Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using shRNA-mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with two potent allosteric MEK inhibitors (PD0325901 or trametinib/GSK1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The PI3K inhibitor GDC-0941 was ineffective as a single agent, and did not augment the activity of PD0325901. All mice ultimately succumbed from progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors.
MAGUK Inverted 2 (MAGI-2) is a PTEN-interacting scaffold protein implicated in cancer on the basis of rare, recurrent genomic translocations and deletions in various tumors. In the renal glomerulus, MAGI-2 is exclusively expressed in podocytes, specialized cells forming part of the glomerular filter, where it interacts with the slit diaphragm protein nephrin. To further explore MAGI-2 function, we generated Magi-2-KO mice through homologous recombination by targeting an exon common to all three alternative splice variants. Magi-2 null mice presented with progressive proteinuria as early as 2 wk postnatally, which coincided with loss of nephrin expression in the glomeruli. Magi-2-null kidneys revealed diffuse podocyte foot process effacement and focal podocyte hypertrophy by 3 wk of age, as well as progressive podocyte loss. By 5.5 wk, coinciding with a near-complete loss of podocytes, Magi-2-null mice developed diffuse glomerular extracapillary epithelial cell proliferations, and died of renal failure by 3 mo of age. As confirmed by immunohistochemical analysis, the proliferative cell populations in glomerular lesions were exclusively composed of activated parietal epithelial cells (PECs). Our results reveal that MAGI-2 is required for the integrity of the kidney filter and podocyte survival. Moreover, we demonstrate that PECs can be activated to form glomerular lesions resembling a noninflammatory glomerulopathy with extensive extracapillary proliferation, sometimes resembling crescents, following rapid and severe podocyte loss.
Double aza-Michael addition of n-butylamine to the two acrylamide groups of acyclic N(2),N(6)-bis(6-acrylamidopyridin-2-yl)pyridine-2,6-dicarboxamide gives the corresponding macrocycle, . has potential coordination pockets associated with the 2,6-dicarboxamide (head) and the butylamine (tail) regions of the macrocycle. Depending on the conditions employed, macrocyclic complexes with palladium(ii) coordinated to either the tail or the head of the macrocycle can be isolated. Thus, treatment of with [PdCl2(NCPh)2] and sodium acetate, or [Pd(OAc)2] gives the closely related "tail-coordinated" complexes [PdCl(H3L)] () or [Pd(OAc)(H3L)] (), respectively. However, employment of the bases 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or pyridine during the treatment of with [Pd(OAc)2] results in the "head-coordinated" complexes [Pd(NH2R)(H2L)] (NH2R = N-(3-aminopropyl)caprolactam, which is formed by hydrolysis of DBU) () or [Pd(OH2)(H2L)] (), respectively. Translocation of the palladium ion from the macrocycle tail in to the head occurs on treatment with either DBU or N-(3-aminopropyl)caprolactam. In both cases the product is formed. The aqua ligand in is labile and easily displaced by the N-donor ligands n-butylamine, N-(3-aminopropyl)caprolactam or DBU to give the corresponding complexes [Pd(NH2(n)Bu)(H2L)] (), (), or [Pd(DBU)(H2L)] (). The data suggest that hydrolysis of DBU to produce the N-(3-aminopropyl)caprolactam ligand in is catalysed by the acetic acid formed during ligand metallation rather than by coordination to palladium. The X-ray crystal structures of , , and are reported.
Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) of plasma that has been depleted of abundant proteins and fractionated at the peptide level into six to eight fractions is a proven method for quantifying proteins present at low nanogram-per-milliliter levels. A drawback of fraction-MRM is the increased analysis time due to the generation of multiple fractions per biological sample. We now report that the use of heated, long, fused silica columns (>30 cm) packed with 1.9 ?m of packing material can reduce or eliminate the need for fractionation prior to LC-MRM-MS without a significant loss of sensitivity or precision relative to fraction-MRM. We empirically determined the optimal column length, temperature, gradient duration, and sample load for such assays and used these conditions to study detection sensitivity and assay precision. In addition to increased peak capacity, longer columns packed with smaller beads tolerated a 4- to 6-fold increase in analyte load without a loss of robustness or reproducibility. The longer columns also provided a 4-fold improvement in median limit-of-quantitation values with increased assay precision relative to the standard 12 cm columns packed with 3 ?m material. Overall, the optimized chromatography provided an approximately 3-fold increase in analysis throughput with excellent robustness and less than a 2-fold reduction in quantitative sensitivity relative to fraction-MRM. The value of the system for increased multiplexing was demonstrated by the ability to configure an 800-plex MRM-MS assay, run in a single analysis, comprising 2400 transitions with retention time scheduling to monitor 400 unlabeled and heavy labeled peptide pairs.
The last 20 years have seen a shift from the view that publics need to be educated so that they trust science and its governance to the recognition that publics possess important local knowledge and the capacity to understand technical information sufficiently to participate in policy decisions. There are now a variety of approaches to increasing the role of publics and advocacy groups in the policy and governance of science and biotechnology. This article considers recent experiences that demonstrate that it is possible to bring together those with policy making responsibility and diverse publics to co-produce policy and standards of practice that are technically informed, incorporate wide social perspectives and explicitly involve publics in key decisions. Further, the process of deliberation involving publics is capable of being incorporated into governance structures to enhance the capacity to respond to emerging issues with levels of public engagement that are proportionate to the issues.
Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-? (PLC-?), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.
The observations of the exceptionally bright gamma-ray burst (GRB) 130427A by the Large Area Telescope aboard the Fermi Gamma-ray Space Telescope provide constraints on the nature of these unique astrophysical sources. GRB 130427A had the largest fluence, highest-energy photon (95 GeV), longest ?-ray duration (20 hours), and one of the largest isotropic energy releases ever observed from a GRB. Temporal and spectral analyses of GRB 130427A challenge the widely accepted model that the nonthermal high-energy emission in the afterglow phase of GRBs is synchrotron emission radiated by electrons accelerated at an external shock.
Gamma-ray burst (GRB) 130427A is one of the most energetic GRBs ever observed. The initial pulse up to 2.5 seconds is possibly the brightest well-isolated pulse observed to date. A fine time resolution spectral analysis shows power-law decays of the peak energy from the onset of the pulse, consistent with models of internal synchrotron shock pulses. However, a strongly correlated power-law behavior is observed between the luminosity and the spectral peak energy that is inconsistent with curvature effects arising in the relativistic outflow. It is difficult for any of the existing models to account for all of the observed spectral and temporal behaviors simultaneously.
The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts.
We report a mass spectrometry-based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.
Since the initial publication of this chapter in 2004, additional methodologies have been developed which could improve and/or complement the original retroviral-mediated insertional mutagenesis. Retroviral vectors have also been shown to be useful for goals other than mutagenesis. In addition, retroviral-mediated insertional mutagenesis has been applied to zebrafish for use in reverse genetics as well as forward screening. Finally, the insertional mutant collection described herein has been screened by a number of labs to find a host of mutants (with genes already identified) with developmental and/or growth defects affecting the eye, liver, skin, craniofacial skeleton, kidney, myeloid cells, hematopoietic stem cells, and axon pathfinding, as well as mutants with defects in the cell cycle or DNA damage response, altered aging properties, and modulated cardiac repolarization. The major complementary approaches and new uses of this technique include:
Biobanks are increasingly hailed as powerful tools to advance health research. The social and ethical challenges associated with the implementation and operation of biobanks are equally well-documented. One of the proposed solutions to these challenges involves trading off a reduction in the specificity of informed consent protocols with an increased emphasis on governance. However, little work has gone into formulating what such governance might look like. In this paper, we suggest four general principles that should inform biobank governance and illustrate the enactment of these principles in a proposed governance model for a particular population-scale biobank, the British Columbia (BC) Generations Project. We begin by outlining four principles that we see as necessary for informing sustainable and effective governance of biobanks: (1) recognition of research participants and publics as a collective body, (2) trustworthiness, (3) adaptive management, and (4) fit between the nature of a particular biobank and the specific structural elements of governance adopted. Using the BC Generations Project as a case study, we then offer as a working model for further discussion the outlines of a proposed governance structure enacting these principles. Ultimately, our goal is to design an adaptive governance approach that can protect participant interests as well as promote effective translational health sciences.
We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/?l in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.
Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA. Patients and Methods: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression.
Providing technical and experiential information without overwhelming participants perspectives presents a major challenge to public involvement in policy decisions. This article reports the design and analysis of a case study on incorporating expert and stakeholder knowledge without including them as deliberators, while supporting deliberative participants ability to introduce and critically assess different perspectives. Analysis of audio-recorded deliberations illustrates how expert and stakeholder knowledge was cited, criticized and incorporated into deliberations. In conclusion, separating experts and stakeholders from deliberations may be an important prima facie principle when the goal is to enhance citizen representation on technical issues and related policy.
This paper addresses the dilemmas of participant sampling and recruitment for deliberative science policy projects. Results are drawn from a deliberative public event that was held in April and May, 2007. The research objective of The BC Biobank Deliberation was to assess deliberative democracy as an approach to legitimate policy advice from a subset of British Columbians concerning the secondary use of human tissues for prospective genomic and genetic research. The overall goal was to have participants identify key values that should guide a biobank in British Columbia. This paper assesses our teams group decision-making processes concerning participant sampling for the 2007 event. Results presented here should allow the reader to critically examine our teams choices and could also be used to assist advocates of deliberative democracy and others who may wish to propose similar events in the future.
Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.
As acknowledged in the literature, public consultation related to biobanks has been largely oriented to assuring and informing rather than seeking considered input. In April and May of 2007, the authors participated in running a deliberative public engagement event in British Columbia, Canada, which sought to enhance public input related to the governance of biobanks. The topic of the event was Biobanking in British Columbia (BC) and at the event a random-digit dialed demographically stratified sample of 21 participants deliberated on what values and interests ought to be considered in the regulation and use of biobanks for health research. In this paper, we report results related to debate over the place of informed consent in biobank research. Drawing on a pre/post-survey and qualitative analysis of event transcripts, we show that participants indicated strong support for biobanks, for a general reduction in concern for withdrawal of samples, and placed a strong emphasis on the need for review of biobanks research that is independent of funders and researchers. In this context, there was persistent disagreement about when consent was required for new research activities.
Protein biomarker candidates from discovery proteomics must be quantitatively verified in patient samples before they can progress to clinical validation. Here we demonstrate that peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (SISCAPA-MRM) can be used to configure assays with performance suitable for candidate biomarker verification. As proof of principle, we configured SISCAPA assays for troponin I (cTnI), an established biomarker of cardiac injury, and interleukin 33 (IL-33), an emerging immunological and cardiovascular marker for which robust immunoassays are currently not available.
Medical research to improve health care faces a major problem in the relatively limited availability of adequately annotated and collected biospecimens. This limitation is creating a growing gap between the pace of scientific advances and successful exploitation of this knowledge. Biobanks are an important conduit for transfer of biospecimens (tissues, blood, body fluids) and related health data to research. They have evolved outside of the historical source of tissue biospecimens, clinical pathology archives. Research biobanks have developed advanced standards, protocols, databases, and mechanisms to interface with researchers seeking biospecimens. However, biobanks are often limited in their capacity and ability to ensure quality in the face of increasing demand. Our strategy to enhance both capacity and quality in research biobanking is to create a new framework that repatriates the activity of biospecimen accrual for biobanks to clinical pathology.
Computer-assisted oral anticoagulant dosage is being increasingly used to meet growing demands for oral anticoagulation. The DAWN AC is one of the most widely used computer-dosage programs. Evidence of its value and that of other computer programs has been based previously only on laboratory evidence of "time in target INR range" (TIR) not on clinical safety in practice. A five-year international randomised clinical study of computer assistance with the DAWN AC program compared with manual dosage in 2,631 patients has been performed at 13 centres with established expertise in oral anticoagulation mainly in the EU. Safety assessment have been based on the comparison of bleeding or thrombotic events with DAWN AC compared with manual dosage in a randomised study. Safety of the DAWN AC program has been demonstrated. Clinical events of bleeding and thrombosis were almost identical with the experienced manual dosage group. Therapeutic control improved with DAWN AC to 66.8% from 63.4% TIR. The program failed to provide a dosage recommendation on only 5.7% of occasions. At a group of experienced centres with a special interest in oral anticoagulation, the DAWN AC computer-dosage program proved as safe clinically as manual dosage by experienced medical staff. With DAWN AC, laboratory control was improved, the difference being highly significant. The results should reassure hospitals and community clinics that the DAWN AC program is safe and facilitate greater and longer provision of warfarin treatment where required.
This paper reports on the design, implementation, and results of a structured public deliberation on human tissue biobanking conducted in Vancouver, Canada, in 2009. This study builds on previous work on the use of deliberative democratic principles and methods to engage publics on the social and ethical implications of human tissue biobanking. In a significant refinement of methods, we focus on providing public input to institutional practice and governance of biobanks using a tailored workbook structure to guide participants discussion. Our focus is on the local context and practices of a particular institution, the BC BioLibrary. However, elements of both the methodological innovations and the ethical guidance implied by our findings are generalisable for biobanking internationally. Recommendations from the deliberative forum include issues of informed consent, privacy protections, collection of biospecimens, governance of biobanks, and how to manage the process of introduction between biobanks and potential donors. Notable findings include public support for research use of anonymised un-consented tissue samples when these come from archived collections, but lack of support when they are collected prospectively.
A limited battle involving the nuclear arsenals of India and Pakistan would have significant climatic impacts upon agricultural crop production in the United States; corn, soybeans, and winter wheat yields would be significantly reduced in the Corn Belt region of the US. The most severe impacts would occur during the second year after the modeled nuclear battle.
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