Multiplatform Approach Research Articles

Development of an integrated multiplatform approach for assessing brush management conservation efforts in semiarid rangelands

Millions of dollars have been spent on brush management, or removal of unwanted woody vegetation, as a conservation practice to control the presence of woody species. Land managers need an inexpensive means of monitoring the effects of brush management conservation methods for decreasing degradation in rangeland systems. In this study, free, publically available, high-resolution (1 m) imagery from the National Agricultural Imagery Program (NAIP) and moderate-resolution (30 m) Landsat-5 Thematic Mapper (TM) imagery were combined to produce a large-scale technique for mapping woody cover. High-resolution imagery-based estimates of woody cover were found to be reasonable (RMSE=3.8%, MAE=2.9%) surrogates for ground-based woody cover. An equation for TM-derived woody cover was developed. TM scenes of woody cover (TMWC) were produced and validated using NAIP and ground-based data. Results showed that the developed relation produced viable (RMSE=8.5%, MAE=6.4%) maps of woody cover that could be used to successfully track the occurrence of brush removal, as well as monitor the presence or lack of subsequent reemergence. This work provides land managers with an operational means of determining where to allocate resources to implement brush management, as well as a cost-effective method of monitoring the effects of their efforts.

Abstract P5-04-08: Expression of novel immunotherapeutic targets in luminal breast cancer patients

Abstract Background: The development of novel chemotherapeutic agents has significantly improved the prognosis and survival of patients with breast cancer. ER positive or luminal tumors represent around two thirds of all breast cancers and these cancers are comprised of different histologies including differing gene expression and mutational profiles. This study examined biomarkers involved in immune evasion including PD-L1 and its association with other biological pathways as potential treatment options for luminal breast cancer patients. Methods: We analyzed 1311 breast samples using a multiplatform approach including whole genome mRNA expression (HumanHT-12 v4 BeadChip Illumina Inc., San Diego, CA), protein expression (immunohistochemistry), gene copy number changes (in situ hybridization) and gene sequencing and an additional 304 breast samples were tested for PD-1 and PD-L1 by IHC. The mRNA expression data was based on whole tumor and represents cell type heterogeneity. Heat map analysis was done to look at differential gene expression between the PD-L1 high vs low luminal breast cancers. Results: Based on expression of ER, PR and HER2 by IHC, we subdivided the data into sub- cohorts. Elevated mRNA expression of immune markers including PD-L1, CTLA4, B7H-3, and IDO1 was noted in the ER+HER2- luminal population including ER+ PR- and ER+PR+ cohort. Positive correlation was found between PD-L1 and other immune regulators including CTLA-4, B7-H3 and IDO1 (Spearman correlations of 0.49, 0.36 and 0.46). Protein expression of PD-L1 was found to be specific to the HER2 negative cohort with no expression in the HER2 positive cohort regardless of ER status. Within the ER+HER2- cohort, PD-L1 expression was 5% {ER+PR+ was 5% (4/81) and the ER+PR- was 6% (2/34)}. In contrast, PD-L1 expression was higher in the triple negative cohort, 17% (13/75). The expression of PD-1 on the other hand was present throughout the different cohorts. PD-1 expression ranged from 43% in the ER+HER2- cohort {41% (14/34) in ER+PR-HER2-; 43% (35/81) in ER+PR+HER2-} to 33% in the ER+HER2+ cohort {44% (4/9) in ER+PR-Her2+; and 17% (1/6) in ER+PR+HER2+}. In the ER-HER2+ cohort PD-L1 expression was 75%{6/8 in ER-PR-HER2+ and 0/0 in ER-PR+HER+} and 63%(47/75) in the triple negative cohort. Pathway analysis of the PD-L1 negative vs positive luminal population identified 127 genes involved in various cancer pathways including EGFR and VEGF signaling network (P=7.47e-09). Conclusions: The expression of immune regulatory targets in the breast cancer population suggests that immune- targeted therapies with anti PD-1/PD-L1, CTLA4, B7-H3 and IDO1 may be effective especially in the luminal cohort. Our study further shows that ER+HER2- breast cancer patients may be better candidates for immunotherapy with checkpoint inhibitors as compared to ER+HER2+ due to lack of PD-L1 expression in the HER2 positive cohort. Further validation of our findings is ongoing. Citation Format: Gargi D Basu, Anatole Ghazalpour, Randal Vader, Sandeep Reddy, Karen Anderson, Ann McCullough, Barbara Pockaj. Expression of novel immunotherapeutic targets in luminal breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-08.

Systemic alterations in the metabolome of diabetic NOD mice delineate increased oxidative stress accompanied by reduced inflammation and hypertriglyceremia.

Nonobese diabetic (NOD) mice are a commonly used model of type 1 diabetes (T1D). However, not all animals will develop overt diabetes despite undergoing similar autoimmune insult. In this study, a comprehensive metabolomic approach, consisting of gas chromatography time-of-flight (GC-TOF) mass spectrometry (MS), ultra-high-performance liquid chromatography-accurate mass quadruple time-of-flight (UHPLC-qTOF) MS and targeted UHPLC-tandem mass spectrometry-based methodologies, was used to capture metabolic alterations in the metabolome and lipidome of plasma from NOD mice progressing or not progressing to T1D. Using this multi-platform approach, we identified >1,000 circulating lipids and metabolites in male and female progressor and nonprogressor animals (n = 71). Statistical and multivariate analyses were used to identify age- and sex-independent metabolic markers, which best differentiated metabolic profiles of progressors and nonprogressors. Key T1D-associated perturbations were related with 1) increases in oxidation products glucono-δ-lactone and galactonic acid and reductions in cysteine, methionine and threonic acid, suggesting increased oxidative stress; 2) reductions in circulating polyunsaturated fatty acids and lipid signaling mediators, most notably arachidonic acid (AA) and AA-derived eicosanoids, implying impaired states of systemic inflammation; 3) elevations in circulating triacylglyercides reflective of hypertriglyceridemia; and 4) reductions in major structural lipids, most notably lysophosphatidylcholines and phosphatidylcholines. Taken together, our results highlight the systemic perturbations that accompany a loss of glycemic control and development of overt T1D.

A multi-platform metabolomics approach demonstrates changes in energy metabolism and the transsulfuration pathway in Chironomus tepperi following exposure to zinc

Measuring biological responses in resident biota is a commonly used approach to monitoring polluted habitats. The challenge is to choose sensitive and, ideally, stressor-specific endpoints that reflect the responses of the ecosystem. Metabolomics is a potentially useful approach for identifying sensitive and consistent responses since it provides a holistic view to understanding the effects of exposure to chemicals upon the physiological functioning of organisms. In this study, we exposed the aquatic non-biting midge, Chironomus tepperi, to two concentrations of zinc chloride and measured global changes in polar metabolite levels using an untargeted gas chromatography–mass spectrometry (GC–MS) analysis and a targeted liquid chromatography–mass spectrometry (LC–MS) analysis of amine-containing metabolites. These data were correlated with changes in the expression of a number of target genes. Zinc exposure resulted in a reduction in levels of intermediates in carbohydrate metabolism (i.e., glucose 6-phosphate, fructose 6-phosphate and disaccharides) and an increase in a number of TCA cycle intermediates. Zinc exposure also resulted in decreases in concentrations of the amine containing metabolites, lanthionine, methionine and cystathionine, and an increase in metallothionein gene expression. Methionine and cystathionine are intermediates in the transsulfuration pathway which is involved in the conversion of methionine to cysteine. These responses provide an understanding of the pathways affected by zinc toxicity, and how these effects are different to other heavy metals such as cadmium and copper. The use of complementary metabolomics analytical approaches was particularly useful for understanding the effects of zinc exposure and importantly, identified a suite of candidate biomarkers of zinc exposure useful for the development of biomonitoring programs.

Experimentation and Imitation: The Journey to Elle 360

It is widely acknowledged that the media industries are facing an unprecedented period of disruption within the internet age. Whilst avoiding technological determinism, it is important to recognize the scale of the changes being faced and the altering landscape which media businesses, particularly those with a strong legacy, are forced to operate in (Chan-Olmsted and Chang, 2003; Küng, 2008; Küng, Picard and Towse, 2008; Küng, 2013). This paper focuses on the initial findings from a case study of Elle, UK and the title’s explicit engagement with multi-platform which has involved multiple and wide ranging product, process and content innovations. The paper focuses on an analysis of documentary evidence and semi-structured interviews with Hearst Magazines UK and Elle UK senior employees. With regard to adjusting to a multiplatform approach to publishing, Hearst UK is considered to have innovated more quickly than rivals (Halliday and Sweney, 2013) and Elle UK has engaged with a multi-platform strategy, ‘Elle 360’ for the past 18 months. Despite a public discourse of platform neutrality, evidence from the initial research suggests that the economic strategies, as a corollary work practices and content decisions, continue to preference the print magazine above emerging and alternative platforms. This is suggested to be a result of the fact that the print magazine remains the dominant source of revenue despite wider experimentation. The paper concludes by identifying tensions in the continuing experimentation with new business models and maintaining the delicate balancing act of editorial integrity.

Impact of lake breezes on ozone and nitrogen oxides in the Greater Toronto Area

Meteorological and air quality datasets from summertime (May to September, 2010–2012) were analysed in order to assess the influence of lake-breeze circulations on pollutant levels in the Greater Toronto Area (GTA). While previous estimates of the frequency of summer days experiencing lake breezes range between 25 and 32 % for the GTA, a simple algorithm using surface meteorological observations suggested Lake Ontario breezes occurred on 56% of summer days, whereas a more reliable multiplatform approach yielded a frequency of 74%. Data from five air quality stations across the GTA were used to compare air quality on days during which a lake-breeze circulation formed (“lake breeze days”) versus days when one did not (“non-lake breeze days”). Average daytime O3 maxima were 13.6–14.8 ppb higher on lake breeze days relative to non-lake breeze days. Furthermore, the Ontario Ambient Air Quality Criteria (AAQC) for 1-h average O3 (80 ppb) and 8-h average O3 (65 ppb) were exceeded only on lake breeze days and occurred on a total of 30 and 54 days throughout the study period, respectively. A causal link between lake-breeze circulations and enhanced O3 was identified by examining several days in which only some of the air quality sites were inside the lake-breeze circulation. O3 mixing ratios at sites located within the circulation were at least 30 ppb higher than sites outside the circulation, despite similar temperatures, cloud conditions and synoptic regimes across the region. Rapid O3 increases were concurrent with the arrival of the lake-breeze front, suggesting O3-rich air from over the lake is being advected inland throughout the day. Lake-breeze circulations were found to have less impact on nitrogen oxide (NOx) levels. Morning NOx was greater on lake breeze days, probably due to the stagnant conditions favourable for lake breeze formation. During the late afternoon, only inland sites experience increased NOx on lake breeze days, likely as a result of being downwind from near-shore city centres.

Biomarker patterns of localized and metastatic prostate cancer.

192 Background: Presence or absence of metastases is a determining factor for prostate cancer prognosis. Common sites of metastasis include bone (B), lymph nodes (LN) or visceral organs (V). We sought to determine theranostic biomarker differences between primary (P) and metastatic (M) specimens, with subset analysis for differences between metastatic sites. Methods: 497 prostate cancer cases referred to Caris Life Sciences between 2009 thru 2014 were evaluated. Specific testing was performed and included a multiplatform approach: sequencing (Sanger, NGS), protein expression (IHC) and gene amplification (CISH/FISH). Results: 58% (287/497) were M specimens, of which 23% (66), 24% (68) and 52% (149) were from B, LN and V, respectively. Significant differences for EGFR amplification/overexpression, low MGMT expression, TOPO1/2A overexpression and higher PTEN mutation rate were found in the M subgroup. These alterations indicate a role for EGFR/PTEN in progression of prostate cancer, and potential role of MAPK/PAM-targeted therapies, alkylating agents and topoisomerase inhibitors in M disease. Mutational profiles of the M subgroup are more genetically unstable exhibiting mutations in 60% of genes tested (notable events include APC & β-Catenin, PTEN, TP53 and BRCA1/2) vs. 30% observed in the P subgroup (p=0.0067). In the subset analysis, EGFR amplification was higher in B (29%) and V (24%) compared to LN (13%); (not significant). Low TS expression was more frequent in B vs. LN (p=0.004), TOPO2A overexpression was higher in V vs. B (p=0.0001) and there was a trend for PD1 positive infiltrating lymphocytes being more abundant in LN vs B (p=0.09). Interestingly, cMET overexpression (7/230) and amplification (1/162) were rarely observed across the cohort, providing support for the surprising failure of cabozantinib in castration-resistant prostate cancer. Conclusions: Our data indicate a role for multiplatform profiling to identify therapeutic options that are appropriate for subsets of patients. Molecular changes associated with M disease including EGFR, PTEN, MGMT and TOPO1/2A, present opportunities for developing therapeutic strategies which may include a combination of targeted therapies, as well as traditional cytotoxic chemotherapies.

Molecularly-guided therapeutic options beyond tyrosine kinase inhibitors (TKIs) for gastrointestinal stromal tumors (GIST).

58 Background: GISTs are characterized by KIT/PDGFRA mutations. A range of multi-targeted tyrosine kinase inhibitors (TKIs) are available for treatment, however, resistance mechanisms inevitably emerge. Recent data (Boichuk, et al 2014) suggests the potential efficacy of various cytotoxic therapies that were identified as being able to effectively kill TKI-responsive and -resistant GIST cells. We sought to investigate the theranostic markers associated with non-TKI therapy options for their potential role in treatment of GIST. Methods: 147 GIST cases were evaluated. A multiplatform approach of biomarker testing was used and included a combination of sequencing (NGS, Sanger), protein expression (IHC) and gene amplification (ISH). Results: Multidrug resistance phenotype was found in 52-68% (PGP, MRP1). Tubulin-binding agents (taxanes, vinca alkaloids) may be of potential use due to the high frequency of low TUBB3 expression (72% or 39/54). Anthracyclines and topoisomerase inhibitors may be of potential benefit in 1/3 of patients based on expression of TOPO2A (32% or 32/99) and TOPO1 (34% or 37/110). Cytotoxic agents used in non-GIST solid tumors, may also be considered, based on high frequency of low expressin of MGMT (47% or 57/122), TS (70% or 76/109) and RRM1 (79% or 88/111). PTEN was intact (positive expression) in the majority of GIST (87%). Nine patients were examined for PD1/PDL1: 56% exhibited positive tumor infiltrating lymphocytes and 33% exhibited PDL1 tumor expression. Only one amplification event was observed: cMET (0/53), HER2 (0/69), EGFR (0/16), PIK3CA (0/1) and TOP2A (1/11). Mutational screening using a hot spot cancer panel (and Sanger sequencing for some genes) resulted in the detection of variants in only 10 genes, excluding KIT (97/132) and PDGFRA (5/55). Variants were detected in the following genes, in decreasing order of frequency: RB1, APC and JAK3 (2/55; 2/55; 2/57), PIK3CA (2/69) and ABL1, cMET, EGFR, KDR, VHL and BRAF (1/55, 1/57, 1/57, 1/57, 1/52, 1/78). Conclusions: A multi-platform approach of theranostic biomarkers identified therapies beyond TKIs for GIST. Various cytotoxics and non-KIT/PDGFRA targeted therapies were identified based on protein expression or gene variations.

Comprehensive multiplatform biomarker analysis of 206 squamous cell anal cancers.

603 Background: Anal squamous cell carcinoma (Anal SCC) is a rare, HPV-associated malignancy accounting for 2.4% of digestive system cancers. In most cases, these malignancies are detected in the early stages and successfully managed with chemoradiation therapy. Uncommonly, these cancers recur or present with metastases. In this setting, cisplatin and 5-fluorouracil represent the only endorsed regimen. Once beyond standard therapy, few therapeutic options exist for those patients with aggressive disease. The purpose of this study is to identify other novel, potential targets and therapeutic options for this disease, utilizing a multiplatform approach. Methods: 206 anal SCC specimenswere tested via a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ) consisting of gene sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]) and gene amplification (CISH or FISH). 6 cases were documented as positive for HPV or HIV; status was not provided on the remaining 200. Results: Key results are shown in the table below, shown as percent change/total cases. Conclusions: Multiplatform tumor profiling identified a low incidence of gene mutations. Protein expression aberrations identified potential treatment options not routinely considered, such as topoisomerase inhibitors and taxanes. Mutations in PIK3CA, Akt1, and FBXW7 as well as PTEN loss indicate potential for targeting the PI3 kinase pathway. Additionally, immunomodulatory agents may be a therapeutic option, based on PD-1 expression levels. Targeting the ErbB-family receptors, namely with anti-EGFR agents or newer generation pan-HER - inhibitors, may represent another option, given EGFR and HER2 amplification as well as EGFR overexpression. It should be mentioned that differences in anal carcinomas whose etiology is of viral origin may present different treatment options based on the driver mutations. [Table: see text]

Cross-platform metabolic profiling: application to the aquatic model organism Lymnaea stagnalis.

The freshwater pond snail Lymnaea stagnalis is used in several studies on molecular and behavioral neurobiology and ecotoxicology showing its successful application as a model organism. In the present study, a cross-platform metabolomic approach has been evaluated to characterize the organ molecular phenotypes of L. stagnalis central nervous system (CNS), digestive gland (DG), and albumen gland (AG). Two types of tissue disruption methods were evaluated of which beads beating was the preferred method. To obtain a broad picture of the hydrophilic and lipophilic metabolome, two complementary analytical platforms have been employed: liquid chromatography (LC) and gas chromatography (GC) coupled to high-resolution mass spectrometry. Furthermore, to increase the power to separate small polar metabolites, hydrophilic interaction liquid chromatography (HILIC) was applied. The analytical platform performances have been evaluated based on the metabolome coverage, number of molecular features, reproducibility, and multivariate data analysis (MVDA) clustering. This multiplatform approach is a starting point for future global metabolic profiling applications on L. stagnalis.

Three Traits of the Islamic State’s Information Warfare

The success of the Islamic State throughout the ‘fertile crescent’ is a striking example of a modern insurgency, and information operations have played a central role in the group's strategy. Drawing on primary sources, Haroro J Ingram analyses three traits of the Islamic State's information warfare: the use of a multidimensional, multi-platform approach that simultaneously targets ‘friends and foes’ to enhance the reach, relevance and resonance of its messaging; the synchronisation of narrative and action to maximise operational and strategic ‘effects’ in the field; and the centrality of the Islamic State ‘brand’ to its entire campaign.

Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation.

Giardia duodenalis is a protozoan parasite of the small intestine in vertebrates, including humans. Assemblage A of G. duodenalis is one of the two discrete subtypes that infects humans, and is considered a zoonotic assemblage. Two G. duodenalis Assemblage A strains BRIS/95/HEPU/2041 and BRIS/83/HEPU/106, constituting virulent and control strains respectively, were analyzed in one of the first comparative shotgun proteomic studies performed in this parasite. Protein extracts were prepared using a multiplatform approach with both an in-gel and in-solution sample preparation to enable us to assess the complementarity for future Giardia proteomic studies. Protein analysis revealed that BRIS/95/HEPU/2041 possessed a wider and more varied repertoire of variant surface proteins (VSPs), which are hypothesized to be involved in host adaptation, immune evasion, and virulence. A total of 35 VSPs were identified, with three common to both strains, six unique to BRIS/82/HEPU/106, and twenty-six unique to BRIS/95/HEPU/2041. Additionally, up to 25.6% of all differentially expressed proteins in BRIS/95/HEPU/2041 belonged to the VSP family, a trend not seen in the control BRIS/83/HEPU/106. Greater antigen variation in BRIS/95/HEPU/2041 may explain aspects of virulence phenotypes in G. duodenalis, with a highly diverse population capable of evading host immune responses.

Tumor Biomarker Evaluation of 6785 Patients for Combination Treatment Strategies in NSCLC: Translational Research

Non-small cell lung cancer (NSCLC) exhibits activation of multiple tumor pathways. Presence of multiple aberrations may account for drug resistance as well as strategies for combination therapies. We examined concurrent aberrations of biomarkers in NSCLC to present an overview of potential patient cohorts who may benefit from such combinations. 6785 NSCLC cases were evaluated. Specific testing was performed and included a multiplatform approach: sequencing (Sanger, NGS), protein expression (IHC), and gene amplification (CISH/FISH). The EGFR mutation (MT) rate in this population was 12.7% (135/1059), of which 57% (55/96) overexpressed EGFR (IHC) and 61% (169/277) had EGFR gene amplification (FISH). This demonstrates the dependence on the EGFR pathway and potential importance of dual inhibition with cetuximab and EGFR TKIs. Sixty-six percent (63/96) and 7% (6/90) of EGFR MT patients were MET high (IHC) and amplified (CISH), respectively, suggesting potential benefit from dual targeting of EGFR and MET. Interestingly, TP53 mutations were observed in 54% (70/129) of EGFR MT which has important implications for resistance to EGFR TKIs and possible cross-resistance to radiation therapy. ALK translocations were observed in 101 of 3611 (2.8%) patients, among which 19% (7/36), 3% (1/29) and 2% (1/48) carried concurrent EGFR, MET and HER2 amplification (ISH), respectively, suggesting the potential for combining crizotinib with agents such as cetuximab, onartuzumab, or trastuzumab. A BRAF mutation was observed in 3.3% (34/1061), among which EGFR and MET expression was high by IHC (both have been implicated in resistance to BRAFi in other tumor types) in 58% (15/26) and 48% (12/25), respectively, indicating benefit from combination of newly approved dabrafenib with cetuximab or onartuzumab. This is one of the largest analyses in the United States of biomarkers in NSCLC. Our study shows the nonexclusive activation of multiple pathways. These results also show the importance of multiplatform testing as both protein, gene copy number, and genetic alterations were detected.

Genetic Modifiers of Neurofibromatosis Type 1-Associated Café-au-Lait Macule Count Identified Using Multi-platform Analysis

Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count.

Qualitative multiplatform microanalysis of individual heterogeneous atmospheric particles from high-volume air samples.

High-resolution microscopic analysis of individual atmospheric particles can be difficult, because the filters upon which particles are captured are often not suitable as substrates for microscopic analysis. Described here is a multiplatform approach for microscopically assessing chemical and optical properties of individual heterogeneous urban dust particles captured on fibrous filters during high-volume air sampling. First, particles embedded in fibrous filters are transferred to polished silicon or germanium wafers with electrostatically assisted high-speed centrifugation. Particles are clustered in an array of deposit areas, which allows for easily locating the same particle with different microscopy instruments. Second, particles with light-absorbing and/or light-scattering behavior are identified for further study from bright-field and dark-field light-microscopy modes, respectively. Third, particles identified from light microscopy are compositionally mapped at high definition with field-emission scanning electron microscopy and energy-dispersive X-ray spectroscopy. Fourth, compositionally mapped particles are further analyzed with focused ion-beam (FIB) tomography, whereby a series of thin slices from a particle are imaged, and the resulting image stack is used to construct a three-dimensional model of the particle. Finally, particle chemistry is assessed over two distinct regions of a thin FIB slice of a particle with energy-filtered transmission electron microscopy (TEM) and electron energy-loss spectroscopy associated with scanning transmission electron microscopy (STEM).

Illustration of the emerging capabilities of SARAL/AltiKa in the coastal zone using a multi-platform approach

Satellite altimetry measurements from SARAL-AltiKa are analysed in the coastal ocean using the results of the G-AltiKa mission (1–5 August 2013), which combined altimeter, HF radar and glider data coincident with the satellite track, south-west of Ibiza Island, in the Western Mediterranean Sea.The absolute dynamic topography, computed from 1Hz and 40Hz altimetry data, and the dynamic height, computed from glider temperature and salinity, both exhibited a weak signal with amplitudes of order 2cm. The resulting geostrophic and HF radar velocities along the track depicted a north-westward coastal current with a maximal velocity larger than 20cms−1.This demonstrates that the AltiKa altimeter is able to resolve SLA signals of more than 2cm, and gradients in those signals over several tens of kilometres. After filtering, the 40Hz data depicted a signal consistent with the other platforms, up to a distance of order 10km from the coast.

570P - Thymidylate Synthase Over-Expression Underlies the Observed Lack of 5-Fu Therapy Benefit for Msi-H Colorectal Cancers

ABSTRACT Aim: 5-FU based treatment remains the standard of care for the adjuvant treatment of colorectal carcinoma (CRC), in combination with oxaliplatin and irinotecan. CRC is molecularly heterogenous disease and patients with microsatellite stable (MSS) CRC have been previously shown to benefit from 5-FU based therapies while patients with high microsatellite instability (MSI-H) CRC did not. We investigated the expression thymidylate synthase, the targeted enzyme for 5-FU and topoisomerase I (Topo1), the targeted enzyme for irinotecan, in molecularly defined cohort of CRC. Methods: 106 patients with CRC (86 MSS and 20 MSI-H) were molecularly profiled using multiplatform approach [Caris Life Sciences] including immunohsitochemistry, PCR microsatellite analysis and gene sequencing (NGS) . Results: TS over-expression (over the ≥1+ and ≥10% threshold, and frequently >2+ and >50%) was observed in all 20 MSI-H cases (100%). In contrast, MSS cases overexpressed TS in only 31/86 (36%) of the cases (p Conclusions: Two most common molecular subtypes of colorectal cancer exhibit different expression patterns of TS and Topo1; overexpression of TS may explain the observed lack of benefit of 5-FU based therapy in MSI-H CRC patients. Additional genetic and molecular biomarkers could provide guidance to alternative chemotherapy modalities. Disclosure: Z. Gatalica: Stock options Caris Life Sciences; D. Bryant: Stock options Caris Life Sciences All other authors have declared no conflicts of interest.

Breast milk metabolome characterization in a single-phase extraction, multiplatform analytical approach.

Breast milk (BM) is a biofluid that has a fundamental role in early life nutrition and has direct impact on growth, neurodevelopment, and health. Global metabolic profiling is increasingly being utilized to characterize complex metabolic changes in biological samples. However, in order to achieve broad metabolite coverage, it is necessary to employ more than one analytical platform, typically requiring multiple sample preparation protocols. In an effort to improve analytical efficiency and retain comprehensive coverage of the metabolome, a new extraction methodology was developed that successfully retains metabolites from BM in a single-phase using an optimized methyl-tert-butyl ether solvent system. We conducted this single-phase extraction procedure on a representative pool of BM, and characterized the metabolic composition using LC-QTOF-MS and GC-Q-MS for polar and lipidic metabolites. To ensure that the extraction method was reproducible and fit-for-purpose, the analytical procedure was evaluated on both platforms using 18 metabolites selected to cover a range of chromatographic retention times and biochemical classes. Having validated the method, the metabolic signature of BM composition was mapped as a metabolic reaction network highlighting interconnected biological pathways and showing that the LC-MS and GC-MS platforms targeted largely different domains of the network. Subsequently, the same protocol was applied to ascertain compositional differences between BM at week 1 (n = 10) and 4 weeks (n = 9) post-partum. This single-phase approach is more efficient in terms of time, simplicity, cost, and sample volume than the existing two-phase methods and will be suited to high-throughput metabolic profiling studies of BM.

Global Emergency Medicine Journal Club: Social Media Responses to the March 2014 Annals of Emergency Medicine Journal Club on Targeted Temperature Management

In March 2014, Annals of Emergency Medicine continued a successful collaboration with an academic Web site, Academic Life in Emergency Medicine (ALiEM), to host another Global Emergency Medicine Journal Club session featuring the 2013 New England Journal of Medicine article "Targeted Temperature Management at 33°C (91.4°F) Versus 36°C (96.8°F) After Cardiac Arrest" by Nielsen etal. This online journal club used Twitter conversations, a live videocast with the authors, and detailed discussions on the ALiEM Web site's comment section. This summary article details the community discussion, shared insights, and analytic data generated using this novel, multiplatform approach.

Commentary: Innovations in programming for HIV among adolescents: towards an AIDS-free generation.

Ultimately achieving an AIDS-free generation might rely more than we thought on developing adolescent leaders as agents of change -- not simply behavior change but societal and legal change. Innovative programming may be expanding adolescents’ social networks and engagement increasing their HIV-specific knowledge and increasing demand for HIV testing and treatment; but what is to meet this demand; and how will the effects of these programs be rigorously measured? Technology companies and mobile host sites are not clinicians or experts in evaluation. Even dizzying advances in technology are useless if adolescents have no access to them or are not able to access health promoting information services or networks. Moving forward technology and social innovators -- young and old -- must task themselves with removing barriers to testing and treatment that will reduce the sheer numbers of HIV infections and AIDS deaths in this population. Innovations must support adolescents’ access to a wider array of confidential testing options available in the community and through the health sector link them to biomedical and other prevention services of proven efficacy such as antiretroviral therapy medical male circumcision and to specific harm reduction interventions. In the years to come it is network disruption -- of entrenched social mores stigma attitudes and discriminatory practices -- that will constitute true innovation. (Excerpts)