The objective is to describe the implementation and outcomes of a structured communication module used to supplement case-based simulated resuscitation training in an emergency medicine (EM) clerkship.We supplemented two case-based simulated resuscitation scenarios (cardiac arrest and blunt trauma) with role-play in order to teach medical students how to deliver news of death and poor prognosis to family of the critically ill or injured simulated patient. Quantitative outcomes were assessed with pre and post-clerkship surveys. Secondarily, students completed a written self-reflection  to further explore learner experiences with communication around resuscitation. Qualitative analysis identified themes from written self-reflections.A total of 120 medical students completed the pre and post-clerkship surveys. Majority of respondents reported that they had witnessed or role-played the delivery of difficult news, but only few had real-life experience of delivering news of death  and poor prognosis . This communication module led to statistically significant increased scores for comfort, confidence, and knowledge with communicating difficult news of death and poor prognosis. Pre-post scores increased for those agreeing with statements (somewhat/very much) for delivery of news of poor prognosis: comfort 69% to 81%, confidence 66% to 81% and knowledge 76% to 90% as well as for statements regarding delivery of news of death: comfort 52% to 68%, confidence 57% to 76% and knowledge 76% to 90%. Respondents report that patient resuscitations  generated a variety of strong emotional responses such as anxiety, stress, grief and feelings of loss and failure.A structured communication module supplements simulated resuscitation training in an EM clerkship and leads to a self-reported increase in knowledge, comfort, and competence in communicating difficult news of death and poor prognosis to family. Educators may need to seek ways to address the strong emotions generated in learners with real and simulated patient resuscitations. We use a hybrid simulation curriculum where case-based simulated resuscitation scenarios are supplemented with a structured communication that immediately follows the technical training. Our overall goal is to teach medical students in the EM clerkship how to compassionately communicate with families of critically ill and dying patients. We describe here the curriculum implementation. The main outcomes are pre-post self-assessments by learners to rate their knowledge, competence, and comfort with delivering difficult news to family. The secondary aims of our project are to 1) encourage students to use written reflective inquiry for communication skills development and 2) analyze these written learner experiences with the goal to inform future case scenario and curriculum design.The educational project was conducted in a large academic, urban, tertiary care teaching hospital with approximately 100,000 ED patient visits per year. The study period was from July 2011 through May 2012. The target population consisted of fourth-year medical students on a four-week mandatory EM clerkship. The faculty facilitators were EM board certified and one facilitator was also certified in Hospice and Palliative Medicine. The institutional review board approved the study. The curriculum design and implementation was supported by the Y.C. Ho/Helen and Michael Chiang Foundation administered and guided by the expert faculty at the Harvard Medical School Center for Palliative Care.The curriculum is grounded in Kolb’s experiential theory in which the learner ‘touches all the bases’ of thinking, experiencing, acting and reflecting . Concret23The communication and self-reflection educational module supplements pre-existing technical resuscitation training in the clerkship. The technical component consisted of two case-based simulated resuscitation scenarios (one addresses ACLS and the other ATLS skills). Trainees practice on a high-fidelity human adult simulation mannequin. Both simulation case scenarios with objectives as well as step-by-step outlines and competencies are available via MedEd portal.16We developed a pre- and post-clerkship survey. Pre-clerkship questions asked the student about their experiences in prior clinical rotations with delivering difficult news of death and/or poor prognosis . In addition, the pre-clerkship survey asked students to rate their baseline knowledge, comfort, and confidence with communicating difficult news of death and poor prognosis on a scale of 1–4 .The post-clerkship survey asked learners to rate their knowledge, comfort, and confidence regarding delivery of difficult news after completion of the simulation and communication role-play learning activity.Since our secondary aims were to encourage reflective inquiry and to explore learner needs/experiences via written self-reflection we also added questions to address this in the pre-post surveys. We asked for the pre-EM clerkship medical student experiences with written self-reflection and its perceived value. The students were then also asked their perception of value of a written self-reflection after completion of the exercise.The questions were piloted for readability, clarity, and content with a small group of the EM faculty as well as medical students. The comments from the pilot were reviewed and final survey was determined by author consensus.Anonymous pre-clerkship surveys were administered to all senior medical students during orientation on the first day of the EM clerkship as a paper and pencil survey. Students placed the completed pre-clerkship survey (with no student identifiers) into an envelope, sealed it, and wrote a self-assigned, easy to remember number on the envelope. Post-clerkship surveys were similarly administered on the last day of rotation. The students identified and opened their sealed envelope and returned the stapled completed pre- and post-clerkship surveys together. We randomly assigned the returned surveys a study number and entered corresponding data into an Excel spreadsheet. Every third survey was audited (RN) to ensure accuracy of data entry. No research incentives were provided to participants.Descriptive statistics, such as percentages, means, and medians, are provided below. We compared pre- post-clerkship dichotomous data using McNemar chi-square tests. We analyzed comparisons of pre- and post-clerkship Likert like scale ratings with Wilcoxon signed-ranks tests. P-values for comparisons of means and medians were obtained via paired-samples t-tests.We performed qualitative analyses on experiences reported by students via the written reflections.26Out of the 160 senior medical students completing their fourth-year at our institution, 120 answered both the pre- and post-clerkship surveys on their EM clerkship rotations . EighteeFew students  reported receiving specific training on closed-loop communication. A vast majority recalled witnessing and/or role-playing communication of difficult news of death and poor prognosis. However, few students reported personally delivering difficult news of death  and poor prognosis .After completion of the simulation and communication role-play learning activity, there was a statistically significant increase in scores related to comfort, confidence, and knowledge regarding communicating difficult news of poor prognosis and patient death  and 3. TOf the 119 student respondents, a majority  felt that the clinical ED faculty served as either somewhat or very positive role models for communicating difficult news .Prior to starting the EM clerkship an overwhelming majority of respondents  stated that they had not received specific training on written self-reflection. Similarly, a majority of students  perceived self-reflection to be a valuable tool for personal growth. Comparisons of pre- and post-clerkship survey responses revealed a statistically significant decrease in the perceived value of written self-reflection .Of the 110 written self-reflections analyzed, we explored themes expressed for real and/or simulated resuscitations under three main categories: “things that went well and why;” “things that did not go well and why;” and finally any student emotional responses identified. In general, among “things that went well,” students had positive comments about the simulation and described ED resuscitations as good role models to learn team organization and efficiency; “ED codes run as a well-oiled machine;” “ED team knew their roles well;” and “Everyone worked well together.” Students also listed that the observed ED clinician to family communication interactions were positive experiences; “Took time to explain, prompt effort to contact family.” However, among “things that did not go well,” the chaotic resuscitation setting, negative emotions, and some of the negative healthcare worker to healthcare worker communication interactions during resuscitations were cited.Students listed strong emotional responses to both real and/or simulated resuscitation encounters:Anxiety and stress around the overwhelming nature of resuscitation; “….the day when the responsibility will fall on me;” Feelings of a personal sense of loss, grief and of own mortality; “I am that age…that could have been me”Sense of failure when patient died after resuscitation attempt; “You work to save a life and failed”Feelings of worthlessness and abandonment; “I felt….lost,.…overlooked,….useless.”Teaching the essential communication skills of how to deliver difficult news of poor prognosis and death to family can be accomplished by including role-play communication exercises with simulated resuscitation scenarios. Role-play can be incorporated without a significant impact on session time and faculty resources. Major outcomes of our study included the following: 1) Most medical students reported limited real-life experiences with communicating difficult news; 2) The communication learning activities increased knowledge, comfort and confidence with communicating news of death and poor prognosis. When we explored student written self-reflections with intent to inform future curricula we found as a secondary outcome that resuscitations  generated a variety of strong emotional responses in student learners.Communication skills are taught in the pre-clerkship years to prepare students for clinical encounters in all undergraduate medical school curricula. However, these skills may decline by graduation if not reinforced.The secondary aims of our project were to 1) encourage students to use written reflective inquiry for communication skills development and 2) explore these written learner experiences with the goal to inform future case scenario and curriculum design. Self-reflection exercises have also been shown to foster development of reflective capacity and enhance life-long learning and professionalism when used appropriately.43Patient resuscitations  generated an emotional response in the learner. Frequently expressed learner emotions included anxiety, grief, sense of loss, and sense of failure. The emotions of professional failure and other personal reactions related to death and dying have been previously described.In summary, we propose that communication modules should ideally routinely supplement ACLS and ATLS simulation resuscitation scenarios. We feel that this is an opportune time to closely integrate and reinforce communication skills with family members and encourage trainee self-reflection.Limitations of our study include the use of survey tools that were not previously validated, but internally developed by experts in EM and palliative care. Though the project was conducted at a single institution, the educational module is feasible to implement and adaptable across other settings. We target learners in a mandatory clerkship and results may differ with students on electives who may have a special interest in resuscitation or communication skills. We only evaluate pre-clerkship and post-clerkship self-report data (four weeks later). This self-report methodology is dependent on student perception and does not allow for objective measurement of knowledge and skills acquisition. Since the graduating senior students were not followed as they went on to the various residencies (with varied emphasis on communication skills training by discipline) we are unable to provide long-term data. As stated above, we did not provide one-on-one feedback to the learners on their written self-reflections and this may have led to the decline in scores related to the perceived value of the written exercise. This specific self-reflection format therefore may not have been constructed to provide maximal benefit to students.Simulated resuscitation case-based scenarios present an opportunity to closely integrate teaching of communication and self-reflection skills. A communication module with role-play increased trainee knowledge, comfort, and competence regarding communication of difficult news of poor prognosis and death after resuscitation. Educators may need to seek ways to address the strong emotions generated in learners with real and simulated patient resuscitations.


The high-resolution refinement of docked protein-protein complexes can provide valuable structural and mechanistic insight into protein complex formation complementing experiment. Monte Carlo (MC) based approaches are frequently applied to sample putative interaction geometries of proteins including also possible conformational changes of the binding partners. In order to explore efficiency improvements of the MC sampling, several enhanced sampling techniques, including temperature or Hamiltonian replica exchange and well-tempered ensemble approaches, have been combined with the MC method and were evaluated on 20 protein complexes using unbound partner structures. The well-tempered ensemble method combined with a 2-dimensional temperature and Hamiltonian replica exchange scheme (WTE-H-REMC) was identified as the most efficient search strategy. Comparison with prolonged MC searches indicates that the WTE-H-REMC approach requires approximately 5 times fewer MC steps to identify near native docking geometries compared to conventional MC searches. Protein-protein interactions are integral to many mechanisms of cellular activities, ranging from enzyme catalysis and inhibition to signal transduction and gene regulation. Atomic-level structural knowledge is essential to understand the function of protein-protein complexes in biological processes. However, experimental structure determination of protein-protein complexes is often difficult and for many interactions the corresponding complex structures are lacking ,2. CompuDocking programs often employ a two-stage protocol ,9. FirstFor high-resolution refinement, most protocols require a starting configuration that is already fairly close to the native structure. The likely reason for this strong dependence on the starting structure is the energy landscape’s ruggedness, which frustrates the sampling and renders the global energy minimum hard to reach . AccordiFocus of this work is to improve the sampling for high-resolution docking based on the MC approach. In the MC method, random translational and rotational moves or conformational changes of the partner structures are applied on the configuration in a step-wise manner using the Metropolis criterion for acceptance of a move. Advantages of the MC method are the generation of a physically relevant canonical ensemble of docking configurations, use of arbitrary energy functions that can contain discontinuities and for the possibility to include various levels of structural flexibility. However, an exhaustive high-resolution sampling of the conformational space with the MC method can be computationally demanding. In general, the docking success of MC docking is limited by the sampling of putative complex geometries and by the accuracy of the energy function used for scoring predicted complexes.Parallel tempering or replica exchange techniques promise to overcome these challenges and have received wide-spread interest in recent years –25. The Metadynamics is another popular enhanced sampling method, in which sampling is facilitated by a history-dependent biasing potential. It is constructed as the sum of Gaussian functions deposited along the trajectory in the collective variable space ,38. ChooRosettaDock and various extensions based on advanced sampling techniques. In particular, we tested four different protocols, standard Monte Carlo (MC), Temperature Replica Exchange Monte Carlo (REMC), well-tempered ensemble temperature Replica Exchange Monte Carlo (WTE-REMC), and well-tempered ensemble two dimensional Hamiltonian Replica Exchange Monte Carlo (WTE-H-REMC). The approaches were systematically evaluated on protein-protein complexes using unbound partner structures and starting in each case from the same starting placements. Overall best performance was achieved with the WTE-H-REMC method at the same computational cost compared to the alternative protocols.In the present study we have compared the efficiency of a standard MC protocol for high resolution protein-protein docking using RosettaDock as given by the weight-set docking , and the same I_sc range of [-15.0 Rosetta score units]. For each target, the tested protocols are grouped together and the corresponding protocol is indicated in the score-axis label on the left side. The snapshot number is color-coded, with dark blue and dark red dots corresponding to decoys sampled at the beginning and towards the end of the sampling interval, respectively. Figure B. Same as Figure A in S1 File but for the docking searches up to 2x106MC steps. Figure C. Same as Figure A in S1 File but for the docking refinement simulation with 107MC steps. Figure D. Scatter plot of interaction score (I_sc) vs. ligand RMSD (L_rmsd) for the additional 10 targets with 3x105MC steps. All the panels have the same L_rmsd range of [0.30], and the same I_sc range of [-15.0]. For each target, the tested protocols are grouped together and the corresponding protocol is indicated in the score-axis label on the left side. The snapshots number is color-coded, with dark blue and dark red dots corresponding to decoys sampled at the beginning and the end, respectively. Figure E. Same as Figure D in S1 File but for docking searches up to 2x106MC steps. Figure F. Same as Figure D in S1 File but for docking searches with 107MC steps.(PDF)Click here for additional data file.


Recent studies have indicated the use of gonadotropin-releasing hormone antagonists (GnRH-ant) as an adjuvant treatment to prevent premature luteinization (PL) and improve the clinical outcomes in patients undergoing controlled ovarian stimulation (COS) with intrauterine insemination (IUI). However, the results of these studies are conflicting.We conducted a systematic review and meta-analysis of randomized trials aiming to compare the clinical efficacy of GnRH-ant in COS/IUI cycles. Twelve studies were identified that met inclusion criteria and comprised 2,577 cycles assigned to COS/IUI combined GnRH-ant or COS/IUI alone.Meta-analysis results suggested that GnRH-ant can significantly increase the clinical pregnancy rate (CPR)  and decrease the PL rate  in COS/IUI cycles. Subgroup analysis results suggested statistically significant improvement in the CPR in non-PCOS patients  but not in the PCOS population  and multiple mature follicle cycles . There were no difference in the miscarriage and multiple pregnancy rates between the groups.This meta-analysis suggested that GnRH-ant can reduce the incidence of PL and increase the CPR when used in COS/IUI cycles, and it was especially useful for non-PCOS patients. However, evidence to support its use in PCOS patients is still insufficient Intrauterine insemination (IUI) combined with controlled ovarian stimulation (COS) with low-dose recombinant FSH (rFSH) is widely used in the treatment of unexplained, endometriosis or male factor infertility because of the higher pregnancy rates associated with this approach compared to IUI cycles without COS or those with clomiphene citrate stimulation Gonadotropin-releasing hormone antagonists (GnRH-ant) have been successfully used in IVF cycles to prevent premature LH surge for many years The Medline, EMBASE, Cochrane Library databases, ClinicalTrials.gov and the World Health Organization International Trials Registry Platform search portal were searched with no time limit applied to any database. A combination of Medical Subject Headings (MeSH) and text key words were used to generate three subsets of citations: for studies of GnRH-ant (using the key words “gonadotropin-releasing hormone antagonist” or “Ganirelix” or “Cetrorelix” or “Cetrotide”), COS (“controlled ovarian stimulation” or “ovarian stimulation” or “gonadotropin” or “FSH”), and IUI (“intrauterine insemination”). These subsets were combined using “AND” to generate a subset of citations relevant to the research question. The last updated search was performed in November 2013. In addition, the citation lists of relevant publications, review articles, abstracts of scientific meetings and included studies were manually searched to identify other potentially eligible studies. Studies published in languages other than English were not considered for inclusion. The study selection was undertaken by two of the authors of this work (YL and YYZ).Criteria for inclusion in the analysis were established before the literature search. Inclusion criteria were as follows: (1) published studies, (2) enrolled study participants were subfertile and for whom COS/IUI treatment was indicated, (3) patients had been treated with GnRH-ant (GnRH-ant group) concurrently with COS/IUI and were compared to patients treated with COS/IUI alone (control group). Two reviewers (YL and YYZ) used these criteria to review each article identified independently.A study was excluded if: (1) the trial was not a RCT, (2) the patients accepted other assisted reproductive techniques instead of IUI after COS, such as IVF-ET, (3) the report was repeated or included identical patients in two studies (only the most recent article was included).Systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Two reviewers (YL and YYZ) completed the data extraction and quality assessment independently. When necessary, the reviewers wrote to the authors to obtain extra information and raw data. Inconsistencies between reviewers' data were resolved through discussion until a consensus was achieved. Outcome data from each study were extracted in 2×2 tables by YL and YYZ.The clinical pregnancy rate (CPR) was the primary outcome of interest, and the PL, miscarriage, multiple pregnancies, and ovarian hyperstimulation syndrome (OHSS) rates were used as secondary outcomes. The CPR and PL were calculated on the basis of the number of cycles randomized across all studies, even if some cycles were cancelled or dropped out after randomization. PL was defined as a premature rise in the LH combined with a rise in the serum progesterone level. However, the threshold values of premature LH and progesterone increases differed slightly across the reviewed studies, because there are no unified criteria for these values at present.To ascertain the validity of each study, the methodological quality of all the selected trials was analyzed on the basis of information reported in the original published papers. The study quality was scrutinized by checking the adequacy of randomization, allocation concealment, the use of blinding, the completeness of follow-up, and outcome reporting according to the Cochrane Collaboration's tool for assessing risk of bias I2 statistic to quantify heterogeneity across studies I2≤50%, the variation between the studies was considered to be homogenous, and the fixed effects model was adopted for analysis. If I2≥50%, there was significant heterogeneity between studies, the random effects model was adopted for analysis. The causes of heterogeneity were explored by investigating the variations in features of the population, exposure, and study quality.Dichotomous outcomes were expressed as an odds ratio (OR) with 95% confidence intervals (CI). Heterogeneity of treatment effects was evaluated statistically using the Subgroup analyses were performed according to the study population and number of mature follicles on the human chorionic gonadotrophin (hCG) day. Statistical analyses were carried out using RevMan 5.0 software .The literature search yielded 81 citations. Of these, 61 were excluded after reading the title and the abstract. Twenty full articles were retrieved for review. On examination of the manuscripts, eight studies were excluded Then, 1,256 cycles were assigned to the GnRH-ant group and 1,321 cycles to the control group. Ten studies used computer-generated randomization 2 = 43%, p = 0.06). Therefore, we chose the fixed effects model to synthesize data. The results of the meta-analysis showed a 42% increase in CPR for GnRH-ant treatment compared with the control group , indicating that there was no statistical inconsistency among these trials.Eight studies reported the PL rate Five studies reported the OHSS rate All studies reported the total dose of rFSH and the duration of therapy This review aimed to explore the efficacy of GnRH-ant when used as an adjuvant to COS/IUI cycles. The results of our meta-analysis showed that GnRH-ant can significantly decrease the rate of PL and cause a 42% increase in the CPR in COS/IUI treatment. Of the RCTs included in this analysis, eight included infertile women with normal ovulation, with conditions including unexplained subfertility, mild male factor infertility, or stage I–II endometriosis; two studies included women with PCOS who were infertile due to anovulation; and two studies included all the previously mentioned causes of infertility. Because ovarian hyper-response to gonadotropins and PL were especially encountered in PCOS patients, we performed subgroup analyses on the basis of the PCOS and non-PCOS populations. Results of the subgroup analysis for non-PCOS patients showed that GnRH-ant administration in COS/IUI cycles could significantly increase the pregnancy rate. This result was consistent with that of the meta-analysis performed by Kosmas et al. in 2008 In this systematic review, subgroup analysis was carried out according to the mature follicle number on the hCG day. Theoretically, GnRH-ant should be more effective in improving the pregnancy rate in women with multiple follicles because such patients face a higher risk of PL. However, only two studies have reported the pregnancy rate in patients with different numbers of mature follicles on the hCG day. No significant difference was found between the GnRH-ant and control groups in single mature follicle cycles. The result for cycles with two or more mature follicles on the hCG day suggested that GnRH-ant tends to improve the clinical pregnancy outcome, although this was not statistically significant. Gomez et al. Prospective data have shown that premature LH surges occur in a significant percentage of COS cycles (22–43%), interfering with the optimal timing of the insemination or even resulting in cycle cancelation Two previous meta-analyses have investigated whether GnRH-ant administration significantly benefited the clinical outcome in COS/IUI programs. Kosmas et al. This meta-analysis has several strengths and weaknesses. It consisted of a total of 12 RCTs with 2,577 cycles. The relatively large number of included trials and participants strengthen the validity of the results of our meta-analysis. However, there was heterogeneity among the studies. The initial gonadotropin dose and ovarian simulation protocols were different across the reviewed studies, which may result in a difference in the mature follicle number, the risk of PL, and the inconsistent pregnancy rates encountered in the different studies. Furthermore, only two studies reported the use of GnRH-ant in PCOS patients undergoing COS/IUI in subgroup analysis. Therefore, the results pertaining to PCOS patients should be interpreted with caution, and more RCTs are required in the future to arrive at a more robust conclusion. Finally, it is important to highlight methodological weaknesses in existing studies, such as lack of double-blinding, and the absence of primary clinical outcome reporting (e.g. live birth rate). Such shortcomings may introduce performance and reporting bias to this review.We concluded that GnRH-ant can effectively lower the incidence of PL and improve the CPR in patients without PCOS. In view of the scant evidence available in literature published till date, however, the role of GnRH-ant in PCOS patients undergoing COS/IUI is still unclear. In the future, well-designed RCTs, which are sufficiently powered and study different populations, should be conducted to reach useful conclusions about the use of GnRH-ant in patients undergoing COS/IUI treatment.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


The National Lung Matrix Trial is currently the largest stratified medicine study in lung cancer. Utilizing a next-generation sequencing screening platform and an adaptive umbrella trial design, we will explore the activity of multiple biomarker/targeted therapy options in order to expand the precision medicine opportunities for patients with non-small-cell lung cancer. The management of NSCLC has been transformed by stratified medicine. The National Lung Matrix Trial (NLMT) is a UK-wide study exploring the activity of rationally selected biomarker/targeted therapy combinations.The Cancer Research UK (CRUK) Stratified Medicine Programme 2 is undertaking the large volume national molecular pre-screening which integrates with the NLMT. At study initiation, there are eight drugs being used to target 18 molecular cohorts. The aim is to determine whether there is sufficient signal of activity in any drug–biomarker combination to warrant further investigation. A Bayesian adaptive design that gives a more realistic approach to decision making and flexibility to make conclusions without fixing the sample size was chosen. The screening platform is an adaptable 28-gene Nextera next-generation sequencing platform designed by Illumina, covering the range of molecular abnormalities being targeted. The adaptive design allows new biomarker–drug combination cohorts to be incorporated by substantial amendment. The pre-clinical justification for each biomarker–drug combination has been rigorously assessed creating molecular exclusion rules and a trumping strategy in patients harbouring concomitant actionable genetic abnormalities. Discrete routes of pathway activation or inactivation determined by cancer genome aberrations are treated as separate cohorts. Key translational analyses include the deep genomic analysis of pre- and post-treatment biopsies, the establishment of patient-derived xenograft models and longitudinal ctDNA collection, in order to define predictive biomarkers, mechanisms of resistance and early markers of response and relapse.The SMP2 platform will provide large scale genetic screening to inform entry into the NLMT, a trial explicitly aimed at discovering novel actionable cohorts in NSCLC.38344105. EML4-ALK fusions [ALK fusion-positive disease. Alongside, these therapeutic advances have been a change in the regulatory landscape; the provisional registration of crizotinib was based on high signals of activity in non-randomized, single-arm studies [The management of patients with non-small-cell lung cancer (NSCLC) has been transformed in the past 10 years. The identification of EGFR-activating mutations as a predictive biomarker for the use of EGFR tyrosine kinase inhibitors ushered in the era of stratified medicine in NSCLC [ fusions  and the  studies . A serie studies . EfficieROS fusions [The NLMT is a multi-arm non-randomized non-comparative phase II umbrella trial in which patients are allocated to the appropriate targeted therapy according to the molecular genotype of their cancer. The trial includes a common set of outcome measures for all molecularly defined cohorts with flexibility to select a cohort-specific primary end point. In most cases, response rate is the primary outcome but for agents whose mode of action is likely to be principally cytostatic, progression-free survival (PFS) is preferred. Although randomized trials make it possible to tease out the predictive and prognostic effects of putative biomarkers for therapies, we are looking here for robust signals of activity such as one would expect from a bona fide targeted therapy. For example, the recent demonstration of a 72% response rate and a 19-month median PFS in patients treated with crizotinib harbouring  fusions  is very There is an option within the trial protocol to test any of the given targeted therapies on biomarker-negative patients (i.e. those with no actionable genetic change) if there is evidence of significant activity in the biomarker-positive population. This allows validation of the specificity of the putative biomarker for that drug but may also detect biomarker-negative patients who have impressive responses to the drug and whose tumours can then be analysed to detect abnormalities that may be additional important positive predictive biomarkers of that drug.The NLMT will be run at all 18 UK Experimental Cancer Medicine Centres (ECMC) with each centre operating a hub and spoke model with patients being referred in from nearby hospitals to the centre. The pace of advances in stratified medicine in lung cancer is such that signal of activity programmes must be nimble, flexible and able to respond promptly to new biomarkers drug combinations being considered. As such, the trial allows for new arms to be entered via substantial amendment, if the international expert review panel and Trial Management Group are convinced of the strength of the pre-clinical data supporting the clinical combination of the biomarker and targeted agent. This will significantly reduce the time from concept to clinical study.One of the limitations for the development of targeted agents in small patient populations has been the conventional approach to tumour testing which could be described as ‘one drug–one test.’ This limitation will be overcome in the NLMT by implementing an umbrella trial design. This approach will facilitate a transition from a drug-centred approach that has asked ‘can a specific therapeutic agent be given to a patient’, to a patient-centred molecular testing approach which allows the treating physician to ask ‘what is best therapeutic agent for my patient?’Screening of patients' tumour biopsies through the Stratified Medicine Programme 2 is performed on a next-generation sequencing (NGS) panel developed and validated by Illumina, carried out in one of three dedicated genotyping centres (Technology Hubs). At present, 28 genes are interrogated but the platform is adaptable to allow new genomic biomarkers to be added. Alignment of tumour DNA reads against germline is mandatory. Careful examination of genetic databases, pre-clinical and clinical data have generated a comprehensive tiering system which ensures that only oncogenically pertinent abnormalities are actioned. Table 50 <10 nM for all three AKT isoforms. Using a cut-off of GI50 <3 µm, 23% of a large cell-line panel were sensitive to inhibition by AZD5363 and three quarters of these had PIK3CA mutations, PTEN loss or HER2 amplification [In vivo activity was demonstrated in both PIK3CA mutant and PTEN inactivated xenograft models. In a study using the PI3K inhibitor GDC-0941, 3/3 PIK3CA amplified cell lines  were highly sensitive to inhibition and there was no co-occurrence of other obvious oncogenic drivers [AZD5363 is potent ATP-competitive AKT inhibitor with ICfication . In vivoEach separate mechanism resulting in AKT activation is treated as a separate cohort. This is an important design feature of the trial. It is unlikely that the activities of targeted agents will be the same for each mechanism of deregulation and treating them all in one cohort may miss the granularity of this response differential. Furthermore, mechanisms of resistance may vary in patients who have an initial response to therapy according to the mechanism of initial AKT activation. Discrete cohort testing allows the specifics of resistance in each molecular cohort to be defined.KRAS wild type. KRAS was shown to be a negative predictive biomarker for AZD5363 response [RAS mutation may still inhibit 4E-BP1 even if AKT is inhibited  but failed to meet the primary end point for continuation [FGFR mutations will be eligible. Liao et al. engineered NIH3T3 cells to express the range of separate mutations represented in the squamous cell lung cancer TGCA [AZD4547 is potent inhibitor of FGFR 1, 2 and 3 with ICectively . Initialinuation . Not allcer TGCA . Only muin vitro across tumour types compared with rapalogues. mTORC1 inhibition reduces S6K1 activation which negatively represses mTORC2; thus, mTORC1 inhibition releases mTORC2 from S6K1-mediated inhibition and activates AKT via mTORC2-mediated phosphorylation of Ser473 [TSC1, TSC2 or LKB1 (STK11) tumour mutations will be eligible for treatment with AZD2014.AZD2014 is an ATP-competitive, selective mTOR kinase inhibitor targeting both mTORC1 (rapamycin-sensitive) and mTORC2 (rapamycin-insensitive) complexes , 17. AZDf Ser473  . A recent clinical study has demonstrated that mTOR inhibitors have significant activity in TSC mutant disease. A patient with anaplastic thyroid cancer who relapsed after surgery and subsequent chemoradiation had an 18-month response to the mTORC1 inhibitor everolimus. Sequencing this patient's tumour DNA revealed a mutation .LKB1 mutant cells resulted in reduced anchorage independent cell growth and migration [LKB1 wild-type models but reduced tumour number and volume in LKB1 mutant models.Loss of LKB1 function phenocopies TSC2 mutation  were the most sensitive to palbociclib, and no other analysed biomarkers pertaining to CDK4/6 signalling were informative [CDKN2A was the most significant gene.p16, a product of the ormative . ExpressKRAS mutant adenocarcinoma represents a significant molecular cohort of lung adenocarcinoma. It was demonstrated that loss of CDK4 activity was synthetically lethal with KRAS mutation in lung adenocarcinoma [in vivo with knock-down of the other CDKs and was not seen in other mutant KRAS expressing tissues including the pancreas. Palbociclib was shown to have significant activity in KRAS mutant genetically engineered mouse models. The mechanism of synthetic lethality appeared to be due to the re-induction of senescence. KRAS mutation causes oncogene-induced senescence and for the development of the full malignant phenotype, KRAS mutant cells must bypass senescence. Thus, CDK4 inhibition is expected to abrogate this bypass in patients whose lung cancers harbour an intact Rb signalling pathway and a KRAS mutation. Activated RAS induces the formation of senescence-associated heterochromatin foci by activating GSK3β which phosphorylates the histone chaperone HIRA facilitating its localization to PML nuclear bodies [AKT activation inhibits RAS-mediated oncogene-induced senescence in part through the inhibitory phosphorylation of GSK3β at serine 9 [arcinoma . This efr bodies . AKT actserine 9 . Thus, aMET amplification [ROS1-rearranged lung cancer [Crizotinib is a potent inhibitor of both MET and ROS1. There is already clear evidence of activity of crizotinib in patients with tumour fication  or ROS1-g cancer . These aEGFR mutation-positive tumours progressing after first-line treatment on an EGFR TKI become resistant via the acquisition of the secondary gatekeeper mutation T790M. AZD9291 is an oral, potent, selective, irreversible inhibitor of both EGFR-TKI-sensitizing and T790M resistance mutations. In 127 assessable patients with T790M disease, a response rate of 61% and median PFS of 9.6 months was observed [About 50%–60% of patients with observed . PatientKRAS mutant adenocarcinoma of the lung [NF1 mutations were found to be significantly represented in cancers not harbouring abnormalities of RAS, RAF or receptor tyrosine kinases [NF1 as an important driver event in oncogene-negative adenocarcinoma. NF1 is a RAS GAP ; in a cohort of PN patients aged 3–18, all patients treated with single-agent selumetinib demonstrated volumetric reduction in the size of their PNs [Selumetinib is a potent, selective, allosteric MEK inhibitor. In a randomized phase II study, it was demonstrated that a combination of selumetinib plus docetaxel significantly improved response rate and PFS when compared with docetaxel alone as second-line therapy for patients with the lung . This co kinases . The autheir PNs .NRAS mutant lung cancer cell lines were sensitive to single-agent selumetinib and MEK signalling appeared to be significantly more important than PI3K/AKT activation given the lack of effect of GDC-0941 in these NRAS mutant lung cancer cell lines. Thus, we will treat patients with NRAS mutant tumours as a separate cohort with this combination.In patients whose tumours harbour NRAS mutations, there appear to be no other obvious driver mutations . 5/6 NRAA secondary objective of the trial is to offer a therapeutic option for patients with successful screening in the trial but without specific eligibility for one of the targeted genomic aberrations at that moment in time. The first in a planned series of drugs that we are testing in this cohort is MEDI4736, an anti-PDL1 monoclonal antibody with clear evidence of activity in patients with NSCLC .The NLMT is an ambitious adaptive programme which seeks to increase the number of actionable genetic abnormalities in NSCLC. Key translational components include voluntary pre-treatment biopsies to identify potential predictive biomarkers post-treatment biopsies to ascertain mechanisms of resistance, and the development of PDX models wherever possible. ctDNA is being collected pre-, during and post-treatment. Repeat biopsies can be difficult to obtain: the recent identification of the C797S mutation in the plasma of patients with T790M mutation-positive disease treated with AZD9291 in second line or beyond demonstrates the utility of liquid biopsies to identify mechanisms of resistance to targeted therapies .MET. Finally, the NLMT is clearly not the only such programme in this therapy space. The NCI-MATCH co-operative group trial currently has 10 arms: some cohorts are in common with NLMT but NCI-MATCH also covers BRAF mutations (dabrafenib/trametinib), HER2 mutations (afatinib), HER-2 amplifications (Ado-trastuzumab emtansine) and c-kit mutations (sunitinib). Integrating the data from this trial together with data from NLMT, other key trials such as SAFIR-02 (NCT02117167) and LUNGMAP (NCT02154490), and further data arising from national genomic screening initiatives such as The Lung Cancer Genome Project  and LC-SCRUM (Japan), will allow the development of a global database of outcomes on personalized medicines in NSCLC.Ensuring the sustainability of NLMT is important in order to maximize the information that can be obtained from this programme. We are in discussion with a number of other potential pharma partners and have already discussed with our current pharma collaborators plans for treatment of patients with p53 loss and patients with rarer but potentially actionable mutations such as those with ATM loss and exon 14 slipping mutations in Cancer Research UK (C22436/A18392 and C11497/A19363), AstraZeneca/MedImmune and Pfizer . Note that the Pharma Partners are providing core funding into the Stratified Medicine Programme in addition to the compounds.This work is supported by GM receives research funding from AstraZeneca and Cancer Research UK. SP is non-compensated consultant to AstraZeneca and Pfizer. LB has received personal fees from Eli Lilly and Roche outside of the submitted work. All remaining authors have declared no conflicts of interest.


The X-ray structures of viruses and viral proteins currently available are providing high-resolution snapshots of viral molecular machineries, expanding our vision of the virus world and giving crucial information on potential targets for future antiviral therapies. For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes. Unless the nucleic acid enters alone, viral genome replication and expression will require uncoating of the capsid and release of the internalized viral nucleic acid. Cell recognition, entry and uncoating frequently overlap and rely on quite different mechanisms depending on the virus species and host type.Picornaviridae family that includes a large number of human and animal pathogens. In these viruses, uncoating can be mediated by receptor binding, by low pH, or by the cooperative effect of both factors, and appears to be linked to the inherent stability and dynamics of the capsid. The X-ray and cryo-electron micrography structures of different HRV2 uncoating intermediates revealed the structural alterations that take place in the virus architecture, allowing the delivery of the genomic RNA: the acidic pH induces the conversion of the native HRV2 virions into an expanded, porous uncoating intermediate called an A-particle. This conversion is accompanied by major changes in the RNA organization and interactions at the inner capsid wall that would facilitate the subsequent RNA egress through large capsid disruptions produced at the particle twofold symmetry axes  and Human Rhinoviruses (HRV) both belonging to the et al., 2013Enveloped animal viruses also use a two-step process to release their genetic material into the cell: first they bind to specific cell-surface receptors anchored to the target cell membrane and then they induce fusion of the viral and cell membranes. Binding to the cell-surface receptor in these viruses is mediated by a viral glycoprotein embedded in the viral lipid envelope which specifically interacts with some cellular molecule, which typically is a membrane-associated glycoprotein, carbohydrate or glycolipid. Detailed knowledge of virus–receptor interactions is essential to understand different aspects that determine viral tropism, spread and pathogenesis. During the last few years, a large number of high-resolution structures of viral proteins in complex with their specific receptors have been reported, shedding light on these aspects. Very recently, Wang and co-workers characterized the interactions between the newly identified Middle East respiratory syndrome coronavirus and its target cell by solving the structure of the receptor-binding domain of the viral envelope spike glycoprotein bound to its cellular receptor, the dipeptidyl peptidase 4  appears to preferentially enter cancer cells by interacting with a 54 kDa cell-surface protein, vimentin  to ensure genome replication within the infected cell. These enzymes are major targets for the development of antiviral compounds against the corresponding pathogens. A number of studies have revealed the structures of different RdRPs and provided a mechanistic insight to the RNA-synthesis process in the case of single-stranded positive-sense RNA viruses and double-stranded RNA viruses. However, such information is still lacking for single-stranded negative-polarity RNA viruses. The most complete sets of structural data are from Picornaviruses and Caliciviruses. These structures provide high-resolution snapshots of the conformations adopted by the enzyme in the different steps of the catalytic cycle. Ng and colleagues provide structural and biochemical evidence in Norovirus for a range of conformational states of the polymerase associated with RNA-template–primer binding, NTP binding, catalysis, RNA translocation and pyrophosphate release  and several elements on the cellular membrane that can get attached to the virus. Most viruses establish contacts with cellular receptors which could be sialic acid bound molecules or proteins on the cell surface. Frequently, the first virus–cell contact is mediated by adhesion receptors that allow reversible attachment of the virus to particular cells or organs, mainly by means of electrostatic interactions. This initial interaction could lack specificity and is employed to enhance the encounter between virus and entry receptors, which irreversibly bind the virus, allowing them access to the intracellular milieu either by direct endocytosis/pinocytosis or by inducing fusion or penetration of the viral genome into the cell. Characterization of the virus–receptor interactions has been a focus of sustained interest and many outstanding contributions of high-resolution X-ray structures of viral proteins in complex with their specific receptors have been reported during the last few years. Haemagglutinin (HA)–receptor interaction was extensively studied in different influenza virus subtypes. Influenza HA is synthesized in the infected cell as a polypeptide precursor (HA0) of about 550 amino acids that is proteolytically cleaved to generate the HA1  and HA2  chains that remain covalently linked by a disulfide bond. At the newly created HA2 N-terminus there is a stretch of hydrophobic amino acids, called the fusion peptide, which is inserted into the membrane during fusion (see the next section). The overall structure of the influenza HA resembles an elongated spike sticking out of the membrane. The distal head, formed exclusively by HA1 sequences, bears the receptor-binding site, formed by a shallow pocket exposed on an outward-forming surface. Different residues within this pocket mediate viral attachment to the α2,3- or α2,6-linked sialic acid moieties in avian or human cellular surfaces, respectively, as observed by X-ray crystallography in different complexes with receptor analogues. The sialic acid binding pocket of HA1 is formed by a loop–helix–loop structure composed of the 130-loop, 190-helix and 220-loop Fig. 1. et al., 2012et al., 2013A repertoire of crystal structures of HA–receptor complexes from different influenza outbreaks has recently been reported, providing a picture of the evolutionary path for the emergence of the H3N2 Influenza strain between 1968 and 2002 , which is a single-stranded, negative-sense, enveloped RNA virus. Three seminal works provide a landscape of atomic interactions between MV haemagglutinin (MV-H) and the protein receptors. The structure of MV-H bound to human CD46 fragment . The spike glycoprotein (S) of the CoV particle forms characteristic surface projections that are employed to interact with the target cell receptors. The determinants of CoV tropism are located in the distal globular domain of this viral membrane glycoprotein, which mediates attachment of the virus to the cell-surface molecules. In fact, the structure of the porcine respiratory coronavirus (PRCV) receptor-binding domain of the S protein in complex with its cell-receptor protein, the aminopeptidase N (pAPN), shows that the conformation of the receptor-binding edge in the envelope S protein contains the determinants of their receptor-binding specificity Fig. 1. This woet al.  identified relevant residues that support the viral–receptor interaction that will potentially guide therapeutic strategies against this virus.Very recently, Wang  al. 2013 determin3.The viral fusion glycoproteins structurally characterized to date fall into three classes, although the membrane-fusion pathway seems to be very similar for all the enveloped viruses studied so far.et al., 1981et al., 1994et al., 2005et al., 2013Class I fusogens are characterized by a seven-residue periodicity of nonpolar amino acids  that give rise to a central parallel trimeric α-helical coiled coil along the long axis of a rod-shaped molecule Fig. 2. The beset al., 1995via a C-terminal TM anchor, which is linked by a flexible ‘stem’ region to the ectodomain  quasi-equivalent assemblies  and alphaviruses (protein E1), which share many key characteristics  from Rift Valley fever virus (RVFV) reveal a class II fusion protein fold  Fig. 2, anotherly Fig. 2. The stret al., 2013et al., 2007et al., 2006et al., 2012Class III proteins are trimeric before and after fusion and share structural characteristics with both class I and class II fusion glycoproteins Fig. 2. A classN-ethylmaleimide-sensitive factor (NSF) attachment protein receptors]  and long but simple helical viruses (filamentous phages), to large tailed viruses (e.g. the dsDNA phages T4 or ϕ29). Bacteriophages show widely diverse structures and types of nucleic acid genomes; they have helical or icosahedral capsids and may not include a lipid envelope. Phages range in structural complexity from very small and simple non-enveloped icosahedral viruses  and/or OmpC molecules, serving for primary host recognition. It also has six short tail fibres attached to its periphery. Upon binding of the long tail fibres, a signal is transmitted to the baseplate causing the six short tail fibres to extend and bind irreversibly to the LPS, resulting in a conformational rearrangement where the baseplate switches from a dome-shaped form to a star-shaped conformation which, in turn, triggers the contraction of the tail sheath causing the specialized tip of the inner tail tube to puncture the outer membrane. The genome then passes through the tail tube into the cytoplasm DNA bacteriophage ϕX174 possesses a mechanism of DNA translocation similar to that described for the tailed phages. However, the ϕX174 ‘tail’ protrudes from the virion only at the time of infection  are the catalytic components of the RNA replication and transcription machineries and the central players in the life cycle of RNA viruses. RdRPs belong to the superfamily of template-directed nucleic acid polymerases, including DNA-dependent DNA polymerases (DdDP), DNA-dependent RNA polymerases and reverse transcriptases (RT). All theses enzymes share a cupped right-hand structure, including fingers, palm and thumb domains, and catalyse phosphodiester bond formation through a conserved two-metal-ion mechanism  VPg protein lines the RNA-binding cleft of the corresponding RdRP  determined in more than ten different crystal forms in the presence and absence of divalent metal cations, nucleoside triphosphates, inhibitors and primer-template duplex RNAs. These structural comparisons revealed that, in addition to the active-site closure, the NV RdRP exhibits two other key changes: a rotation of the central helix in the thumb domain by 22°, resulting in the formation of a binding pocket for the primer RNA strand, and the displacement of the C-terminal tail region away from the central active-site groove, which also allows the rotation of the thumb helix Fig. 4.in vitro  leads to the activation of the protease (Satheshkumar et al., 2005pro and the polymerase domain 3Dpol, also exhibits protease activity similar to 3Cpro, but the polymerase is active only as 3Dpol after the elimination of the 3Cpro domain (Harris et al., 1992et al., 2000The catalytic function of viral proteases has been found to be regulated by other domains that may be attached covalently to the N- or C-terminus of the protease. These regulatory mechanisms also serve to control the time and place of proteolytic activity (Babé & Craik, 1997et al., 1988ab initio methods of phase determination using isomorphous replacement or anomalous dispersion. In most cases, only the catalytic residues are conserved. The Coronaviral proteases contain an additional domain consisting mainly of α-helices at the C-terminus which is essential for dimerization (Anand et al., 2002et al., 2003Another feature of viral proteases is their extreme sequence diversity, not only amongst themselves but also with respect to cellular proteases. No significant sequence identity has been found between viral proteases and cellular proteases, though the structural fold might be maintained (Bazan & Fletterick, 1988et al., 1996et al., 1997pro encoded by the HRV genome cleaves the eukaryotic initiation factor eIF-4G of the host, preventing host protein synthesis (Gradi et al., 1998et al., 1988et al., 2000Many viral proteases perform more than one role. Hence, there are certain features in these proteases that confer unique sequence and structural properties compared to normal cellular proteases. The proteases of the alphaviruses have a compact structure resulting from shortening of loops such that their assembly into capsids is facilitated (Choi 7.in situ diffraction and X-ray free-electron lasers, that will allow the possibility of single-virus-particle analysis, will undoubtedly influence our research on structural virology in the near future.Since the first virus structures were determined at almost atomic resolution, about 35 years ago, virus X-ray crystallography has continued to provide an ever deeper and wider understanding of the virus world. However, many of the questions still remaining in virus structure and function will require an even bolder use of X-ray studies in combination with new experimental approaches, such as high-resolution electron microscopy and single-molecule techniques. Technological developments associated with synchrotron radiation with the advent of


DGAT) genes encode the main enzymes needed for TAG biosynthesis. Different types of DGAT genes  have been identified in plants. DGAT1 and DGAT2 are well characterized, but little is known about DGAT3 and DAcT genes in most plant species. The understanding of TAG biosynthesis enzymatic steps and its transcriptional regulation in plants is important to help improve the content and composition of nutritional and industrial oils. DAcT or Diacylglycerol-acetyltransferase was previously identified in Euonymus alatus and it seems to be related to DGAT enzymes. This enzyme catalyzes the condensation of acetil-CoA to diacylglycerol for the formation of 1,2-diacyl-3-acetyl-sn-glycerols or simply "ac-TAGs". These sn-3 acetylated diacylglycerol oils are abundant in Euonymus and display a 30% reduction on viscosity. DGAT3 enzymes were first reported in peanut and are unique due to their cytoplasmic localization due to the lack of transmembrane domains.Triacylglycerols (TAGs) are the main seed storage lipids of plants. TAGs chemichal properties are largely dependent on their fatty acid composition. Diacylglycerol Acyltransferase (Ricinus communis). The cDNA sequences from Euonymus alatus and Arachis hypogaea were used as queries in the blastx and tblastx programs to search for DAcT (Diacylglycerol acetyltransferase) and DGAT3 (soluble Diacylglycerol acyltransferase) from different plant species. The sequences identified as RcDAcT and RcDGAT3 in the Ricinus communis genome were used to design RT-qPCR primers to evaluate their expression profile in developing seeds. For the functional characterization of these proteins, full cDNA sequences were amplified with gene-specific primers and cloned in pENTR-D. These entry clones were C-terminally fused to the YFP and CFP coding sequences. Arabidopsis mesophyll protoplasts were transformed with the YFP construction to identify DGAT3 and DAcT subcellular localization. Via Floral-dip, we transformed castor bean DGAT3-CFP in Arabidopsis to characterize its role in lipid metabolism and to obtain a purified protein for enzymatic assays. We also transformed yeast lipid synthesis mutants to confirm the role of these proteins in TAG accumulation by complementation assay.Our goal is to identify and characterize DGAT3 and DAcT genes of castor bean (Arabidopsis transgenic lines overexpressing castor bean DGAT3-CFP and evaluating the capacity of DGAT3 and DAcT genes to complement yeast lipid-synthesis mutants.We have successfully identified 4 different DAcT paralogs and one DGAT3 ortholog in castor bean. We have already characterized the expression pattern of the DGAT3 gene in castor bean in five different stages of castor bean seed development where we verified an active expression of this gene; however, by RT-qPCR approach, it was not possible to identify substantial expression for DAcT genes in castor bean developing seeds. The DAcTA-YFP fusion protein seems to be localized to the ER membrane as the other previously described DGATs are, on the other hand, DGAT3-YFP seems to localize to cytoplasmic structures which we believe to be oil bodies. Currently, we are characterizing


The results revealed that ADM was upregulated in human ICC tissues (73/133) compared with that in healthy controls. ADM expression was positively correlated with shorter overall survival (P<0.01). The characteristics of EMT were induced in vitro by adenoviral transduction of ADM into HuCCT1 cells, resulting in the downregulation of E-cadherin and ZO-1, and the concomitant upregulation of N-cadherin and vimentin. Knockdown of ADM by short hairpin RNA in HUH28 cells expressing high levels of ADM was associated with the reversal of EMT. Functional studies revealed that ADM regulated the activation of ZEB1, which subsequently mediated EMT. The results of the present study suggest that ADM plays an important role in ICC metastasis, and that ADM signaling of EMT may represent a valuable therapeutic target in cancer patients.Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its etiology and molecular pathogenesis remain largely unknown. The present study aimed to investigate the association between adrenomedullin (ADM) and epithelial-mesenchymal transition (EMT) in ICC and to elucidate the underlying signaling pathway. We evaluated the clinical significance of ADM in 133 ICC patients using tissue microarray analysis of ICC tissues. We also investigated the mechanisms of ADM in ICC EMT-mediated metastasis in cholangiocarci-noma cell lines  Intrahepatic cholangiocarcinoma (ICC) is defined as a chol-angiocarcinoma located proximally to the second-degree bile ducts. It is an aggressive neoplasm associated with extremely poor survival. The incidence and mortality of ICC have increased worldwide over the past two decades –3. SurgiThis study included 80 men and 53 women with an average age of 55.6±12.8 years , who had undergone curative resection at the First Affiliated Hospital of Zhengzhou University  from January 2005 to December 2008. The diagnosis of ICC was confirmed by morphological criteria, immunohistochemical (IHC) staining and clinical findings. None of the patients had received any preoperative anticancer treatment. The present study was approved by the Human Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and informed consent was obtained from all patients according to the committee's regulations and the Declaration of Helsinki. The clinical characteristics of the patients with ICC are presented in Total RNA was extracted from ICC and liver tissues using TRIzol reagent . The quality and concentration of the isolated RNA were measured with a NanoDrop ND-1000 spectrophotometer . First-strand cDNA was synthesized using both oligo-dT primers and random 6-mers and PrimeScript RT Enzyme Mix I according to the manufacturer's instructions .−ΔΔCT method (RT-PCR was performed using specific TaqMan probes and primer sets to examine ADM RNA expression levels. TATA-box-binding protein (TBP), which is considered to be a reliable reference gene for quantitative PCR normalization in HCC specimens, was used as a control . CommercT method .A tissue microarray was constructed as described previously . Samplesµg/ml.Full-length human ADM cDNA was amplified by PCR and cloned into the pEGFP-N1 expression vector  to construct pEGFP-N1-ADM, and then transfected into HuCCT1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with pEGFP-N1 were used as a negative control. Stable ADM-expressing clones were selected using geneticin  at a concentration of 500 µg/ml).Lentivirus containing short hairpin RNAs targeting ADM was purchased from GeneCopoeia  and transfected into HUH28 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with the empty vector were used as controls. Stable clones were selected using puromycin . Each assay was repeated 3 times. For wound-healing assays, monolayers of cells were wounded by scraping with a plastic pipette tip followed by rinsing several times with medium to remove dislodged cells. Cells that had migrated into the wound area were photographed. For invasion assays, 2×105 cells were plated into the upper chamber of a polycarbonate Transwell filter chamber coated with Matrigel  and incubated for 48 h. The cells that migrated to the underside of the membrane were stained with Giemsa  and counted under a microscope .Cellular proliferation was assayed in cells seeded at a density of 5×10Survival was analyzed using the Kaplan-Meier estimate, and values were compared using the log-rank test. Independent prognostic factors were identified by multivariate survival analysis using a Cox proportional hazards model. The median value was used to determine the cut-off value for high vs. low expression of ADM. Statistical analyses were performed with SPSS 16.0 for Windows . Statistical significance was accepted for P<0.05 for all tests.The significance of ADM expression in ICC patients was investigated by IHC . ADM staWe investigated the clinical significance of ADM overex-pression in ICC by tissue microarray analysis of ICC tissues from 133 patients who underwent resection . Kaplan-To confirm the involvement of ADM in the growth of ICC cells, we transfected HuCCT1 cells with ADM and silenced HUH28 cells with short hairpin RNA, respectively. ADM expression was determined by western blotting . The tumThe roles of ADM in tumor cell migration and invasion were investigated by wound-healing and Transwell invasion assays. ADM-transfected cells achieved faster closure of the scratched 'wounds' than this rate in the control cells . FurtherEMT is one of the key events in tumor invasion and metastasis. We therefore investigated the effects of ADM on EMT by analyzing the expression levels of EMT markers and EMT-related transcription factors, and by morphological changes in the ICC cell lines. Western blotting demonstrated decreased expression levels of the epithelial markers E-cadherin and ZO-1, and increased expression of the mesenchymal markers vimentin and N-cadherin, and the EMT-related transcription factors ZEB1 and ZEB2 in the ADM-transfected cells , compareADM expression has been demonstrated in many human malignant cells ,15, but EMT is considered to be a critical mechanism involved in cancer metastasis ,17; howein vitro findings and clinical applicability, such animal experiments were not performed in the present study because of a lack of time and experimental conditions. Second, we focused mainly on patients with resectable ICC at a single institution, and further multicenter studies are needed to validate the findings.Our study had some limitations. First, although animal models offer an opportunity to bridge the gap between in vitro. High ADM levels in clinical ICC specimens correlated with poor prognosis. These findings suggest that ADM should be evaluated as a potential novel therapeutic target in ICC with the aim of improving the currently poor outcome of this disease.In conclusion, overexpression of ADM in human ICC cells led to increased growth, invasion and metastasis


We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured  Ever since human embryonic stem cells (hES) cells were first isolated from the inner cell mass of a human blastocyst in vitro differentiation is that hPS cells are inherently highly heterogeneous . Then, we describe main aspects of the tool itself: the automated imaging of the slide and high magnification imaging of colony regions (Section 3), the image processing of colony and cellular images (Section 4), and the construction of the data into seamless cellular maps that are consistent across adjacent fields (Section 5).hESC CHB-8  cells weFor immunofluorescent staining, cells were stained according to Lu et al., 2014. Primary antibodies used were Oct4 (Abcam 19857), Nanog (Abcam 21624), phospho-H3 (Millipore 06-570), and cPARP (Epitomics 1074-1). Secondary antibodies were Alexa Fluor 594 anti-goat IgG or Alexa Fluor 488 anti-rabbit IgG (both from Life Technologies). EdU staining was performed according to manufacturer's instructions (Life Technologies). We found that placing the dish on a shaker resulted in the most even antibody distribution. For single molecule mRNA FISH (smFISH) hybridization, Custom Stellaris mRNA probes were obtained from Biosearch Technologies. Hybridization was performed following a published protocol We utilized a Nikon Eclipse Ti inverted microscope equipped with a Prior motorized stage and a high-resolution CoolSNAP HQ2 CCD camera. We wrote custom software in MATLAB to control the microscope, camera, shutters, and stage hardware. This software utilizes the Micro-Manager API We constructed a program that allows multi-scale imaging of ESCs in different regions across the slide. A detailed walkthrough of this software is provided in The second major element of the program is region selection. In selecting regions, the user has the option of generating a lower-magnification pre-scan imaged with transmitted phase light and/or fluorescent channels. The pre-scan sub-GUI is illustrated in The software offers many flexible options for the high-resolution scan. Users may select any level of magnification, including both dry and oil-immersion objective lenses; any combination of fluorescent filters and exposure times; and may opt for an overlap between adjacent fields. Additionally, we implemented the ability to image Z-stacks, which may be useful for many applications such as detection of single molecule mRNA FISH probes . BecauseThis system provides powerful and flexible controls to image and analyze the heterogeneous population context of hPS cell culture at multiple scales, from molecular level, to cell, to colony regions, to colonies. Because it seamlessly integrates imaging with analysis, including automatic region selection based on colony segmentation, our setup is an improvement over currently available solutions to study long-range relationships of single hPS cells in culture.We mosaicked constituent field images into complete regions, taking advantage of the submicron accuracy of our Prior ProScan III stage. Using a combination of local neighborhood Gaussian blurring and thresholding using Otsu's method Field images were loaded into the segmentation module for processing and feature extraction. CellProfiler The analysis and informatics pipeline is easily portable. As an example, images acquired with Laser Scanning Cytometry , a non-confocal laser scanning microscope, can also be processed; the tool is thus capable of multimodal data fusion, at least at the classification or decision level.The software outputs either a nested MATLAB data structure or a CSV file. The data include the colony label and address of each cell, linked with the properties of those colonies , allowing for intra-colony and inter-colony analysis.We wrote custom scripts to export data into the FCS Express 4 Image Cytometry format (De Novo Software), allowing flexible manipulation of image and feature data.The virtual slide abstracts the important information about cellular space without having to deal with massive, cumbersome image files; an attribute particularly important when data are multimodal. The segmentation label matrix for each field is loaded into the region label matrix. Field labels are converted to global labels. For each field, the feature data are loaded and collated in to a data array structure addressed by the region number, field coordinates, and feature of interest. The centroid of each cell is derived from the segmentation results. Those values were combined with the colony identification step to yield an “address” of the cell within the context of its neighbors and the colony in which it resides.At a higher scale, our system incorporates a method for generating seamless maps of cellular fields .. We appWe analyzed quantitative relationships of individual cell properties to their location within the colony and colony-wide features such as size, shape, and texture. These properties include cell cycle state, expression of pluripotency markers such as Oct4 and Nanog, and shape and size of the individual nuclei. In addition, we used this tool to examine relationship of cell cycle variation as a function of colony properties and cellular position.Cell cycle regulation and pluripotency are closely linked; changes in pluripotency are coupled to changes in cell cycle progression One of the features derived from our informatics pipeline is the distance of a cell from the edge of the colony it resides in. Using the distance transform illustrated in The multiple scale capabilities helped uncover a noteworthy pattern : the freTo check whether this was primarily a phenomenon of cells belong to colonies of a certain size, we divided cells into groups on the basis of they belonged to a small, medium, or large-sized colony. We then similarly assessed the cell cycle percentages of those different colony types in each of the identified Cell Layers. We found that cells of each colony size were enriched in S-phase in Cell Layer 1 relative to Cell Layers 2 and 3, though the enrichment was slightly stronger in small colonies ., probabFurther understanding of the biological meaning of the enrichment of G2 phase cells at colony edge would require use of other methodologies, and measurement across different hES cell lines and culture conditions. However, it is a useful demonstration of the applicability of the tool we have described herein: novel insights on cell cycle progression by cell cycle determination of individual cells and analyzing their distribution relative to colony space across multiple colonies. Cellular decision-making is inherently stochastic and influenced by the cellular micro-environment, necessitating a high-throughput system capable of measuring and abstracting those relationships.We previously reported that cellular differentiation state has a profound impact on DNA damage regulation Looking at a higher level scale, the colony-level behavior of the cells within a single condition (after 1 day of RA differentiation), we found that instead of cells in all colonies responding in a uniform fashion to the external signals, there were clear regional behaviors from one colony to another and sometimes different patches within the same colony. The cell density classifier from our image analysis tool did a reasonable job at separating cells into subpopulations. The high-density cell regions had a lower Oct4 distribution than the low-density cell regions , and theTo test if the platform is amenable to datasets at the smallest scale of light microscope, we applied the imaging capture and analysis pipeline to single-molecule mRNA FISH (smFISH) data where each FISH spot corresponds to the diffraction-limited image of a RNA molecule with a size less than the resolution of the light microscope. To detect RNA spots computationally, we developed an algorithm that uses a morphological opening filter to remove out-of-focus light, followed by a Laplacian of Gaussian filter to sharpen the image and detect candidate foci x, y, using adjacent overlapped fields  image field calculated in the top (t), bottom (b), left (l), or right (r) margins using the threshold level x,ys. Minimizing the error function across all grids by locally or adaptively adjusting thresholds provides continuity and consistency as we move from one grid to another. The objective function uses a 2-norm, but a 1-norm may also be used for greater robustness to outliers. The minimization provided a matrix of local or grid specific threshold values S = . An initial estimate for 0,S, S was obtained by thresholding the combined data for all fields and setting as constant. With this equalized spot detection, we are able to quantitate the FISH spots in each cell with high confidence, and follow the dynamic down regulation of Oct4 levels during differentiation of hES cells Click here for additional data file.S2 FigCell cycle and Nanog distribution in hES colonies. (A) Density scatter plot of integrated DAPI intensity versus mean EdU intensity, generated using FCS Express. Three gates were chosen to categorize cells based on their cell cycle state: G1, S, and G2/M. (B) Normalized histograms of mean Nanog intensity for the G1 (red), S (magenta), and G2/M (blue) subpopulations. (C) Differential spatial distribution of Nanog positive and negative cells in hES colonies (D). Histogram distribution of mean Nanog intensity of cells on periphery (green), Cell Layer 1 (yellow), Cell Layer 2 (cyan), and Cell Layer 3 (black) of the colonies.(TIF)Click here for additional data file.S3 FigInter-colony heterogeneity of cell cycle distribution. (A) Histogram of cellular distance from edge in cells belonging to differently-sized colonies. The maximum cellular distance from the edge of the colony to the center was used to separate colonies into small (<150 um), medium (150–300 um) and large (>300 um) sizes. (B) Distributions of cell cycle phases for each cell layer in small, medium, and large-sized colonies. The inter-colony heterogeneity is not significant except for a slight enrichment in S-phase cells in Cell Layer 1 of small colonies. Error bars represent 95% of bootstrap samples.(TIF)Click here for additional data file.S4 FigPhenotypic changes of hES cells during RA induced differentiation. Cells undergoing RA-induced differentiation were stained for Oct4 and DAPI and analyzed with the pipeline. (A) Oct4 level goes down; and (B) more cells are in G1-phase as cells differentiate.(TIF)Click here for additional data file.S5 FigAssessment of segmentation consistency and staining variation. (A) Exemplary image of colony with 1 day RA differentiation and no NCS exposure. Entire sample contained 24,629 cells in 15 colonies divided into 410 sub-colony windows. Windows were 250 µm (width) by 192 µm (height) in size. (B) Integrated DAPI intensity over regional windows versus the number of segmented nuclei within the window. The relationship is linear over most densities, but is less linear at high densities where segmenting individual cells is more difficult and poorly segmented nuclei are discarded. Trend line is binned average +/− standard deviation. (C) Number of segmented nuclei within a window versus the average nuclear p53 content in that window. With no NCS treatment, nuclear p53 levels do not change as a function of cell density. The relationship is constant over the range of most densities. (D) In contrast to p53, nuclear Oct4 content decreases as a function of cell density.(TIF)Click here for additional data file.S1 TextDetailed Description of Program Operation. Description of the source code availability, motivation, detailed image acquisition and analysis steps with 5 supporting figures .(DOCX)Click here for additional data file.


Puma concolor coryi, to evaluate recently developed and traditional estimators.Global positioning system (GPS) technology for monitoring home range and movements of wildlife has resulted in prohibitively large sample sizes of locations for traditional estimators of home range. We used area-under-the-curve to explore the fit of 8 estimators of home range to data collected with both GPS and concurrent very high frequency (VHF) technology on a terrestrial mammal, the Florida panther Area-under-the-curve was the highest for Florida panthers equipped with Global Positioning System (GPS) technology compared to VHF technology. For our study animal, estimators of home range that incorporated a temporal component to estimation performed better than traditional first- and second-generation estimators.Comparisons of fit of home range contours with locations collected would suggest that use of VHF technology is not as accurate as GPS technology to estimate size of home range for large mammals. Estimators of home range collected with GPS technology performed better than those estimated with VHF technology regardless of estimator used. Furthermore, estimators that incorporate a temporal component (third-generation estimators) appeared to be the most reliable regardless of whether kernel-based or Brownian bridge-based algorithms were used and in comparison to first- and second-generation estimators. We defined third-generation estimators of home range as any estimator that incorporates time, space, animal-specific parameters, and habitat. Such estimators would include movement-based kernel density, Brownian bridge movement models, and dynamic Brownian bridge movement models among others that have yet to be evaluated.The online version of this article (doi:10.1186/s40462-015-0039-4) contains supplementary material, which is available to authorized users. Recent advances in global positioning system (GPS) technology for monitoring wildlife have revolutionized data collection for spatial analysis of movements, home range, and resource selection. These datasets acquired with GPS technology are more copious and locations are more precise when compared to locational data collected using very high frequency (VHF) systems. Although published studies have reported on the reliability of home range estimators using datasets collected with VHF technology ,2, few hConcurrent with advances in GPS technology, alternative methods for estimation of home range have been developed to accommodate large numbers of auto-correlated relocations from GPS datasets. Amongst these are first-generation methods such as kernel density estimators that have proven capable of providing home ranges using large GPS datasets KDE; -5), alth, alth5])Traditionally, the suggested estimator of home range was based on simulated datasets ,17 that Puma concolor coryi) with GPS technology and concurrent VHF technology to explore relationships between 8 estimators of home range. Specifically, our objectives were to: (1) determine if AUC differed for estimators of home range between locations collected with GPS versus concurrent VHF technology and (2) assess factors that influence AUC for estimators of home range across a range of individual animals using GPS technology.Recently, area-under-the-curve (AUC) was assessed as an analytical means of choosing the most appropriate estimator of home range for an avian and mammalian species . The AUCOur study area encompassed a large portion of the range of the breeding population of panthers in South Florida Figure  south ofWe used trained hounds to track and tree adult and subadult panthers for subsequent capture and radiocollaring by project personnel during concurrent research projects associated with management and conservation of panther from 2005 to 2013 ,20. We dTo collect concurrent VHF locations of GPS-collared panthers, we used a Cessna 172  equipped with a pair of directional antennas that were attached to a radio receiver via coaxial cable to estimate location of a VHF beacon in the GPS collar by selectively listening to radio signals from either or both antennas mounted to the struts of the wings and homing in on signal strength. We demarcated these locations using an application on a laptop computer synchronized with a GPS and loaded with satellite imagery to obtain Universal Transverse Mercator coordinates in flight. We conducted most telemetry flights between 0700 hours and 1100 hours 3 times per week . We estimated the location of collars at fixed locations unknown to the observer  during flights and determined VHF aerial telemetry location error to be 124 m .We collared 31 independent-aged panthers  between February 2005 and February 2013 , r (sphere of influence), or a (adaptive sphere of influence) of nearest neighbors [k and the square root of the number of relocations for each individual for the value of k .We estimated utilization distributions with LOCO that produces bounded home ranges using a generalization of the minimum convex polygon method. Minimum convex polygon construction is applied to a subset of localized data in space using either eighbors ,9. The hWe estimated utilization distributions with the single-linkage cluster (SLCA) method that links 3 locations in clusters that minimizes the mean joining distance . The cluWe estimated utilization distributions with computation of the Delaunay triangulation to create characteristic hull (CHAR) polygons using a set of relocations then ordering triangles from smallest to largest . The CHAWe estimated utilization distributions using the fixed-KDE method because fixed kernel was considered most accurate compared with adaptive kernel ,24. We sWe also estimated KDE using the bivariate plug-in bandwidth (PKDE) that performs well even when analyzing dependent data that is especially common from animals with locations collected with GPS technology . First- We estimated utilization distributions with biased random bridges using the movement-based kernel density estimator (MKDE) that can incorporate time, distance, and habitat into estimates of home range ,15. UnliWe estimated utilization distributions using the BBMM that requires (1) sequential location data, (2) estimated error associated with location data, and (3) grid-cell size assigned for the output utilization distribution . The BBMWe estimated utilization distributions using the dBBMM that requires the same parameters as BBMM . The varadehabitatHR , ks , BBMM , and mo . We mod .hourly), (2) 2–4 hours (four), and (3) 7–14 hours (seven). Study area was categorical and used as a proxy for habitat interference in acquiring a GPS location or influencing accuracy and represented a continuum of generally more open marsh landscape in South Florida to more upland and wetland forested habitats in the northern portion of the breeding range  of home ranges estimated by GPS technology. Fixed effects were covariates that have been suggested to influence the accuracy of estimating home range that included 5 covariates: estimator type, fix success, study area, GPS collection schedule, and number of locations used to estimate home range ,30. We s effects .Table 1x2 = 573.99, df =14, P < 0.001) with the highest AUC consistently occurring for GPS compared to VHF technology  to estimate home range using third-generation estimators should be critically reviewed based on our comparison using concurrent GPS location data  from the same animals. The AUC differed for estimators of home range determined using data collected with GPS compared to estimates of home range derived from concurrent VHF locations, which were typically estimated with 10% fewer locations , GPS error, and movement-specific parameters that could vary by individual -14. LocaA caveat in our study is that we evaluated annual home ranges but we chose not to evaluate the influence of the extent of location data across the landscape and resulting estimation of home range on AUC. The variability in our data for some estimators may be attributed to using annual home range that incorporate animal-specific movements , when they are actually present, as opposed to shorter-duration seasonal home ranges. This issue may have resulted in poorer estimates for location-based KDE or polygon-derived estimators than third-generation estimators due to over-estimation of home ranges as previously reported  and these variables can be directly accounted for within third-generation estimators. Development of these third-generation estimators were a result of GPS datasets and previous research suggesting that location-specific parameters and landscape characteristics influenced accuracy of estimates of size of home range ,34,37. O


Helicobacter pylori infection is one of the most frequent causes of dyspepsia in the general population. As gastro-intestinal symptoms are a common complaint among HIV-positive persons, the purpose of the present study is to assess the role of Helicobacter pylori infection in this category of patients.Helicobacter pylori infection by stool antigen detection: group A – 36 dyspeptic subjects and group B – 5 patients without dyspeptic complaints. We compared the two groups from the point of view of the frequency of Helicobacter pylori infection. Statistical analysis of data was performed with the help of GraphPad programme.We performed a retrospective, case-control study on two groups of HIV-infected patients admitted to the Infectious Diseases Clinic I, Tîrgu-Mureş and tested for Helicobacter pylori infection, while none of the patients in group B was diagnosed with Helicobacter pylori infection .4 (11.11%) HIV-positive patients with dyspeptic symptoms tested positive for Helicobacter pylori, the absence of a statistically significant association between Helicobacter pylori infection and dyspepsia among HIV-positive patients may suggest that other etiologies should be searched in HIV-infected patients with gastro-intestinal complaints.Although none of the asymptomatic patients in our study was diagnosed with


Systemic lupus erythromatosus (SLE) is a complex and systemic autoimmune disease. It is characterized by diverse clinical symptoms, revealing widespread immune-mediated damage. The common clinical features diagnosed in patients with SLE comprise of skin and joint diseases, hematological abnormalities, renal disease and neuropsychiatric complications. Although the etiology of SLE is still unknown, genetic factors are likely to be important in susceptibility to SLE and influence presentation of disease heterogeneity and production of autoantibody in affected subjects.There are several evidences that Human leukocyte antigens are associated with SLE. Herein, we studied HLA-DRB1 alleles to detect the association of these alleles in Iranian children with juvenile onset SLE.This study consists of 31 children with systemic lupus erythematosus and 56 healthy controls. Genomic DNA was extracted and HLA typing was performed by Polymerase Chain Reaction with Sequence-Specific Primers technique (PCR-SSP).HLA- DRB1*01, HLA- DRB1*04, HLA-DRB1*11 and HLA- DRB1*13 were detected to be the most frequent alleles associated with SLE in Iranian children. The frequency of HLA-DRB1*08 was not significantly different in both groups. HLA-DRB1*07 had a higher rate of repetition in the control group than patients with SLE.We reached the conclusion that there was a significant difference in the frequency of some alleles between patients and controls which could be related to susceptibility to SLE. This difference may help to predict the onset of lupus in children.None declared.