastPop is more efficient as it finished ancestry inference for 19661 samples in 16 min compared with 21–24 h required by Structure. FastPop also provided scores based on SNP weights so the scores of reference population can be applied to other studies provided the same set of markers are used. We also present application of the method for studying four continental populations .We developed an algorithm that can infer ancestries on data involving two or more intercontinental origins. It is efficient for analyzing large datasets. Additionally the PCA derived scores can be applied to multiple data sets to ensure the same ancestry analysis is applied to all studies.The online version of this article (doi:10.1186/s12859-016-0965-1) contains supplementary material, which is available to authorized users. Genome wide association (GWA) studies usually evaluate data from thousands of individuals. Identifying the subpopulations within the data set and inferring biogeographic origins of the samples are important steps in the conduct of any study. Not allowing for population substructure in the analysis will introduce false positives . FurtherWe selected 2318 SNPs across the whole genome based on having a large fixation index (FST) value among European, African and Asian populations for PCA analysis. We conducted PCA analysis of 505 Hapmap samples with European, African or Asian ancestry along with a collection of 19661 additional samples of unknown ancestry. To perform PCA, we use the eigendecomposition method, which parses the covariance relationships among markers.Define For the mean-centered data matrix, we need to compute the mean on each SNP,Then, we generate the mean-centered data matrix asN > P). We construct the covariance matrix asAssume that there are larger number of samples than number of SNPs  and the matrix λi.In PCA, the ZN × k = YN × P × PP × k (1). Where k = 2 or 3 is adequate for capturing ethnic similarities when considering 3 or 4 continental origins respectively. The eigenvectors areFor our purposes we find that selection of SNPs to maximize interancestral variability ensures that the first few eigenvectors capture interpopulation variation. We therefore obtain a low dimensional projection, score matrix Once we select the first k eigenvectors, named as SNP weights, which we would like to keep among the principal components computed from the discovery data, we can predict new scores in the new data using pre-computed SNP weights.Let PP × k from the original analysis, compute the new score matrix, Z*N × k = WN × P × PP × k (2). For prediction of new score matrix, we recommend that the SNP weights should be generated from large samples (N> > P) to avoid variance shrinkage if the eigenvectors will be applied to a subsequent data set [Using the SNP weights data set .We started with three well characterized continental populations, studied by the HapMap consortium of European, Asian, and African descent, since characterizing ancestry for these populations is often a major goal in genome-wide association studies. The centroid of each known Hapmap population was characterized by the multivariate mean of the first and second score Fig. . A trianhttp://hapmap.ncbi.nlm.nih.gov/downloads/genotypes/hapmap3_r3/plink_format/.The approach can also be generalized to include additional populations. When we plotted the first three PC scores for individuals coming from four distinct populations , we observed a three-dimensional tetrahedron instead of a two dimensional triangle for three populations, which suggested we could extend the triangle algorithm to a tetrahedron-based algorithm to incorporate four populations. When applied to four populations, a similar approach to that taken for 3 populations is employed. We first define regions for which the closest point to an ancestry tetrahedron is a vertex, and for those individuals, a single continental ancestry is assigned. For points outside the tetrahedron but closest to a side, we project to the nearest face of the tetrahedron (as described in the Supplementary methods) and then use equation 4 to estimate the proportions of ancestry for each of three origins for that face. For points inside the tetrahedron, an extension of equation 4 to encompass a mixture of 4 populations is applied. In this case, the projections are to each of the four faces of the tetrahedron. For interior points, once the sample was mapped to the two-dimensional face  is modeled by a characteristic set of allele frequencies and the main modeling assumptions are Hardy-Weinberg equilibrium within populations and complete linkage equilibrium between loci within populations . StructuPCA has become a standard procedure in population genetics study for substructure analysis. The eigenvectors from PCA are easy to use for population adjustment in GWA studies. However it lacks the ability to provide clear information for ancestral origin. To fill this gap, we developed an efficient tool for inference of ancestry with PCA scores as the input. The PCA scores generated by FastPop can be used to identify ancestries of individuals or could be used to adjust for population structure in association analysis. The scores are based on SNP weights so the scores of reference population such as Hapmap samples can be applied to other studies provided the same set of markers are used. This characteristic is attractive especially in large consortia studies when multiple independent studies may be analyzed by individual laboratories. The PCA scores generated in one site can be adopted by other sites thus to reduce the repetitive work and ensure consistency among analyses. In current GWA studies, sample sizes keep increasing and now involve tens of thousands individuals. FastPop can be more efficiently implement than Structure in analyzing data with a large sample size.FastPop provides the estimated centroids from a training set considering the users may have a small data set and may require a golden standard for the centroid positions for the populations. Theoretically, the triangle model in FastPop will work without training samples. When the sample size of a data comprising of three populations is big enough, we can calculate the centroids position for each population based on the principal component values from the study samples instead of deriving centroid positions from Hapmap samples. We also tested this idea by inferring ancestry for 19661 study samples without using Hapmap samples, and the correlations of the results between FastPop and Structure were still > 95 %. For this approach, one needs to define a set of centroids for defining ancestral origins.As a further comparative analysis, we also evaluated linear discriminative analysis (LDA) method applied to PCA scores as input to predict ancestry. Compared to LDA, FastPop had better performance in terms of the estimated proportions, consistent performance across different cut off values for decisions and a lower excess positive rate for Europeans. We are using the term ‘excess positives’ here to denote the classification of individuals who may have multiple ancestries into a single ancestry group by LDA  – the Institutional Review Board (IRB) at Dartmouth College. All human subjects involved in this study consented to research involving genetic analysis. All individuals signed Institutional Review Board approved consent documents related to genetic analysis of germline samples. Samples were deidentified prior to analysis. All the analysis was performed at Dartmouth College.Additional file 1:Supplementary Materials. (DOCX 35 kb)


Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc. Many human genetic disorders are caused by structural variants such as deletions, duplications, and inversions of genomic regions involving several genes. Many deletion syndromes caused by deletions spanning several million base pairs, have severe phenotypes, for example, DiGeorge and Smith–Magenis syndromes. Large duplications are somewhat more benign, unless the duplicated region is very large, such as that seen in Down's syndrome. Inversions are often asymptomatic because relatively few genes are affected, although very large heterozygous inversions can result in infertility.loxP recombination , inversions and duplications extending to several 100 kb in mammalian cell lines and up to 1 Mb in zebrafish have been generated  because the arrangements in this region should be non‐lethal and Tyr provides the ability to score re‐arrangements phenotypically  of these carried an imprecise deletion, and 3 (4%) had correctly incorporated the bridging oligonucleotide in the repair process  and Grm5 (545 kb) as well as the whole region from Nox4 to Grm5 including Tyr (1.15 Mb) .Several junction points in the proximity of the nge Fig. . Two paiMore than half of the injected embryos developed to term. The resultant founder mice were genotyped for the full spectrum of variants, using a variety of polymerase chain reaction (PCR) assays Fig. . Mice wiAllelic heterogeneity was also observed. Given the loss of sequence at some of the breakpoints, some inversions were scored with one breakpoint only. Many of the founders were mosaics (Supporting Information Table 1). Mice could be identified with both an inversion as well as a deletion allele, others were observed with wild type, inversion and deletion alleles, and yet others were mosaic with an inversion, a deletion, and indels which could be detected at one or both breakpoints on a non‐rearranged chromosome. Sequencing of DNA samples from the mice revealed the expected allelic heterogeneity at the breakpoints observed in the outcome of the experiments with the 9.4 and 64.8 kb deletions. The co‐injected oligonucleotide led to the generation of mice with a defined deletion for all of the intervals tested at an overall rate of 9%.Upon breeding at least 50% transmitted the deletion or inversion to the next generation independent of the rearrangement size (Supporting Information Table 2). Fluorescent in situ hybridization was conducted on some of the F1 pups which confirmed the rearrangements detected by PCR analysis .To further understand the contribution of the different gRNAs in the generation of the rearrangements we determined the cutting efficiency of each gRNA by analysis of the INDEL rate. The allele structure in the founder mice is complex due to mosaicism; therefore, we breed the founders to segregate their chromosomes and examined the gRNA target sites in mice which did not carry rearrangements (Supporting Information Table 3). INDEL efficiencies ranging from 8% to 90% were observed. In cases where cutting is highly efficient, for instance Grm5‐3 in which 18 out of 19 mice carried an INDEL, we observed additional INDELS on the same chromosome generated by the co‐injected gRNAs. In some cases up to three sites on the same chromosome have INDELs. Thus, even in cases where cutting is highly efficient, this frequently does not resolve in a rearrangement.We have examined the 1.15 Mb inversion breakpoints to identify which gRNA sites are responsible for the inversion breakpoints. On one side in 3 out of 10 cases the inversion utilized the outer gRNA with an INDEL efficiency of 14% rather than the adjacent gRNA with an INDEL efficiency of 90% . We find a similar situation on the other breakpoints. On this basis we can conclude that different combinations of gRNAs contribute to the generation of the inversions. Moreover, although the more efficient gRNA was used most frequently, this was not saturating because some inversions were generated by the less efficient gRNA.et al., et al., The CRISPR/Cas9 system is a very efficient and comparatively fast method to generate genome edited mice with small modifications such as insertion–deletions of a few nucleotides and the introduction of short tags via direct injection in zygotes . PCR amplification of a breakpoint requires that the primer sites are retained at the junctions, which may in some cases be degraded beyond the primer binding sites. In many cases the founder mice were mosaic, most easily detected from the observation of small patches of albino hair in otherwise black mice. This is reminiscent of chimaeras resulting from ES cell injection at the blastocyst stage (3.5 days). Given the limited number of DNA target molecules available and the vast excess of both Cas9 and gRNA, cutting and re‐joining must occur for an extended period of time, until the site is destroyed by an INDEL or a structural variant. Analysis of somatic DNA from the ear and germ line transmission detects up to 4–5 alleles which suggests that Cas9 is active to the 2–4 cell stage at least.The generation of an inversion or deletion requires two break and re‐joining events on the same chromosome, which using dual gRNAs is efficiently achieved. The occurrence of combinations of deletions and inversions in single founder mice, often with mosaicism with an unmodified and/or homologue, illustrates that the overall efficiency of the process is high. To generate founder mice with this genotype, the CRISPR/Cas9 process must be active on either homologues or two sister chromatids simultaneously and/or cutting and re‐joining may continue after the first zygotic division. We reasoned that the provision of two pairs of gRNAs targeted to each breakpoint, would extend the temporal window for achieving the desired modification by providing the opportunity for two complete cycles of error‐prone repair before the target sites are destroyed. Analysis of the resultant mice confirms that different combinations of the gRNAs are able to contribute to the generation of the structural variant, not just the most efficient ones. In some cases variants are found in which the less efficient gRNA has been used to generate the break point, but the same chromosome contains an INDEL generated by the more efficient gRNA. We cannot resolve if the INDEL was generated before or after the re‐arrangement, but it is not unreasonable to conclude that because of its efficiency its target site was rapidly destroyed by INDEL formation thereby excluding this gRNA from contributing to the process. We can further conclude that although two gRNAs are sufficient more events are generated with four.In these experiments we sought to generate deletions with nucleotide precision through the provision of a repair template in the form of a single strand oligonucleotide. The sequence of the oligonucleotide was designed to omit the Cas9 cleavage sites to prevent re‐cleavage of the repaired chromosome by continued CRISPR/Cas9 cutting. Precise deletion junctions were successfully generated for all but one of the intervals attempted in this study, although this was most efficient for the 1 Mb deletion. While we do not understand the reason for this, the proximity of the broken ends may favor simple NHEJ for smaller genetic intervals. The oligonucleotide directed the repair of the break in 17 out of 53 32%) of the mice born. One potential reason for this is exonuclease activity may resect the broken DNA at the break point beyond the limit of the sequence in the oligonucleotide. Overall, an equal number of deletions (precise plus imprecise) and inversions were generated  and in vitro transcribed using mMessage mMachine T7 Ultra kit and MEGAshortscript T7 kits (Life Technologies), respectively. Both, Cas9 mRNA and gRNA, were purified using the MEGAclear kit (Life Technologies) and eluted in RNase‐free water. The quality of the RNA was analyzed using Agilent RNA 6000 Nano kit  and Qubit RNA HS assay kit (Life Technologies). The bridging oligonucleotides were dissolved in RNAse‐free water to a concentration of 1,000 ng/μL.For Cas9 RNA production, the T7/Cas9 plasmid was linearized with et al., Four‐ to five‐week‐old C57BL/6NTac females were super‐ovulated by intraperitoneal (IP) injection of 5 IU of pregnant mare's serum (PMSG) at 12:00 to 13.00 hours  followed 48 hours later by an IP injection of 5 IU human chorionic gonadotrophin (hCG) and mated overnight with C57BL/6NTac stud males. The next morning the females were checked for the presence of a vaginal copulation plug as evidence of successful mating, oviducts were dissected at approximately 21–22 hours post HCG and cumulus oocyte complexes from these were released and treated with hyaluronidase as previously described  and 100 ng/μL oligonucleotide were mixed in RNase free water, backfilled into an injection needle with positive balancing pressure and injected into the cytoplasm of fertilized 1‐cell embryos held in FHM medium. A successful injection was indicated by visible movement in the cytoplasm . Injected embryos were briefly cultured and viable embryos were transferred the same day by oviducal embryo transfer into a 0.5 days post coital pseudo‐pregnant female F1 (CBA/C57BL/6J) recipients . The PCR products were examined on gels and sequenced to ascertain the integrity of inversions, duplications, and deletions. Resulting positive F0 founders were bred to examine transmission of the detected alterations  and after 44 hours blocked in metaphase with 0.1 μg/mL KaryoMax Colcemid (Gibco Life Technologies) for 2 hours prior to harvesting chromosomes. These were hybridized with probes using a standard FISH protocol  labeled with Aminoallyl‐dUTP‐Texas Red and RP24‐102H24  Aminoallyl‐dUTP‐Cy5 (Jena‐Bioscience) within the genome edited region. The control probes RP23‐84N6  labeled with Aminoallyl‐dUTP‐XX‐Atto‐488 and RP23‐335D4  labeled with Aminoallyl‐dUTP‐Cy3, lying outside the genome edited region (Supporting Information Table 6).The BAC clones were prepared with a BAC DNA kit (Sigma‐Aldrich), amplified using a whole genome amplification kit . Using 10–100 ng of purified genomic BAC DNA the WGA‐2 products were then directly labeled using a GenomePlex Reamplification kit  with Aminoallyl‐dUTP‐Cy3, Aminoallyl‐dUTP‐Cy5, Aminoallyl‐dUTP‐Texas Red, and Aminoallyl‐dUTP‐XX‐Atto‐488, respectively. Hybridization and washes were carried out by standard procedures.FISH images were captured and processed using the SmartCapture  digital imaging system using a Zeiss microscope  equipped with narrow bandpass filters for Cy5, Cy3, Texas Red, FITC and DAPI fluorescence, a cooled CCD camera (Hamamatsu).KB designed and performed most experiments. BD developed and performed the cytoplasmic injections. RB and FY designed and performed the fluorescent insitu hybridization. AB devised and coordinated the project, and together with KB, designed experiments, interpreted data and wrote the manuscript.Supporting Information Figure 1.Click here for additional data file.Supporting Information Figure 2.Click here for additional data file.Supporting Information Figure 3.Click here for additional data file.Supporting Information Figure 4.Click here for additional data file.Supporting Information Tables.Click here for additional data file.


We conducted retrospective analyses of commercial claims and electronic medical record (EMR) databases to understand how treatment patterns for mCRPC have changed in a US‐based real‐world population. Truven Health Analytics MarketScan® (2000–2013) and EMR (2004–2013) databases were used to identify patients with an index prostate cancer diagnosis (ICD‐9 codes 185X or 233.4X) and prescription claims for an mCRPC drug . Regimen analyses for first line of therapy (LOT1), second line of therapy, and beyond were performed among cohorts based on year of first mCRPC drug usage. mCRPC drug usage and treatment duration were compared across cohorts and age groups within each cohort. The commercial claims cohort yielded 3437 evaluable patients. Most men (91%) commencing mCRPC treatment had docetaxel as LOT1 in 2010; this number had declined to 15% in 2013. In 2013, 67% and 9% of patients used abiraterone acetate and enzalutamide, respectively, as LOT1. Among both commercial claims and EMR cohorts, treatment pattern changes were most pronounced in men aged >80 years, and median treatment duration for some mCRPC drugs was shorter than expected based on available clinical trial information. These results demonstrate a shift in mCRPC treatments during the past 5 years, with greater use of newer noncytotoxic treatments than docetaxel. These real‐world data aid in understanding the changing role of chemotherapy in the management of mCRPC.Despite increasing drug treatment options for metastatic castration‐resistant prostate cancer ( For more than three decades, the mainstay of medical therapy for advanced prostate cancer has remained androgen deprivation therapy (ADT) via the medical disruption of the androgen‐signaling pathway Historically, the treatment of metastatic castration‐resistant prostate cancer (mCRPC) has focused on palliation, with modest impact on patients’ survival. In addition to the mainstay of ADT, mitoxantrone was approved in 1996, based primarily on improvement of quality‐of‐life measures, as opposed to prolonged survival More recently, five new agents have been approved by the US Food and Drug Administration (FDA) for mCRPC Table  2, 11, cWith the improved toxicity profile of abiraterone acetate and enzalutamide over docetaxel, there may be a broader population of patients who are eligible for mCRPC treatment with these agents as opposed to chemotherapy. Consequently, a shift to later use of docetaxel may be anticipated. Patterns of real‐world treatment data have been infrequently reported in patients with mCRPC, with limited data on how these new agents are sequenced. We therefore conducted a retrospective analysis of commercial claims and electronic medical record (EMR) data to better understand how treatment patterns for mCRPC have changed.® Arbor, Michigan, US claims databases were used to identify the study population . The database contained claim‐level data of privately insured individuals. Additionally, EMR‐based data (2004–2013) compiled by IMS Health from 327 US oncology practice sites were used to identify patients for a validation cohort . All patient data were de‐identified, in compliance with the Health Insurance Portability and Accountability Act (HIPAA) regulations.Truven Health Analytics MarketScanInternational Classification of Diseases, ninth revision, clinical modification (ICD‐9‐CM) diagnosis code for prostate cancer (ICD‐9 codes 185 or 233.4) occurring between 2000 and 2013. Patients were excluded if they had <180 days of eligibility history prior to first prostate cancer diagnosis date; any other diagnosis of prostate cancer within these 180 days; or other primary malignancies within 5 years prior to the index prostate cancer diagnosis.Male patients aged ≥44 years were required to have a medical claim indicating an Patients with mCRPC were defined based on exposure to ≥1 drugs approved by the FDA for mCRPC . Yearly and multiyear cohorts were based on the year of first mCRPC drug usage, which was the date captured for the patient's first pharmacy claim for an mCRPC drug. We examined claims from 2000 to 2013 for three different multiyear cohorts . Within each multiyear cohort, patients were divided into three age groups: 44–64 years, 65–80 years, and >80 years. The >80 years age group cutoff was chosen to determine changes in treatment patterns for the very old, as only a small proportion of mCRPC patients aged >80 years receive cytotoxic chemotherapy currently A regimen analysis for first line of therapy (LOT1), second line of therapy (LOT2), and beyond second line of therapy (LOT2+) was carried out among 1‐year and multiyear cohorts. Analytic rules were applied to determine the lines of treatment used for each patient: (1) Claims for an mCRPC drug without any other claim for the same drug in the 90 days prior to and 90 days after a claim were excluded. (2) The mCRPC drug treatment period started on the first date of use to the last date of use; the latter was the earliest date when there was at least a 90‐day gap before the next claim of the same drug. (3) For an oral therapy, the end date for each treatment period was the last claim date in that treatment period plus the number of days’ supply, while that for injectable therapy was the last claim date. (4) Treatment periods of each mCRPC drug were configured such that the entire treatment duration for a patient was divided into multiple intervals, each with a distinct usage of regimens . Each successive interval was deemed a different line of treatment. (5) Detailed rules were applied to blank regimens, based on their duration and if the succeeding regimen was an expanded or reduced form of the preceding regimen. This resulted in blank regimens (no other claim of an mCRPC drug ±90 days from an mCRPC drug claim) being retained in some cases  and disregarded in others.® 9.3. Statistical differences were assessed with the independent sample t‐test and analysis of variance. Differences between cohorts regarding mCRPC drug usage were assessed by the Fisher's exact test and Pearson chi‐square test.Univariate descriptive statistics included the mean (±SD) and median values for continuous variables and relative frequencies for categorical variables. The analysis was carried out using SASA total of 326,907 patients with an index prostate cancer diagnosis from 2000 to 2013 were identified from the commercial claims database . The majority of patients had health insurance through a preferred provider and/or a health services network plan.P < 0.0001 for both LOT1 and LOT2). For the 2000–2003 commercial claims cohort, a total of 175 and 66 patients used an mCRPC drug for LOT1 and LOT2+, respectively . Among the 2010 commercial claims cohort, docetaxel usage was consistently at around 90% in all age subgroups  and docetaxel (100 days) . The use of cabazitaxel was less prominent in the docetaxel‐naïve, setting, in which there was a decreased proportion of prescription claims for cabazitaxel among patients with increasing age.Among patients aged 65–80 years and >80 years, the use of noncytotoxic agents was more prominent among those who were docetaxel‐naïve and there was an increased proportion of abiraterone acetate and enzalutamide usage among docetaxel‐naïve patients with increasing age Fig. . The utiThis retrospective analysis demonstrates that there has been a considerable shift in the treatment patterns of mCRPC since 2010 in accordance with the approval of five new medical therapies for its treatment, which have dramatically increased the treatment options for this patient population.The use of cytotoxic drugs—docetaxel in LOT1 and cabazitaxel in LOT2—has seen a remarkable decline. In contrast, the use of highly effective antiandrogen therapy (HEAT)—abiraterone acetate and enzalutamide—has become the dominant approach in both LOT1 and LOT2 mCRPC settings. Additionally, the median duration of therapy is generally longer with HEAT compared with the cytotoxic agents used for mCRPC, suggesting a net gain in clinical benefit and tolerance to patients who receive it. With the shift to new agents and more options in general, there are several emerging questions about the future treatment of mCRPC: Should treatment start earlier in the course of mCRPC, a disease state that frequently lasts several years? Will more patients be eligible for treatment with better‐tolerated agents? What is the best sequence of therapies? Will combination therapy with new agents be more beneficial than the current sequential therapy?Changes in the treatment patterns in LOT1 were among the most dramatic. Prior to 2010, approved treatment options for mCRPC were limited and first‐line docetaxel was common. From 2010 to 2013, the proportion of men in the commercial claims cohort who used for docetaxel as LOT1 decreased from 91% to 15%, with LOT2 usage increasing modestly during this period, from 4% to 18%, suggesting a decrease and delay in docetaxel use for mCRPC. While the magnitude of these changes is notable, the overall shift from more toxic intravenous therapy to less toxic oral therapy itself is not surprising. During the observation period of this analysis, sipuleucel‐T and radium‐223 were approved for use in the pre‐docetaxel period; abiraterone acetate in the pre‐ and post‐docetaxel settings. This observation is reflective of the changes noted with the use of enzalutamide and cabazitaxel in the post‐docetaxel setting, consistent with their labeling at the time of this data collection.Usage of HEAT as LOT2+ was observed in the 2010 cohort. It should be noted that the yearly cohorts were defined on the basis of the first mCRPC drug usage. Thus, while the 2010 cohort would have had their LOT1 usage in 2010, their LOT2+ usage would have been more likely in 2011 and beyond, during the years when most of the newer agents were approved by the FDA for mCRPC. In addition, a small proportion of the commercial claims data may reflect mCRPC treatment exposure as part of a clinical trial and early access program.The duration of therapy data Fig.  provide Treatment decisions are important in older patients with mCRPC, considering the higher burden of medical comorbidities and physical limitations in this population. In the 2009–2013 cohort, the majority of patients (67%) were aged >80 years, and we observed distinct changes in treatment patterns across younger and older age cohorts. A shift away from treatment with docetaxel from 2010–2013 was most pronounced in men aged >80 years. Along these same lines, an assessment of the absolute use of any mCRPC drug in men aged >80 years showed that there were 132 total mCRPC regimens observed in 2013, compared with 41 total mCRPC regimens in 2010, suggesting an expanding group of eligible patients for this less toxic therapy.Historically, there have been concerns that elderly patients have been underrepresented in prostate cancer clinical trials and that little is known about the real‐world treatment of this age group Limitations of this retrospective analysis include the limited follow‐up of patients, particularly in the 2010–2013 cohort, and the ICD‐9 coding variations across practice types and geographic regions. In addition, the database represents privately insured individuals whose socioeconomic status may differ from that of the general population. Acknowledging these limitations, some internal crosschecking provides reassurance of data fidelity. The median duration of sipuleucel‐T administration (28 days) is consistent with the approved dosing regimen of three doses every 2 weeks. Moreover, the marked change in the use of docetaxel and mitoxantrone following the approval of docetaxel for advanced prostate cancer in 2004 fits expectations and historical trends. Another strength of this analysis is the utilization of both commercial claims and EMR databases, which are separate, and independent data sources to assess mCRPC treatment patterns for the period of dramatic change.These real‐world data provide a greater understanding of contemporary treatment of mCRPC beyond that discernable from clinical trial populations. Although randomized clinical trials are the gold standard by which to demonstrate the efficacy of new drug treatments, the study designs used in clinical trials typically place limitations on provider actions Treatment options for mCRPC have expanded in recent years with the approval of five new agents. There has been a dramatic shift in the treatment of mCRPC patients in LOT1 and LOT2, from cytotoxic chemotherapy (docetaxel) to HEAT. The shift away from cytotoxic therapy was most pronounced in elderly patients, mostly likely due to the better tolerance of HEAT, and this holds the potential of expanding the proportion of men able to receive treatment for mCRPC. Many questions remain, including the optimal timing, sequencing, and potential combination of these new agents. An understanding of the current treatment patterns will be essential to designing the next generation of clinical trials for mCRPC.Thomas W. Flaig has served as consultant/advisor to GTX and Janssen and received research funding from BN ImmunoTherapeutics, Cougar Biotechnology, Dendreon, Genentech, GTx, Janssen Oncology, Medivation, Novartis, Sanofi, and Exelexis. Ravi C. Potluri has served as a consultant/advisor to Janssen. Maneesha Mehra, Mary B. Todd, and Yvette Ng are employees of Janssen Research & Development and hold stock in Johnson & Johnson.Figure S1. mCRPC drug usage proportion among the 2012 and 2013 commercial claims and EMR cohorts.Click here for additional data file.Table S1. Insurance plan types in the mCRPC population: commercial claims databaseClick here for additional data file.Table S2. mCRPC drug usage proportion among the 2000–2003 and 2004–2008 cohorts.Click here for additional data file.Table S3. mCRPC drug usage proportion by 1‐year cohorts from 2010 to 2013 for LOT1 and LOT2+.Click here for additional data file.Table S4. mCRPC Drug Usage Proportion for LOT1 by Age Group and 1‐Year Cohorts From 2010–2013.Click here for additional data file.Table S5. mCRPC drug usage proportion for LOT1 by age group from the 2013 commercial claims and EMR Cohortsa.Click here for additional data file.Table S6. Estimated treatment duration (Days) of mCRPC Drugsa in the 2009–2013 Cohorts.Click here for additional data file.


Trypanosoma brucei. We outline four distinct stages of cytokinesis and show that an asymmetric division fold bisects the two daughter cells, with a cytoplasmic bridge‐like structure connecting the two daughters immediately prior to abscission. Using detection of tyrosinated α‐tubulin as a marker for new or growing microtubules and expression of XMAP215, a plus end binding protein, as a marker for microtubule plus ends we demonstrate spatial asymmetry in the underlying microtubule cytoskeleton throughout the cell division cycle. This leads to inheritance of different microtubule cytoskeletal patterns and demonstrates the major role of microtubules in achieving cytokinesis. RNA interference techniques have led to a large set of mutants, often with variations in phenotype between procyclic and bloodstream life cycle forms. Here, we show morphogenetic differences between these two life cycle forms of this parasite during new flagellum growth and cytokinesis. These discoveries are important tools to explain differences between bloodstream and procyclic form RNAi phenotypes involving organelle mis‐positioning during cell division and cytokinesis defects.Trypanosomes use a microtubule‐focused mechanism for cell morphogenesis and cytokinesis. We used scanning electron and video microscopy of living cells to provide the first detailed description of cell morphogenesis and cytokinesis in the early‐branching eukaryote  Saccharomyces cerevisiae and Schizosaccharomyces pombe  form trypomastigotes indicates that it is not essential for division  and the mammalian bloodstream form, were developed and are widely used in cell and molecular studies of T. brucei biology, life cycle stage differentiation, pathogenicity, and as a model organism for analysis of the flagellum and cytoskeleton. In our previous work, we established descriptions of cell cycle, cytokinesis and cellular morphogenesis in the procyclic form of T. brucei . Analysis of detergent‐extracted cytoskeletons revealed that in the pre‐abscission stage, while many microtubules terminate to form the new posterior end  were clearly evident pre‐abscission and following abscission as the two daughters moved away from each other; thus the two daughters are not identical at abscission. XMAP215 labelling also indicated two distinct organizations of microtubule plus ends found at the posterior of both procyclic , illustrating the significance of this stage in cell division cycle timings.This difference in posterior end structure post‐abscission implies the existence of post‐division morphological changes to produce a rounded posterior end in the old‐flagellum daughter cell. At pre‐abscission the cytoplasmic bridge was sometimes found at a more posterior position, leading to a more ‘end to end’ morphological configuration of the two daughter cells  in our in vitro cultured cell line. The cartoon in Fig. In our SEM analysis we identified an additional morphological cell type in the bloodstream form, where daughter cells were still attached by their posterior ends, but each possessing a second flagellum; indicating they had either prematurely re‐entered the cell cycle before abscission or failed abscission. We called these ‘quadriflagellate doublet cells’ Fig. A and B. Trypanosoma brucei and a comparison of cell morphogenesis in the two major life cycle forms (bloodstream and procyclic) that are widely used in molecular and RNAi studies. We provide the most detailed analysis of microtubule‐driven cytokinesis in this important protist, and the morphogenetic differences between the two life cycle stages.In this paper we have carried out an in‐depth analysis of cytokinesis in T. brucei we show that the position of the division fold and path of ingression of the furrow is positioned asymmetrically along the long axis of cell, and is more asymmetric in the procyclic form. The anterior end of the division fold coincides with the anterior end of new flagellum attachment to the cell body, and we have previously hypothesized that the flagellum attachment zone (FAZ) is involved in defining the point at which furrow ingression starts  composed of two novel kinesins  arising from underlying microtubule organization resulting from resolution of the cytoplasmic bridge. Indeed in mammalian cells asymmetry in newly divided cells is also receiving attention and it is now known that the midbody structure of the cytoplasmic bridge is inherited by only one of the two daughter cells at abscission  results in a partial division furrow ingression  in the procyclic form, but no division furrow (referred to as cleavage furrow) detected in the bloodstream form  heat‐inactivated fetal calf serum as described previously  heat‐inactivated fetal calf serum.Procyclic form et al., Thin section and whole mount cytoskeleton electron microscopy samples were prepared as previously described . Tyrosinated α‐tubulin was labelled with YL1/2  with two kinetoplasts (assessed by DAPI), two flagella (assessed by L8C4) and no more than 4K4N and 4 flagella. This cell type was recorded as quadriflagellate doublet cells.Live cell micrographs and time lapse videomicrographs were captured using a Zeiss Axioplan microscope and a Photometrix Coolsnap HQ CCD camera using Metamorph software. A Bioptechs heated stage was used to maintain procyclic and bloodstream form cells at 28°C and 37°C respectively.et al., T. brucei 427 SiMP (Daniels et al., A cell line expressing Ty::YFP::XMAP215 fusion protein from the endogenous locus was generated using pEnT6P (Kelly Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.