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Introduction: Resistance to trastuzumab in breast cancer is an ongoing challenge. Clinical and biological effects of co-targeting HER2 and mammalian target of rapamycin (mTOR) in patients with HER2-positive early operable breast cancer via the addition of everolimus to preoperative trastuzumab were evaluated in a phase II randomised study., Methods: Patients were randomised 1:1 to receive trastuzumab (4 mg/kg initial dose then 2 mg/kg weekly for 5 weeks) alone or combined with everolimus (10 mg/day for 6 weeks) and then underwent surgery. Tumours were assessed by clinical examination and echography at the baseline and on treatment. The primary end-point was the clinical response rate at 6 weeks. Pathological response and safety were also evaluated. Baseline and surgery tumour samples were assessed by immunohistochemistry and multiplex immunoanalysis for predictive downstream effectors of the PI3K/AKT/mTOR and MAP kinase (MAPK) pathways., Results: Eighty-two patients were enrolled, 41 per arm. The clinical response rates were 34.1% and 43.9% with trastuzumab alone and combined with everolimus, respectively. Pathological response rates were 43.6% and 47.5%, respectively. Addition of everolimus increased toxicity, notably mucositis (82.5% versus 5.0%) and rash (57.5% versus 10.0%), but grade III/IV events were rare. No correlation between response to treatments and baseline candidate biomarkers was identified, except for PIK3CA mutations which were found to predict trastuzumab resistance. Significant changes were seen in several MAPK pathway effectors after combination therapy., Conclusions: The addition of everolimus did not improve the efficacy, but induced MAPK signalling. Combination therapy to overcome pathway cross-talk should be considered to maximise the effectiveness of trastuzumab in this setting. ClinicalTrial.gov Identifier NCT00674414., Competing Interests: Conflict of interest statement M.C. received research funding and served as a speaker/advisor (compensated to the hospital) for AstraZeneca, Novartis, AbbVie, Sanofi, Lilly, Pfizer, Sandoz, Accord, G1 Therapeutic, Pierre Fabre Oncology, Servier and Roche. T.B. served as a speaker/advisor (compensated) for Roche and Novartis. B.P. received research funding, personal fees and non-financial support from Novartis. M.A. received research funding and served as a speaker/advisor (compensated to the hospital) for Novartis, AstraZeneca, Seattle Genetics, AbbVie and Pfizer. V.D. served as a speaker/advisor (compensated) for Pfizer, Novartis, Daiichi Sankyo and Lilly. F.A. received research funding and served as a speaker/advisor (compensated to the hospital) for Roche, AstraZeneca, Daiichi Sankyo, Pfizer, Novartis and Lilly. J-L.M. is a speaker for Roche and Novartis and a translational research board member for Novartis. Unicancer as the study sponsor received research funding (investigator-sponsored study) by Roche and Novartis. All other authors declare no potential conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Planning forest management relies on predicting insect outbreaks such as mountain pine beetle, particularly in the intermediate-term future, e.g., 5-year. Machine-learning algorithms are potential solutions to this challenging problem due to their many successes across a variety of prediction tasks. However, there are many subtle challenges in applying them: identifying the best learning models and the best subset of available covariates (including time lags) and properly evaluating the models to avoid misleading performance-measures. We systematically address these issues in predicting the chance of a mountain pine beetle outbreak in the Cypress Hills area and seek models with the best performance at predicting future 1-, 3-, 5- and 7-year infestations. We train nine machine-learning models, including two generalized boosted regression trees (GBM) that predict future 1- and 3-year infestations with 92% and 88% AUC, and two novel mixed models that predict future 5- and 7-year infestations with 86% and 84% AUC, respectively. We also consider forming the train and test datasets by splitting the original dataset randomly rather than using the appropriate year-based approach and show that this may obtain models that score high on the test dataset but low in practice, resulting in inaccurate performance evaluations. For example, a k -nearest neighbor model with the actual performance of 68% AUC, scores the misleadingly high 78% on a test dataset obtained from a random split, but the more accurate 66% on a year-based split. We then investigate how the prediction accuracy varies with respect to the provided history length of the covariates and find that neural network and naive Bayes, predict more accurately as history-length increases, particularly for future 1- and 3-year predictions, and roughly the same holds with GBM. Our approach is applicable to other invasive species. The resulting predictors can be used in planning forest and pest management and planning sampling locations in field studies., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo -synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo -synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.

The chemotherapeutic drug epirubicin increases the exosomal export of miR-503 in endothelial cells. To understand the mechanisms behind this process, we transfected endothelial cells with miR-503 carrying a biotin tag. Then, we pulled-down the proteins interacting with miR-503 and studied their role in microRNA exosomal export. A total of four different binding partners were identified by mass spectrometry and validated by western blotting and negative controls, among them ANXA2 and hnRNPA2B1. Using knock-down systems combined with pull-down analysis, we determined that epirubicin mediates the export of miR-503 by disrupting the interaction between hnRNPA2B1 and miR-503. Then, both ANXA2 and miR-503 are sorted into exosomes while hnRNPA2B1 is relocated into the nucleus. The combination of these processes culminates in the increased export of miR-503. These results suggest, for the first time, that RNA-binding proteins can negatively regulate the exosomal sorting of microRNAs.

Objective: This study presents trends in organism isolation and antimicrobial resistance in routine microbiology test results from acute-care hospital microbiology laboratories in Vermont., Methods: Organism identifications and antimicrobial susceptibility test results were captured from acute-care hospital laboratories to monitor geographic and temporal trends in resistance and emerging microbial threats with the free WHONET software., Results: Data were provided from 12 acute care hospital laboratories from 2011 through 2018 for 318,833 isolates from 148,994 patients (70% female, 74% outpatient, and 63% urine). Significant differences (p < 0.05) in age, gender, and antimicrobial susceptibility results (e.g. Escherichia coli and levofloxacin) between outpatient and inpatient isolates were identified with temporal increases in certain species (e.g. Aerococcus urinae ) and resistance (e.g. Streptococcus pneumoniae and erythromycin). The use of multi-resistance phenotypes demonstrated significant heterogeneity (p < 0.05) in MRSA strains between facilities, for example Staphylococcus aureus resistant to six priority antimicrobials were found in no critical access hospitals (fewer than 25 inpatient beds) but in all non-critical access hospitals., Conclusions: Comprehensive electronic surveillance of antimicrobial resistance utilizing routine clinical microbiology data with free software tools offers early recognition and tracking of emerging community and healthcare resistance threats at the local and state level.

Mountain pine beetle (MPB) outbreaks have caused major economic losses and ecological consequences in North American pine forests. Ecological and environmental factors impacting MPB life-history and stands susceptibility can help with the detection of MPB infested trees and thereby, improve control. Temperatures, water stress, host characteristics, and beetle pressure are among those ecological and environmental factors. They play different roles on MPB population dynamics at the various stages of an outbreak and these roles can be affected by intensive management. However, to make detailed connections between ecological and environmental variables and MPB outbreak phases, a deeper quantitative analysis on local scales is needed. Here, we used logistic regressions on a highly-detailed and georeferenced data set to determine the factors driving MPB infestations for the different phases of the current isolated MPB outbreak in Cypress Hills. While we showed that the roles of ecological and environmental factors in a forest intensively controlled for MPB are consistent with the literature for uncontrolled forests, we determined how these factors shifted through onset, peak, and collapse phases of the intensively controlled forest. MPB presence mostly depends on nearby beetle pressure, notably for the outbreak peak. However additional weather and host variables are necessary to achieve high predictive ability for MPB outbreak locations. Our results can help managers make appropriate decisions on where and how to focus their effort, depending on which phase the outbreak is in.

The efficacy of direct control methods in bark beetle outbreaks is a disputed topic. While some studies report that control reduces tree mortality, others see little effect. Existing models, linking control rate to beetle population dynamics and tree infestations, give insights, but there is a need to take into account the environment spatial variability and its impact on beetle life cycle. Here, we use natural variability found in a carefully monitored and controlled infestation to simulate outbreak dynamics under different control effort and to explore the impact of control on outbreaks suppression and tree mortality. Our semi-empirical predictive model of the number of infested trees as a function of ecological and environmental variables is coupled to a simulation model for infestation dynamics. We show that even a little control can have a major impact on the number of infested trees after several years of sustained effort. However, a moderate control of 60% is required to reduce the beetle population on the long term. Furthermore, a control rate of 69%-83% is needed to achieve outbreak suppression in under 13 years depending on the abundance of incoming flights from outside sources., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The data presented in this article are related to the research article entitled "Mountain pine beetle outbreak duration and pine mortality depend on direct control effort" [1]. This article provides presence of mountain pine beetle infested trees detected by the Saskatchewan Forest Service on a grid covering the spatial extent of the Saskatchewan portion of the Cypress Hills interprovincial park between 2006 and 2018. For each grid cell, associated ecological and environmental covariates, such as topography, weather and vegetation, are also provided. These data cover the spatio-temporal extent of an almost entire mountain pine beetle outbreak and contribute to the understanding of mountain pine beetle outbreak dynamics., (© 2020 The Authors.)

An understanding of the dynamic structural properties of chromatin requires techniques that allow the profiling of regions of both open and closed chromatin as well as the assessment of nucleosome occupancy. The recently developed MNase accessibility (MACC) technique allows for the simultaneous measurement of chromatin opening and compaction, as well as nucleosome occupancy, on a genome-wide scale in a single assay. This article presents a low-input MACC procedure that considerably extends the utility of the original MACC assay. Low-input MACC generates high-quality data using very low cell numbers (as few as 50 cells per titration point), making it ideal for samples obtained after fluorescence-activated cell sorting or dissection, or in clinical settings. Moreover, low-input MACC has significantly improved several steps of the initial method, offering a more rapid and robust methodology. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Background: The p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated ΔN isoform that inhibits TAp73 and p53 and thus, acts as an oncogene., Results: By employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch. Finally, PpIX showed potent antitumor effect and inhibited the growth of xenograft human tumors in mice., Conclusion: Our findings may in future contribute to the successful repurposing of PpIX into clinical practice.

Background: Video and hobby gaming are immensely popular among adults; however, associations between gaming and health have primarily been investigated in children and adolescents. Furthermore, most research has focused on electronic gaming, despite traditional hobby gaming gaining prominence., Objective: To determine whether the number of platforms used, platform preference, and gaming time are associated with obesity, physical activity, sedentary behavior, and cardiovascular risk factors in an adult gaming population., Methods: We conducted a cross-sectional analysis using data obtained from 292 participants who attended a large Midwestern gaming convention. We collected data using a computer-based questionnaire that comprised questions on gaming behavior, demographics, physical activity (using the International Physical Activity Questionnaire), and health characteristics. In addition, we used multivariable-adjusted linear and logistic regression to model health outcomes as a function of the number of platforms used, platform preference, and weekday and weekend gaming time quartile., Results: After adjusting for covariates, we observed a significant linear trend for increasing odds of being obese and higher weekend sitting time by the number of platforms used (P=.03 for both). The platform preference and weekend gaming time quartile exhibited significant associations with odds of meeting physical activity recommendations (P=.047 and P=.03, respectively). In addition, we observed higher odds of being obese among those reporting that they sat most or all of the time while gaming [odds ratio (OR) 2.69 (95% CI 1.14-6.31) and OR 2.71 (95% CI 1.06-6.93), respectively]., Conclusions: In adult gamers, the number of platforms used, which platforms they prefer to play on, and the amount of time spent gaming on weekends could have significant implications for their odds of being obese and meeting physical activity recommendations., (©James Arnaez, Georgia Frey, Donetta Cothran, Margaret Lion, Andrea Chomistek. Originally published in JMIR Serious Games (http://games.jmir.org), 12.06.2018.)

Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids, and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes are still not completely elucidated. In this study, by the use of next-generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and noncoding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis for studying the involvement of RNA-binding proteins capable of recognizing RNA sequences and their role in the export of RNAs into exosomes., (© 2018 Pérez-Boza et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

To evaluate water use and the supporting water source of a tropical rainforest, a 4-year assessment of evapotranspiration (ET) was conducted in Pasoh Forest Reserve, a lowland dipterocarp forest in Peninsular Malaysia. The eddy covariance method and isotope signals of rain, plant, soil, and stream waters were used to determine forest water sources under different moisture conditions. Four sampling events were conducted to collect soil and plant twig samples in wet, moderate, dry, and very dry conditions for the identification of isotopic signals. Annual ET from 2012 to 2015 was quite stable with an average of 1,182 ± 26 mm, and a substantial daily ET was observed even during drought periods, although some decline was observed, corresponding with volumetric soil water content. During the wet period, water for ET was supplied from the surface soil layer between 0 and 0.5 m, whereas in the dry period, approximately 50% to 90% was supplied from the deeper soil layer below 0.5-m depth, originating from water precipitated several months previously at this forest. Isotope signatures demonstrated that the water sources of the plants, soil, and stream were all different. Water in plants was often different from soil water, probably because plant water came from a different source than water that was strongly bound to the soil particles. Plants showed no preference for soil depth with their size, whereas the existence of storage water in the xylem was suggested. The evapotranspiration at this forest is balanced and maintained using most of the available water sources except for a proportion of rapid response run-off., (Copyright © 2017 John Wiley & Sons, Ltd.)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation. Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Cancer Discov; 7(11); 1336-53. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201 ., (©2017 American Association for Cancer Research.)

Antiglomerular basement membrane glomerulonephritis (anti-GBM GN) is a rare autoimmune disease that is characterized by rapidly progressive glomerulonephritis that may be associated with pulmonary hemorrhage. Anti-GBM GN is caused by autoantibodies (classically type G immunoglobulin) directed against the α3 subunit of type IV collagen. Without any appropriate treatment, the disease is generally fulminant, and patient and kidney survival is poor. The current guidelines recommend the use of plasma exchanges and immunosuppressive drugs. Immunoadsorption (IA) can remove pathogenic IgGs from the circulation and do not require plasma infusions, contrary to plasma exchanges. IA has seldom been used in adult patients with good tolerance and efficiency. We report herein the first pediatric case successfully treated with IA combined with immunosuppressive drugs in a 7-year-old girl who presented acute kidney injury (estimated glomerular filtration rate 38 mL/minute/1.73 m 2 ). A kidney biopsy revealed numerous >80% glomerular crescents and linear IgG deposits along the glomerular basement membrane. Ten IA sessions led to rapid and sustained clearance of autoantibodies and improvement of kidney function until 21 months after onset (glomerular filtration rate 87 mL/minute/1.73 m 2 ). No adverse effect was noted. This report adds to the growing body of evidence suggesting IA as a therapeutic alternative to plasma exchanges in anti-GBM GN. The other 27 published pediatric cases of anti-GBM GN are reviewed., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2017 by the American Academy of Pediatrics.)

We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineage-specific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase. In contrast, in mouse embryonic fibroblasts the coding segment is depleted of nucleosomes, which instead cover the RSS, thereby rendering it inaccessible. Pro-T cells exhibit a pattern intermediate between pro-B cells and mouse embryonic fibroblasts. We also find large-scale variations of nucleosome density over hundreds of kilobases, delineating chromosomal domains within IgH, in a cell type-dependent manner. These findings suggest that developmentally regulated changes in nucleosome location and occupancy, in addition to the known chromatin modifications, play a fundamental role in regulating V(D)J recombination. Nucleosome positioning-which has previously been observed to vary locally at individual enhancers and promoters-may be a more general mechanism by which cells can regulate the accessibility of the genome during development, at scales ranging from several hundred base pairs to many kilobases., Competing Interests: The authors declare no conflict of interest.

Human epidermal growth factor 2 (HER2) is overexpressed in 15-20% of breast carcinomas. The overexpression of HER2 was previously associated with a poor prognosis until the development of the first anti-HER2 therapy, trastuzumab, which drastically improves the prognosis of HER2-overexpressing breast cancers. However, its mechanism of action remains not fully understood. Several studies have proposed that the behavior and mechanism of action of trastuzumab may be drastically altered in vitro and in vivo . The present study assesses the ability of trastuzumab to inhibit the phosphorylation of the key-proteins of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin and Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways in vitro , in breast cancer cell lines and in tumor biopsies obtained from patients treated with trastuzumab preoperative monotherapy as part of the Unicancer GEP04 RADHER phase II clinical trial. HER2-positive SKBR3 and HER2-negative MCF-7 cell lines were exposed to trastuzumab for 72 h. In total, 41 patients received trastuzumab alone for 6 weeks of preoperative treatment. Biopsies were collected at the baseline and at surgery. A total of 19 pairs of associated baseline and surgery tumor specimens were eligible for protein extraction and comparative phosphoprotein expression analysis, prior to and subsequent to treatment. The expression of phosphoproteins was quantitatively assessed using a multiplex immunoassay. In the SKBR3 cell line, a statistically significant decrease of the expression level of phosphorylated (p-)AKT, p-ribosomal protein S6 kinase B1, p-extracellular signal regulated kinase 1/2 and p-mitogen-activated protein kinase kinase 1 was observed after exposure to trastuzumab. In contrast, no statistically significant variations for levels expression of these phosphoproteins were observed in patients following treatment. The lack of downregulation of PI3K and MAPK pathways could probably be explained by the implementation of a predominant immunological mechanism of action for trastuzumab, a type of antibody-dependent cell-mediated toxicity, which has previously been reported in preoperative monotherapy settings. The present study confirms that trastuzumab involves various modes of action when assayed in vitro and used clinically.

In endothelial cells, binding of vascular endothelial growth factor (VEGF) to the receptor VEGFR2 activates multiple signaling pathways that trigger processes such as proliferation, survival, and migration that are necessary for angiogenesis. VEGF-bound VEGFR2 becomes internalized, which is a key step in the proangiogenic signal. We showed that the urokinase plasminogen activator receptor (uPAR) interacted with VEGFR2 and described the mechanism by which this interaction mediated VEGF signaling and promoted angiogenesis. Knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) impaired VEGFR2 signaling, and uPAR deficiency in mice prevented VEGF-induced angiogenesis. Upon exposure of HUVECs to VEGF, uPAR recruited the low-density lipoprotein receptor-related protein 1 (LRP-1) to VEGFR2, which induced VEGFR2 internalization. Thus, the uPAR-VEGFR2 interaction is crucial for VEGF signaling in endothelial cells., (Copyright © 2015, American Association for the Advancement of Science.)

The interaction between tumor cells and their microenvironment is an essential aspect of tumor development. Therefore, understanding how this microenvironment communicates with tumor cells is crucial for the development of new anti-cancer therapies. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression. They are secreted into the extracellular medium in vesicles called exosomes, which allow communication between cells via the transfer of their cargo. Consequently, we hypothesized that circulating endothelial miRNAs could be transferred to tumor cells and modify their phenotype. Using exogenous miRNA, we demonstrated that endothelial cells can transfer miRNA to tumor cells via exosomes. Using miRNA profiling, we identified miR-503, which exhibited downregulated levels in exosomes released from endothelial cells cultured under tumoral conditions. The modulation of miR-503 in breast cancer cells altered their proliferative and invasive capacities. We then identified two targets of miR-503, CCND2 and CCND3. Moreover, we measured increased plasmatic miR-503 in breast cancer patients after neoadjuvant chemotherapy, which could be partly due to increased miRNA secretion by endothelial cells. Taken together, our data are the first to reveal the involvement of the endothelium in the modulation of tumor development via the secretion of circulating miR-503 in response to chemotherapy treatment.

Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective.

The p53 and NFκB sequence-specific transcription factors play crucial roles in cell proliferation and survival with critical, even if typically opposite, effects on cancer progression. To investigate a possible crosstalk between p53 and NFκB driven by chemotherapy-induced responses in the context of an inflammatory microenvironment, we performed a proof of concept study using MCF7 cells. Transcriptome analyses upon single or combined treatments with doxorubicin (Doxo, 1.5μM) and the NFκB inducer TNF-alpha (TNFα, 5ng/ml) revealed 432 up-regulated (log2 FC> 2), and 390 repressed genes (log2 FC< -2) for the Doxo+TNFα treatment. 239 up-regulated and 161 repressed genes were synergistically regulated by the double treatment. Annotation and pathway analyses of Doxo+TNFα selectively up-regulated genes indicated strong enrichment for cell migration terms. A panel of genes was examined by qPCR coupled to p53 activation by Doxo, 5-Fluoruracil and Nutlin-3a, or to p53 or NFκB inhibition. Transcriptome data were confirmed for 12 of 15 selected genes and seven (PLK3, LAMP3, ETV7, UNC5B, NTN1, DUSP5, SNAI1) were synergistically up-regulated after Doxo+TNFα and dependent both on p53 and NFκB. Migration assays consistently showed an increase in motility for MCF7 cells upon Doxo+TNFα. A signature of 29 Doxo+TNFα highly synergistic genes exhibited prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression. We propose that the crosstalk between p53 and NFκB can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy.

The N-terminal fragment of prolactin (16K PRL) inhibits tumor growth by impairing angiogenesis, but the underlying mechanisms are unknown. Here, we found that 16K PRL binds the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1), which is known to contextually promote tumor angiogenesis and growth. Loss of PAI-1 abrogated the antitumoral and antiangiogenic effects of 16K PRL. PAI-1 bound the ternary complex PAI-1-urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR), thereby exerting antiangiogenic effects. By inhibiting the antifibrinolytic activity of PAI-1, 16K PRL also protected mice against thromboembolism and promoted arterial clot lysis. Thus, by signaling through the PAI-1-uPA-uPAR complex, 16K PRL impairs tumor vascularization and growth and, by inhibiting the antifibrinolytic activity of PAI-1, promotes thrombolysis.

Breast cancer is the most common cancer among women with more than 53,000 new cases every year in France. The PI3K/AKT pathway is one of the major pathways involved in mammary tumorigenesis. The first effector of this pathway downstream Human Epidermal growth factor Receptor (HER receptors) is the enzyme phosphatidyl-inositol-3-kinase (PI3K). Some mutations in the gene encoding for the catalytic subunit of this enzyme, the PIK3CA gene, plays an important role, especially in the resistance to targeted therapies used clinically during the last decade. Indeed, the presence of alterations, an overexpression of the PI3K/AKT pathway, or the presence of PIK3CA mutation could explain some resistance to targeted therapies. PIK3CA mutations also appear to have a significant interest in the prediction of response to targeted therapies. Finally, many drugs in development, specifically targeting PI3K or other effectors of the PI3K/AKT pathway are intended to be administered only to patients with tumor bearing a mutation of PIK3CA, which makes the somatic mutations detection more and more important. The aim of this article is to consider biological aspects, clinical significance, diagnostic and theranostic interest of PIK3CA mutations in breast cancer.

MAP kinase signaling proteins have major implications in the molecular oncogenesis of breast cancers and have been extensively investigated as putative targets for therapy. This study reports the investigation of the expression of P38 MAPK and its phosphorylated form (p-P38 MAPK) in clinical specimens of invasive breast carcinomas and their correlation with estrogen receptor (ER) and HER2 expression, as well as MAPK and PI3 kinase-AKT pathway signaling phosphorylated proteins. Expression levels of P38 MAPK and p-P38 MAPK as well as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1 and p-ERK1/2 were quantitatively assessed using multiplex bead immunoassay in frozen specimens from 45 invasive ductal breast cancers. Twenty-nine specimens were ER+, 15 were HER2+ and 10 were triple‑negative breast cancers (TNBCs). P38 MAPK was found to be expressed in all tumor specimens and was significantly (P=0.002) overexpressed in ER+ tumors. P38 MAPK expression was lower in TNBCs than in all of the other tumors. The median expression of p-P38 MAPK was also higher in ER+ tumors while lower in the TNBCs. HER2 status had no effect on P38 MAPK and p-P38 MAPK expression. No variation in the phosphorylation rate of P38 MAPK was observed in relation with ER, HER2 or TNBC status. Significantly higher (P=0.0048) expression of p-AKT was observed in HER2+ tumors. No significant difference in p-MEK1, p-GSK3β and p-S6K expression was found in any other comparisons based on ER and HER2 expression subtypes. Investigation of the expression of multiple phosphorylated signaling proteins can be used for personalized targeted therapy. In invasive breast cancer, the overexpression of P38 MAPK may serve as a biomarker for the evaluation of P38 MAPK inhibitors.

According to the French legislation on medical biology (January 16th, 2010), all biological laboratories must be accredited according to ISO 15189 for at least 50% of their activities before the end of 2016. The extraction of DNA from a sample of interest, whether solid or liquid is one of the critical steps in molecular biology and specifically in somatic or constitutional genetic. The extracted DNA must meet a number of criteria such quality and also be in sufficient concentration to allow molecular biology assays such as the detection of somatic mutations. This paper describes the validation of the extraction and purification of DNA using chromatographic column extraction and quantitative determination by spectrophotometric assay, according to ISO 15189 and the accreditation technical guide in Human Health SH-GTA-04.

Estrogen receptors (ERs) and p53 can interact via cis-elements to regulate the angiogenesis-related VEGFR-1 (FLT1) gene, as we reported previously. Here, we address cooperation between these transcription factors on a global scale. Human breast adenocarcinoma MCF7 cells were exposed to single or combinatorial treatments with the chemotherapeutic agent doxorubicin and the ER ligand 17β-estradiol (E2). Whole-genome transcriptome changes were measured by expression microarrays. Nearly 200 differentially expressed genes were identified that showed limited responsiveness to either doxorubicin treatment or ER ligand alone but were upregulated in a greater than additive manner following combined treatment. Based on exposure to 5-fuorouracil and nutlin-3a, the combined responses were treatment-specific. Among 16 genes chosen for validation using quantitative real-time PCR, seven (INPP5D, TLR5, KRT15, EPHA2, GDNF, NOTCH1, SOX9) were confirmed to be novel direct targets of p53, based on responses in MCF7 cells silenced for p53 or cooperative targets of p53 and ER. Promoter pattern searches and chromatin IP experiments for the INPP5D, TLR5, KRT15 genes supported direct, cis-mediated p53 and/or ER regulation through canonical and noncanonical p53 and ER response elements. Collectively, we establish that combinatorial activation of p53 and ER can induce novel gene expression programs that have implications for cell-cell communications, adhesion, cell differentiation, development and inflammatory responses as well as cancer treatments.

Phosphatidylinositol-3-kinases (PI3K) are essential for cell signaling, proliferation, differentiation and survival. The catalytic subunit of PI3K, encoded by the PIK3CA oncogene, is mutated in 18-45% of breast carcinomas. These mutations, involved in tumorigenic processes, activate the PI3K/AKT/mTOR signaling pathway. Resistance to anti‑human epidermal growth factor receptor, hormonal or anti-PI3K therapies have been described in breast carcinomas bearing activation of the PI3K signaling pathway. The present study reports the evaluation of PIK3CA exon 9 and 20 mutations in 149 invasive breast cancer cases using a validated PCR-high resolution melting assay (PCR-HRM). An amplification refractory mutation system (PCR-ARMS) using allele-specific scorpion primers was used to detect hotspot mutations in exons 9 (c.1624G→A and c.1633G→A) and 20 (c.3140A→G and c.3140A→T) in 118 tumor specimens. No correlation was observed with age at diagnosis, histological type, hormone receptor and HER2 status. PIK3CA exon 9 and 20 mutations were found to be related to Scarff-Bloom-Richardson (SBR) grade with a lower rate of mutations and a higher frequency of exon 9 mutations in SBRI and exon 20 mutations in SBRII/III tumors. No difference was observed in the incidence rates of the two different mutations screened for each exon in any subcategory. A statistically significant correlation was found between PCR-HRM and PCR-ARMS (κ=0.845; P<0.001). PCR-ARMS was found to be more sensitive than PCR-HRM (sensitivity 0.5 and 5-10% of mutated DNA, respectively). We propose that PCR-HRM and PCR-ARMS can be combined for the cost-effective routine clinical identification of PIK3CA mutations for the purpose of personalizing therapy for invasive breast cancers.

Objective: To evaluate the cost-utility of adding tiotropium to usual care versus usual care alone for patients with moderate to very severe chronic obstructive pulmonary disease (COPD) in the UK and Belgium., Methods: A four-state Markov model was developed with three disease severity states (moderate, severe, very severe) and death. Severity was based on post-bronchodilator FEV₁ and transitions were based on outcomes of the Understanding Potential Long Term Impacts on Function with Tiotropium (UPLIFT®) trial. Utilities were derived from EQ-5D scores for a subset of UPLIFT® patients. UK costs were evaluated separately for England (E), and for Scotland, Wales and Northern Ireland (SWNI). Belgian (B) costs were obtained from local sources. Uncertainty was assessed by deterministic and probabilistic sensitivity analysis (PSA)., Results: Adding tiotropium to usual care resulted in an incremental cost per patient of €969 (B), £796 (E), and £812 (SWNI), and incremental QALYs of 0.052 (B), and 0.051 (E, SWNI). The four-year incremental cost-effectiveness ratios (ICER) were €18,617 (B), £15,567 (E) and £15,890 (SWNI) per QALY. Probability of tiotropium being cost-effective at £30,000 (€50,000) per QALY gained was greater than 60%., Conclusions: At willingness to pay thresholds of £(€) 30,000 per QALY gained, adding tiotropium to usual care is cost-effective., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The 16-kDa angiostatic N-terminal fragment of human prolactin (16K hPRL) has been reported to be a new potent anticancer compound. This protein has already proven its efficiency in several mouse tumor models in which it prevented tumor-induced angiogenesis and delayed tumor growth. In addition to angiogenesis, tumors also stimulate the formation of lymphatic vessels, which contribute to tumor cell dissemination and metastasis. However, the role of 16K hPRL in tumor-induced lymphangiogenesis has never been investigated. We establish in vitro that 16K hPRL induces apoptosis and inhibits proliferation, migration, and tube formation of human dermal lymphatic microvascular endothelial cells. In addition, in a B16F10 melanoma mouse model, we found a decreased number of lymphatic vessels in the primary tumor and in the sentinel lymph nodes after 16K hPRL treatment. This decrease is accompanied by a significant diminished expression of lymphangiogenic markers in primary tumors and sentinel lymph nodes as determined by quantitative RT-PCR. These results suggest, for the first time, that 16K hPRL is a lymphangiostatic as well as an angiostatic agent with antitumor properties.

Background: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4., Methodology/principal Findings: Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i) variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1., Conclusions/significance: We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

Background: Angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology., Methodology/principal Findings: Here we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling., Conclusions/significance: Taken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.

Photodynamic Therapy (PDT) is a minimally invasive procedure used for treating a range of neoplastic diseases, which utilises combined action of light and a PDT drug called a photosensitiser. The efficiency of this treatment depends crucially on the properties of the photosensitiser used, namely on its efficient uptake by cells or by the surrounding vasculature, intracellular localisation, minimal dark toxicity and substantial phototoxicity. In this report we compare the spectroscopic properties, cell uptake and in vitro phototoxicity of two novel hydrophilic photosensitisers derived from pyropheophorbide-a (PPa). Both new photosensitisers have the potential to form bioconjugates with antibody fragments for targeted PDT. We find that the photophysical properties of both new photosensitisers are favourable compared to the parent PPa, including enhanced absorption in the red spectral region and substantial singlet oxygen quantum yields. Both molecules show efficient cellular uptake, but display a different intracellular localisation. Both new photosensitisers exhibit no significant dark-toxicity at concentrations of up to 100 microM. The phototoxicity of the two photosensitisers is strikingly different, with one derivative being 13 times more efficient than the parent PPa and another derivative being 18 times less efficient in SKOV3 ovarian cancer cells. We investigate the reasons behind such drastic differences in phototoxicity using confocal fluorescence microscopy and conclude that intracellular localisation is a crucial factor in the photodynamic efficiency of pheophorbide derivatives. These studies highlight the underlying factors behind creating more potent photosensitisers through synthetic manipulation.

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's beta-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis.

Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.

Arachis hypogaea L., commonly known as the peanut or groundnut, is an important and widespread food legume. Because the crop has a narrow genetic base, genetic diversity in A. hypogaea is low and it lacks sources of resistance to many pests and diseases. In contrast, wild diploid Arachis species are genetically diverse and are rich sources of disease resistance genes. The majority of known plant disease resistance genes encode proteins with a nucleotide binding site domain (NBS). In this study, degenerate PCR primers designed to bind to DNA regions encoding conserved motifs within this domain were used to amplify NBS-encoding regions from Arachis spp. The Arachis spp. used were A. hypogaea var. Tatu and wild species that are known to be sources of disease resistance: A. cardenasii, A. duranensis, A. stenosperma and A. simpsonii. A total of 78 complete NBS-encoding regions were isolated, of which 63 had uninterrupted ORFs. Phylogenetic analysis of the Arachis NBS sequences derived in this study and other NBS sequences from Arabidopsis thaliana, Medicago trunculata, Glycine max, Lotus japonicus and Phaseolus vulgaris that are available in public databases This analysis indicates that most Arachis NBS sequences fall within legume-specific clades, some of which appear to have undergone extensive copy number expansions in the legumes. In addition, NBS motifs from A. thaliana and legumes were characterized. Differences in the TIR and non-TIR motifs were identified. The likely effect of these differences on the amplification of NBS-encoding sequences by PCR is discussed.

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.