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Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that the variants of ERβ, namely ERβ1, ERβ2, ERβ4, and ERβ5, contributes to aggressiveness of the TNBC cell line HCC1806 by affecting E-Cadherin expression and chemotherapy sensitivity. We have previously shown that the HCC1806 cell line expresses all the ERβ variants. To investigate their function we used the recently developed dCas9-KRAB system with Guide RNA for both ERβ promoters 0N and 0K, using 3 guide RNA's for each promoter and we found that expression of panERβ decreased from a ct. of 28 to 33 indicating loss of total ERβ. Treatment of HCC1806 cells with chemotherapy in combination with an ERβ agonist, LY500307, is more effective in reducing cancer cell viability. Knockdown of total ERβ in HCC1806 cells using CRISPR-Cas9 technology, decreased the proliferation and increased chemo-sensitivity to docetaxel agent in cells with the 0K promoter knock down, while cells with the 0N promoter knock down were less affected. Furthermore, after ERβ knockdown, the cell morphology of HCC-1806 changed to a more epithelial phenotype and there was increase in the expression of the classical epithelial marker, E-cadherin. N-Cadherin was unchanged in the cells with the 0K promoter knock down, while E-Cadherin was slightly decreased in cells with the 0N promoter knock down. We also found that cells with the 0K promoter knock down migrated less than control cells in a migration assay, while cells with the 0N promoter knock down were less affected. These findings are in agreement with reports showing that the 0K promoter is responsible for expression of the variants ERβ2 and ERβ5, while the 0N promoter expresses more ERβ1. Furthermore, we have earlier shown that ERβ2 and ERβ5 increase aggressiveness of TNBC via several oncogenic signaling pathways. In conclusion, knockdown of ERβ variants in HCC-1806 indicated that the variants may be responsible for conferring oncogenic properties to TNBC such as EMT, cancer cell proliferation and chemo-resistance. Therefore, treating TNBC patients with combination therapy including ERβ agonists, may prove to be extremely beneficial for securing better treatment responses and future pre-clinical studies should design appropriate experiments to confirm these findings in vivo. Citation Format: Faria M, Gustafsson J-A, Strom A. Endogenous expression of ERβ variants contributes towards chemotherapy-resistance in the triple negative breast cancer cell line HCC-1806 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-21.

Provided is the annotated raw data for N-glycan mass spectrometry imaging (MSI) annotations in thin cross-sections of formalin-fixed and paraffin-embedded murine kidney. Relevant meta-data have been provided in this brief and the raw MSI data can be accessed using ProteomeXchange with the PRoteomics IDEntifications (PRIDE) identifier PXD009808. This brief is the first in a set of submissions from our group which will make raw data publicly accessible for existing and future MSI studies. Refereed/Peer-reviewed

The protein corona is a concept central to a range of disciplines exploiting the bio–nano interface. As the literature continues to expand in this field, it is essential to condense and contextualize the in vitro and in vivo proteome databases accumulated over the past decade: a goal which this review intends to achieve for the benefit of nanomedicine and nanobiotechnology. The parameters used for our review are the physicochemical characteristics of the nanoparticles, their surface ligands, the biological matrixfrom which a corona was formed, methods employed, plus the top-ten enriched corona proteins. In addition, the protein coronal networks and their implications in vivo are highlighted for selected studies. Refereed/Peer-reviewed

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona in vivo that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis, we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in fetal bovine serum using two complementary methodologies developed herein: quartz crystal microbalance and "centrifugal capture", coupled with nanoliquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, whereas strong binding with IAPP fibrils occurred for linear proteins or multidomain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets. Refereed/Peer-reviewed

Polyethylene glycol (PEG) is a gold standard against protein fouling. However, recent studies have revealed surprising adverse effects of PEG, namely its immunogenicity and shortened bio-circulation upon repeated dosing. This highlights a crucial need to further examine 'stealth' polymers for controlling the protein 'corona', a new challenge in nanomedicine and bionanotechnology. Poly(2-ethyl-2-oxazoline) (PEtOx) is another primary form of stealth polymer that, despite its excellent hydrophilicity and biocompatibility, has found considerably less applications compared with PEG. Herein, we performed label-free proteomics to compare the associations of linear PEG- and PEtOx-grafted nano-graphene oxide (nGO) sheets with human plasma proteins, complemented by cytotoxicity and haemolysis assays to compare the cellular interactions of these polymers. Our data revealed that nGO-PEG enriched apolipoproteins, while nGO-PEtOx displayed a preferred binding with pro-angiogenic and structural proteins, despite high similarities in their respective top-10 enriched proteins. In addition, nGO-PEG and nGO-PEtOx exhibited similar levels of enrichment of complement proteins. Both PEG and PEtOx markedly reduced nGO toxicity to HEK 293 cells while mitigating nGO haemolysis. This study provides the first detailed profile of the human plasma protein corona associated with PEtOx-grafted nanomaterials and, in light of the distinctions of PEtOx in chemical adaptability, in vivo clearance and immunogenicity, validates the use of PEtOx as a viable stealth alternative to PEG for nanomedicines and bionanotechnologies. Refereed/Peer-reviewed

The electroadsorption of proteins at aqueous−organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous−organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous−organic interface via controlled protein electroadsorption. Four proteins known to be electroactiveat such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken,focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated antiparallel β-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase.This was supported by MALDI ToF MS measurements, which showed the formation of protein-anion complexes for three ofthese proteins, and molecular dynamic simulations, which modeled the structure of lysozyme at an aqueous−organic interface.On the basis of these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins. Refereed/Peer-reviewed

The electroadsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated anti-parallel β-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI ToF MS measurement, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamic simulations, which modelled the structure of lysozyme at an aqueous-organic interface. Based on these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins.

Indole derivatives are a structurally diverse group of compounds found in food, toxins, medicines, and produced by commensal microbiota. On contact with acidic stomach conditions, indoles undergo condensation to generate metabolites that vary in solubility, activity and toxicity as they move through the gut. Here, using halogenated ions, we map promising chemo-preventative indoles, i) 6-bromoisatin (6Br), ii) the mixed indole natural extract (NE) 6Br is found in, and iii) the highly insoluble metabolites formed in vivo using desorption/ionisation on porous silicon-mass spectrometry imaging (DIOS-MSI). The functionalised porous silicon architecture allowed insoluble metabolites to be detected that would otherwise evade most analytical platforms, providing direct evidence for identifying the therapeutic component, 6Br, from the mixed indole NE. As a therapeutic lead, 0.025 mg/g 6Br acts as a chemo-preventative compound in a 12 week genotoxic mouse model; at this dose 6Br significantly reduces epithelial cell proliferation, tumour precursors (aberrant crypt foci; ACF); and tumour numbers while having minimal effects on liver, blood biochemistry and weight parameters compared to controls. The same could not be said for the NE where 6Br originates, which significantly increased liver damage markers. DIOS-MSI revealed a large range of previously unknown insoluble metabolites that could contribute to reduced efficacy and increased toxicity.

The adsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at such interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c and hemoglobin. Following controlled protein electrodeposition onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary traits within the amide I band. The structural variations observed included unfolding to form aggregated anti-parallel beta-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI-ToF MS measurement, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamics simulations, which modelled the structure of lysozyme at an aqueous-organic interface. Based on these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectivly modify proteins.

Reproducibility, or a lack thereof, is an increasingly important topic across many research fields. A key aspect of reproducibility is accurate reporting of both experiments and the resulting data. Herein, we propose a reporting guideline for mass spectrometry imaging (MSI). Previous standards have laid out guidelines sufficient to guarantee a certain quality of reporting; however, they set a high bar and as a consequence can be exhaustive and broad, thus limiting uptake.To help address this lack of uptake, we propose a reporting supplement-Minimum Information About a Mass Spectrometry Imaging Experiment (MIAMSIE)-and its abbreviated reporting standard version, MSIcheck. MIAMSIE is intended to improve author-driven reporting. It is intentionally not exhaustive, but is rather designed for extensibility and could therefore eventually become analogous to existing standards that aim to guarantee reporting quality. Conversely, its abbreviated form MSIcheck is intended as a diagnostic tool focused on key aspects in MSI reporting.We discuss how existing standards influenced MIAMSIE/MSIcheck and how these new approaches could positively impact reporting quality, followed by test implementation of both standards to demonstrate their use. For MIAMSIE, we report on author reviews of four articles and a dataset. For MSIcheck, we show a snapshot review of a one-month subset of the MSI literature that indicated issues with data provision and the reporting of both data analysis steps and calibration settings for MS systems. Although our contribution is MSI specific, we believe the underlying approach could be considered as a general strategy for improving scientific reporting. Refereed/Peer-reviewed

Forest soils are susceptible to anthropogenic acidification. In the past, acid rain was a major contributor to soil acidification, but, now that atmospheric levels of S have dramatically declined, concern has shifted towards biomass-induced acidification, i.e. decreasing soil solution pH due to tree growth and harvesting events that permanently remove base cations (BCs) from forest stands. We use a novel dynamic model, HD-MINTEQ (Husby Dynamic MINTEQ), to investigate possible long-term impacts of two theoretical future harvesting scenarios in the year 2020, a conventional harvest (CH, which removes stems only), and a whole-tree harvest (WTH, which removes 100 % of the above-ground biomass except for stumps) on soil chemistry and weathering rates at three different Swedish forest sites (Aneboda, Gårdsjön, and Kindla). Furthermore, acidification following the harvesting events is compared to the historical acidification that took place during the 20th century due to acid rain. Our results show that historical acidification due to acid rain had a larger impact on pore water chemistry and mineral weathering than tree growth and harvesting, at least if nitrification remained at a low level. However, compared to a no-harvest baseline, WTH and CH significantly impacted soil chemistry. Directly after a harvesting event (CH or WTH), the soil solution pH sharply increased for 5 to 10 years before slowly declining over the remainder of the simulation (until year 2080). WTH acidified soils slightly more than CH, but in certain soil horizons there was practically no difference by the year 2080. Even though the pH in the WTH and CH scenario decreased with time as compared to the no-harvest scenario (NH), they did not drop to the levels observed around the peak of historic acidification (1980–1990), indicating that the pH decrease due to tree growth and harvesting would be less impactful than that of historic atmospheric acidification. Weathering rates differed across locations and horizons in response to historic acidification. In general, the predicted changes in weathering rates were very small, which can be explained by the net effect of decreased pH and increased Al3+, which affected the weathering rate in opposite ways. Similarly, weathering rates after the harvesting scenarios in 2020 remained largely unchanged according to the model.

When the production of a platform well is no longer profitable, two options are available—either perform permanent plug and abandonment (P&A) or reuse the slot by plugging the original well and sidetracking. P&A requires setting barrier elements in the wellbore to prevent hydrocarbon migration between potential flow zones and to the surface. In most cases, it is financially beneficial to perform as much of this work as possible during an intervention phase before rig operations begin, thus saving rig operating expenses. Most conventional barriers are balanced cement plugs, and setting a cement plug requires a drilling rig to handle the workstring through which the cement is placed, crew, and associated equipment [e.g., blowout preventer (BOP)]. However, the rig time spent performing P&A could be used more productively, such as drilling and completing new wellbores. An operating and a service company developed a method for setting the primary P&A cement plug toward the reservoir without a drilling rig. The cement is set through the Christmas tree using a specialized wireline-set packer as a base and wiper plugs installed on a specialized spool piece to isolate the cement and indicate completion of the job. This paper explains the method and equipment and presents three case studies from the Norwegian sector of the North Sea.

Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that the variants of ERβ, namely ERβ1, ERβ2, ERβ4, and ERβ5, regulate aggressiveness of TNBC by regulating hypoxic signaling. RNA-seq of patient derived xenografts (PDX) from TNBC show expression of ERβ4 and ERβ5 variants in more than half of the samples. Furthermore, expression of ERβ4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to mammosphere formation caused transformation and development of mammospheres. By contrast, ERβ1, ERβ2 or ERβ5 were unable to support mammosphere formation. We have previously shown that all variants except ERβ1 stabilizes HIF-1α but only ERβ4 appear to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERβ4. We propose that ERβ variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer. Citation Format: Faria M, Karami S, Granados-Principal S, Dey P, Verma A, Choi DS, Elemento O, Bawa-Khalfe1 T, Chang JC, Gustafsson J-A, Strom AM. The ERβ4 variant induce transformation of the normal breast mammary epithelial cell line MCF-10A; the ERβ variants ERβ2, ERβ4 and ERβ5 increase aggressiveness of TNBC by regulation of hypoxic signaling [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-10.

Ove J. R. Gustafsson, James S. Eddes, Stephan Meding, Shaun R. McColl, Martin K. Oehler and Peter Hoffmann

Rationale Nanostructure-based mass spectrometry imaging (MSI) is a promising technology for molecular imaging of small molecules, without the complex chemical background typically encountered in matrix-assisted molecular imaging approaches. Here, we have enhanced these surfaces with silver (Ag) to provide a second tier of MSI data from a single sample. Methods MSI data was acquired through the application of laser desorption/ionization mass spectrometry to biological samples imprinted onto desorption/ionization on silicon (DIOS) substrates. Following initial analysis, ultra-thin Ag layers were overlaid onto the followed by MSI analysis (Ag-DIOS MSI). This approach was first demonstrated for fingermark small molecules including environmental contaminants and sebum components. Subsequently, this bimodal method was translated to lipids and metabolites in fore-stomach sections from a 6-bromoisatin chemopreventative murine mouse model. Results DIOS MSI allowed mapping of common ions in fingermarks as well as 6-bromoisatin metabolites and lipids in murine fore-stomach. Furthermore, DIOS MSI was complemented by the Ag-DIOS MSI of Ag-adductable lipids such as wax esters in fingermarks and cholesterol in murine fore-stomach. Gastrointestinal acid condensation products of 6-bromoisatin, such as the 6,6'-dibromoindirubin mapped herein, are very challenging to isolate and characterize. By re-analyzing the same tissue imprints, this metabolite was readily detected by DIOS, placed in a tissue-specific spatial context, and subsequently overlaid with additional lipid distributions acquired using Ag-DIOS MSI. Conclusions The ability to place metabolite and lipid classes in a tissue-specific context makes this novel method suited to MSI analyses where the collection of additional information from the same sample maximises resource use, and also maximises the number of annotated small molecules, in particular for metabolites that are typically undetectable with traditional platforms. Copyright © 2017 John Wiley & Sons, Ltd.

Objective: Few studies have investigated the association of childhood IQ and school achievement with suicide. The aim of this study was to investigate the association of childhood IQ with suicide in a cohort of Swedish women and men. Method: 21 809 subjects born in 1948 and 1953 who completed IQ and school tests at age 13 years have been followed until 2003. Information on paternal education and in-patient care for psychosis was linked using the Swedish personal identification number. Results: There were 180 suicides amongst subjects with measured IQ. High IQ was associated with reduced suicide risk among men (OR per unit increase in age-adjusted model 0.90, 95% CI 0.83-0.99), while there was no statistical evidence of an association in women (OR 1.04, 95% CI 0.90-1.20). Among men with a history of psychosis, high IQ was associated with an increased risk of suicide. Conclusion: Low childhood IQ at age 13 years is associated with an increased risk of suicide in men but not in women; however, amongst those with psychosis, low IQ appears to be protective. Language: en

Transcriptional effects of estrogen result from its activation of two estrogen receptor (ER) isoforms; ERalpha that drives proliferation and ERbeta that is antiproliferative. Expression of ERbeta in xenograft tumors from the T47D breast cancer cell line reduces tumor growth and angiogenesis. If ERbeta can halt tumor growth, its introduction into cancers may be a novel therapeutic approach to the treatment of estrogen-responsive cancers. To assess the complete impact of ERbeta on transcription, we have made a full transcriptome analysis of ERalpha- and ERbeta-mediated gene regulation in T47D cell line with Tet-Off regulated ERbeta expression. Of the 35 000 genes and transcripts analysed, 4.1% (1434) were altered by ERalpha activation. Tet withdrawal and subsequent ERbeta expression inhibited the ERalpha regulation of 998 genes and, in addition, altered expression of 152 non-ERalpha-regulated genes. ERalpha-induced and ERbeta-repressed genes were involved in proliferation, steroid/xenobiotic metabolism and ion transport. The ERbeta repressive effect was further confirmed by proliferation assays, where ERbeta was shown to completely oppose the ERalpha-E2 induced proliferation. Additional analysis of ERbeta with a mutated DNA-binding domain revealed that this mutant, at least for a quantity of genes, antagonizes ERalpha even more strongly than ERbeta wt. From an examination of the genes regulated by ERalpha and ERbeta, we suggest that introduction of ERbeta may be an alternative therapeutic approach to the treatment of certain cancers.

Nanostructure imaging mass spectrometry (NIMS) using porous silicon (pSi) is a key technique for molecular imaging of exogenous and endogenous low molecular weight compounds from fingerprints. However, high-mass-accuracy NIMS can be difficult to achieve as time-of-flight (ToF) mass analyzers, which dominate the field, cannot sufficiently compensate for shifts in measured m/z values. Here, we show internal recalibration using a thin layer of silver (Ag) sputter-coated onto functionalized pSi substrates. NIMS peaks for several previously reported fingerprint components were selected and mass accuracy was compared to theoretical values. Mass accuracy was improved by more than an order of magnitude in several cases. This straightforward method should form part of the standard guidelines for NIMS studies for spatial characterization of small molecules. Refereed/Peer-reviewed

Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics. Refereed/Peer-reviewed

Recent clinical studies are indicating that the estrogen receptor β variant β5 (ERβ5) expression correlates to worse prognosis. We wanted to know if expression of ERβ5 is changing the growth behavior of the triple negative cell line SUM159. Estrogen receptor β5 is highly similar to estrogen receptor β1 except for a truncated C-terminus making the remaining ligand binding domain incapable of binding to estrogen. In addition a 4 amino acid unique peptide is added to the C-terminal end. Stably expressing ERβ5 using a transposon integrated tetracycline regulated expression system we find that expression of ERβ5 increases proliferation of the triple negative SUM159 cells especially in reduced serum condition compared to control cells. Since SUM159 have been shown to depend on autocrine stimulation for growth we are suggesting that expression of ERβ5 is affecting the production of autocrine growth factors. Citation Format: Faria M, Tin-U C, Dey P, Gustafsson J-A, Strom AM. Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-04.

Polyethylene glycol (PEG) is widely used as an antifouling and stealth polymer in surface engineering and nanomedicine. However, recent research has revealed adverse effects of bioaccumulation and immunogenicity following the administration of PEG, prompting this proteomic examination of the plasma protein coronae association with superparamagnetic iron oxide nanoparticles (IONPs) grafted with brushed PEG (bPEG) and an alternative, brushed phosphorylcholine (bPC). Using label-free quantitation by liquid chromatography tandem-mass spectrometry, this study determines protein abundances for the in vitro hard coronae of bare, bPC-, and bPEG-grafted IONPs in human plasma. This study also shows unique protein compositions in the plasma coronae of each IONP, including enrichment of coagulation factors and immunogenic complement proteins with bPEG, and enhanced binding of apolipoproteins with bPC. Functional analysis reveals that plasma protein coronae elevate the horseradish peroxidase-like activities of the bPC- and bPEG-IONPs by approximately twofold, an effect likely mediated by the diverse composition and physicochemical properties of the polymers as well as their associated plasma proteins. Taken together, these observations support the rational design of stealth polymers based on a quantitative understanding of the interplay between IONPs and the plasma proteome, and should prove beneficial for the development of materials for nanomedicine, biosensing, and catalysis. Refereed/Peer-reviewed

Recent clinical studies indicate that estrogen receptor β2 (ERβ2) and β5 (ERβ5) expression in TNBC correlates to worse prognosis. We have expressed ERβ2 and ERβ5 in the triple negative cell line SUM159. Estrogen receptors β2 and β5 are highly similar to estrogen receptor β1, except for a truncated C-terminus making the remaining ligand -binding domain incapable of binding to estrogen. In addition, at the C-terminus, ER β2 and β5 have a unique peptide each of 27 and 4 amino acids in length respectively. Stably expressing ERβ2 and ERβ5 using a transposon integrated, tetracycline- regulated expression system increases expression of the drug resistance gene, ABCG2 in the triple negative SUM159 cells. Furthermore, we also find that the cells become more resistant to treatments with paclitaxel. We find that ERβ2 and ERβ5 interact with HIF-1α and HIF-2α and are recruited to HIF response elements (HRE's) in chromatin. In agreement with this, we find that ERβ2 and ERβ5 are recruited to the ABCG2 promoter, which contains HRE's and is known to be a target of HIF signaling. We propose that ERβ2 and ERβ5 play a role in chemo - resistance of TNBC and that targeting these factors may reduce chemo resistance thus preventing tumor-relapses and their associated mortalities. Citation Format: Faria M, Gustafsson J-A, Strom A. Estrogen receptor β2 and β5 increase expression of ABCG2 and drug resistance of the triple- negative breast cancer cell line SUM159 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-10.

Imaging mass spectrometry (IMS) has transformed proteomics by providing an avenue for collecting spatially distributed molecular data. Mass spectrometry data acquired with matrix assisted laser desorption ionization (MALDI) IMS consist of tens of thousands of spectra, measured at regular grid points across the surface of a tissue section. Unlike the more standard liquid chromatography mass spectrometry, MALDI-IMS preserves the spatial information inherent in the tissue. Motivated by the need to differentiate cell populations and tissue types in MALDI-IMS data accurately and efficiently, we propose an integrated cluster and feature extraction approach for such data. We work with the derived binary data representing presence/absence of ions, as this is the essential information in the data. Our approach takes advantage of the spatial structure of the data in a noise removal and initial dimension reduction step and applies $k$-means clustering with the cosine distance to the high-dimensional binary data. The combined smoothing-clustering yields spatially localized clusters that clearly show the correspondence with cancer and various noncancerous tissue types. Feature extraction of the high-dimensional binary data is accomplished with our difference in proportions of occurrence (DIPPS) approach which ranks the variables and selects a set of variables in a data-driven manner. We summarize the best variables in a single image that has a natural interpretation. Application of our method to data from patients with ovarian cancer shows good separation of tissue types and close agreement of our results with tissue types identified by pathologists., Published at http://dx.doi.org/10.1214/15-AOAS870 in the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org)

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3 beta,7 alpha-dihydroxycholest-5-en-26-oic acid (3 beta,7 alpha-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3 beta-hydroxy-7-oxocholest-5-en-26-oic acid (3 beta H,70-CA) promoted maturation of precursors into islet-1(+) cells. Unlike 3 beta,7 alpha-diHCA and 3 beta H,70-CA, 3 beta-hydroxycholest-5-en-26-oic acid (3 beta-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A7, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3 beta-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3 beta,7 alpha-diHCA. Moreover, 3 beta,7 alpha-diHCA prevented the loss of motor neurons induced by 3 beta-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

Fibres from conventional pine kraft pulp (KP), oxygen-delignified KP (KPO) and elemental-chlorine-free (ECF) bleached KP were characterized by scanning probe microscopy (SPM). The surface morphology of lignin isolated from the respective pine KP was studied. The data analysis of the phase-shift data measured by using different tapping mode imaging parameters enabled the study of both the surface amphiphility and stiffness as a function of delignification. The average phase-shift values were observed to correlate to the respective kappa values, when imaging with small tapping amplitude (30 nm), referred to the capability of SPM to follow local amphipilic (adhesion) differences on a wood fibre surface. The roughness (standard deviation) of the average phase-shift values was taken as a measure of the sample homogeneity. The mean morphological roughness was observed to increase with decreasing bulk lignin concentration. The surface of lignin appeared mainly granular but occasionally some linear structures were observed. The surface roughness of different fractions of flow-through residual lignin samples correlated quite well to the molar mass of the respective fraction. Each grain contained hundreds of lignin molecules. Most of the lignin samples were homogeneous as regards friction and phase-contrast images. The surface of the nondelignified pine KP was typically granular. ECF delignification was more effective than oxygen delignification in removing this granular phase. The decrease in the relative amount of the granular phase correlated to a decreasing kappa value of the pulp. The granular phase is thus suggested to correspond to lignin, but it cannot be ruled out that small amounts of hemicellulose, extractives or a mixture of these components are embedded in this phase. The surface concentration of the granular phase of the KPO samples was, according to the phase-contrast images, clearly higher than the bulk lignin concentration, as indicated by the corresponding kappa values.

In combination with androgens, estrogens can induce aberrant growth and malignancy of the prostate gland. Estrogen action is mediated through two receptor subtypes: estrogen receptors alpha (ERalpha) and beta (ERbeta). Wild-type (wt) and transgenic mice lacking a functional ERalpha (alphaERKO) or ERbeta (betaERKO) were treated with the synthetic estrogen diethylstilbestrol (DES). DES induced prostatic squamous metaplasia (SQM) in wt and betaERKO but not in alphaERKO mice, indicating an essential role for ERalpha, but not ERbeta, in the induction of SQM of prostatic epithelium. In order to determine the respective roles of epithelial and stromal ERalpha in this response, the following tissue recombinants were constructed with prostatic epithelia (E) and stroma (S) from wt and ERKO mice: wt-S+wt-E, alphaERKO-S+alphaERKO-E, wt-S+alphaERKO-E, and alphaERKO-S+wt-E. A metaplastic response to DES was observed in wt-S+wt-E tissue recombinants. This response to DES involved multilayering of basal epithelial cells, expression of cytokeratin 10, and up-regulation of the progesterone receptor. Tissue recombinants containing alphaERKO-E and/or -S (alphaERKO-S+alphaERKO-E, wt-S+alphaERKO-E, and alphaERKO-S+wt-E) failed to respond to DES. Therefore, full and uniform epithelial SQM requires ERalpha in the epithelium and stroma. These results provide a novel insight into the cell-cell interactions mediating estrogen action in the prostate via ERalpha.

Clinical case reports have documented disturbances of carbohydrate and lipid metabolism in aromatase deficient and estrogen resistant males. The aim of the present study was to explore the metabolic functions of estrogens in male mice and to dissect the estrogen receptor (ER) specificity of such effects. Total body fat content and serum levels of leptin were followed in ERalpha knockout (ERKO), ERbeta knockout (BERKO), and ERalpha/beta double knockout (DERKO) mice. Neither the total body fat nor serum leptin levels were altered in any group before or during sexual maturation. However, after sexual maturation ERKO and DERKO, but not BERKO, demonstrated a clear increase in total body fat and enhanced serum leptin levels. Serum cholesterol was increased and a qualitative change in the lipoprotein profile, including smaller LDL particles, was observed in ERKO and DERKO mice. In conclusion, ERalpha but not ERbeta-inactivated male mice develop obesity after sexual maturation.

Confirmatory factor analysis (CFA) was used to test models of the relationship between inspection time (IT) and psychometric measures of intelligence (the eleven subtests of the Wechsler Adult Intelligence Scale-Revised). The sample consisted of 134 healthy adults and was broadly representative of the adult United Kingdom population in terms of the distributions of age and social class. A Nested Factors parameterization consisting of g plus three orthogonal group factors with IT loading on all factors provided a reasonably good fit to the data. Variants on this model that incorporated theoretical and empirically derived constraints were tested; constraining IT to have zero loadings on all factors produced a significant deterioration in fit. Constraining IT to load only on General Intelligence (g) and Perceptual Organization did not produce a significant deterioration; nested variants on this latter model in which IT was constrained to load on either of these two factors alone produced a significant deterioration in fit.

The aryl hydrocarbon (or dioxin) receptor (AhR) is a ligand-activated basic helix-loop-helix (bHLH) protein that heterodimerizes with the bHLH protein ARNT (aryl hydrocarbon nuclear translocator) forming a complex that binds to xenobiotic regulatory elements in target gene enhancers. Genetic, biochemical, and molecular biology studies have revealed that the AhR mediates the toxic and biological effects of environmentally persistent dioxins and related compounds. Cloning of the receptor and its DNA-binding partner, ARNT, has facilitated detailed efforts to understand the mechanisms of AhR-mediated signal transduction. These studies have determined that this unique receptor consists of several functional domains and belongs to a subfamily of bHLH proteins that share a conserved motif termed the PAS domain. In addition, recent genetic studies have revealed that expression of the AhR is a requirement for proper embryonal development, which appears to be a common function shared by many other bHLH proteins. This review is a summary of recent molecular studies of AhR-mediated gene regulation.

MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis.However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue micro array cores, from 31 ovarian cancer patients, by LC−MS/MS. The sample preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This result edin 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per sample. From this, a total of 840 proteins and, on average, 297 proteins per sample could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several m/z species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer. Refereed/Peer-reviewed

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the means to map the in situ distribution of tryptic peptides in formalin-fixed clinical tissue samples. The ability to analyze clinical samples is of great importance to further developments in the imaging field. However, there is a requirement in this field of research for additional methods describing the characterization of tryptic peptides by MALDI imaging.This protocol gives highly detailed instructions, with examples, for (1) successfully performing tryptic peptide MALDI imaging on formalin-fixed cancer tissue using a MALDI-TOF/TOF MS instrument, (2) tentatively generating identifications through nLC/MS/MS, and (3) validating these identifications by in situ MS/MS of peptides of interest.This protocol provides a detailed and straightforward description of the methods required for groups new to MALDI imaging to begin analysis of formalin-fixed clinical samples.

One of the hallmarks of acute colitis is loss of epithelial transport. For unknown reasons, many patients still suffer from GI symptoms during remission, indicating a sustained imbalance between absorption and secretion. We hypothesize that the colonic epithelium becomes more reactive to secretagogues to compensate for a failing barrier.Biopsies from ascending colon and sigmoid colon of UC patients in remission and controls were mounted in Ussing chambers. Membrane current (Im) and epithelial capacitance (Cp) were used as markers for anion secretion and mucus exocytosis. Carbachol (1 mmol L(-1) ) and forskolin (10 μmol L(-1) ) were used to study Ca(2+) and cAMP-mediated secretion.Baseline values showed segmental patterns with higher Im in ascending colon and higher Cp in sigmoid colon of both UC patients and controls, but the patterns did not differ between the groups. The Im response to forskolin was increased (+35%) in the ascending colon of UC patients and the Im response to carbachol was decreased (-40%) in the same segment. No group differences were seen in the distal colon for either the forskolin or carbachol-induced Im responses. The Cp response to carbachol was instead up-regulated in the distal colon of UC patients, but remained unaffected in the proximal colon.The proximal colonic mucosa of UC patients in remission seems to shift its reactivity to secretagogues, becoming more sensitive to cAMP-dependent secretion and less sensitive to Ca(2+) -dependent secretion. This phenomenon may contribute to residual diarrhea in this patient group, despite resolution of inflammation.

Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRβ, with overlapping but not identical functions, we investigated whether LXRα and LXRβ exert different effects on intestinal cholesterol absorption.Wild-type (WT), LXRα(-/-) and LXRβ(-/-) mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965.When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα(-/-) mice, but not in the WT or LXRβ(-/-) animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα(-/-) mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα(-/-) mice. A 0.2% cholesterol +GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree.In the intestine, LXRα and LXRβ seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRβ may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRβ-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.

We have used a panel of monoclonal antibodies and a cell-free transcription assay to study the function of the tau 1 transactivation domain of the human glucocorticoid receptor. Three antibodies (monoclonal antibodies 250, 275, and 286) specifically inhibited tau 1-dependent transcription, but had little or no effect on either basal transcription or the activity of an unrelated yeast transcription factor. This inhibition was not due to interference of DNA binding activity, as all three antibodies super shifted tau 1-containing protein-DNA complexes. Epitopes for all three antibodies were localized to a region between amino acids 190 and 200, which lies within the recently defined 41-amino acid core region of tau 1 that is required for transactivation (Dahlman-Wright, K., Almlof, T., McEwan, I.J., Gustafsson, J-A., and Wright, A. P.H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1619-1623). In contrast to the effect on tau 1-dependent transcription none of the antibodies tested antagonized the squelching ability of the tau 1 domain, suggesting that tau 1-mediated transactivation involves interactions in addition to those identified by the squelching assay. Consistent with this, a comparison of the kinetics of tau 1 squelching and inhibition of transactivation by monoclonal antibodies suggested a role for tau 1 mediated transcriptional induction at two or more steps during transcription initiation by RNA polymerase II.

Poor school performance and low IQ are associated with an increased risk of suicide in males; it is uncertain whether cognitive performance is a risk factor for suicide in females and whether severe mental illness influences these associations.Record linkage study of Swedish education, population and census data with mortality and inpatient registers. Data were available for 95,497 males and 91,311 females born in 1972 and 1977 and followed up until 31 December 2005.230 males and 90 females died from suicide over the follow-up period. There were strong inverse associations between school performance at age 16 and subsequent suicide risk in males (fully adjusted hazard ratio (HR) per SD increase in school performance score: 0.6 (95% CI 0.6 to 0.7; p0.001)) but not females: adjusted HR. 1.1 (CI 0.9 to 1.4 p = 0.50). In males there were almost four fold differences in suicide risk between children in the top and bottom fifths of the range of school grade scores. Similar associations were seen with upper secondary school performance (age 18/19 years). There was no strong evidence that deterioration in school performance between ages 16 and 18 was associated with increased suicide risk. Amongst men who developed severe psychiatric illness school performance was not associated with suicide risk.We had limited information on the prevalence of minor psychiatric disorder in cohort members and no data on IQ for the cohort as a whole.Good performance in secondary (age 16) and upper secondary (age 18) school is associated with a reduced risk of suicide in men but not women. This protective effect is not seen amongst those who develop severe psychiatric illness. These differences indicate that the aetiology of suicide differs in males and females and in those with and without severe mental illness.

Full length human glucocorticoid receptor and truncated receptor derivatives lacking the major amino-terminal trans-activating domain were expressed in stably transfected Chinese hamster ovary (CHO) cells. The receptors were co-expressed together with human metallothionein IIa, and the expression levels were amplified in the presence of increasing concentrations of metal. In amplified cells, both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000-520,000 molecules/cell which corresponds to a 10-fold increase in receptor levels as compared to rat liver cells. The hormone (agonist or antagonist) binding properties of the expressed proteins were very similar to those characteristic of authentic glucocorticoid receptors in tissues or cultured cells. Moreover, the expressed proteins specifically recognized a glucocorticoid-response element sequence motif in in vitro protein-DNA binding experiments. The activation of a glucocorticoid-responsive reporter gene by the expressed full length receptor was dramatic (about 75-fold) and strictly ligand-dependent. In contrast, the expressed amino-terminal deletion mutant exhibited considerably weaker functional activity but showed normal hormone-binding properties. Upon exposure to dexamethasone in vivo, the expressed receptor mRNAs and proteins were down-regulated about 2- to 6-fold, indicating that regulatory signals important for autoregulation may be contained within structures corresponding to the ligand and DNA-binding domains. Transcription from the expression vector was not negatively regulated from the hormone, strongly arguing that receptor down-regulation was due to a post-transcriptional mechanism. In conclusion, this expression system should be a useful tool for further structural and functional studies of the receptor, including the biochemistry of its activation from a cryptic to a functional species, and its ligand-dependent autoregulation.

Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS)

By means of double immunolabeling procedures it has been possible to demonstrate glucocorticoid receptor (GR) immunoreactivity (IR) in large numbers of various peptidergic neurons of the brain including neurons containing gastrointestinal peptides, opioid peptides, and peptides with a hypothalamic hormone function. For each peptide system, however, marked heterogeneities exist among brain regions. Thus, in the neocortex and the hippocampal formation most of the brain peptide neurons lack GR IR, while the same types of peptide neurons in the arcuate and paraventricular nucleus [e.g. neuropeptide Y (NPY), somatostatin (SRIF) and the cholecystokinin (CCK) neurons] possess strong GR IR. Furthermore, in the arcuate, parvocellular part of the paraventricular nuclei and the central amygdaloid nucleus practically all the peptidergic neurons are strongly GR IR, while in the lateral hypothalamus, mainly the neurotensin (NT) and galanin (GAL) IR neurons are GR IR. These marked differences among areas probably reflect functional differences dependent upon their participation in stress regulated circuits. All the paraventricular NT, corticotropin-releasing factor (CRF), growth hormone-releasing factor (GRF), thyrotropin-releasing hormone (TRH) and SRIF IR neurons appear to contain GR IR, while the luteinizing hormone-releasing hormone (LHRH) IR neurons lack GR IR, underlying the importance of glucocorticoids (GC) in controlling endocrine function. Finally, the GC may influence pain and mood control mainly via effects on enkephalin (ENK) neurons especially in the basal ganglia (mood) and on all beta-endorphin (beta-END) neurons of the arcuate nucleus, while most of the dynorphin neurons are not directly controlled by GC.

Partially purified preparations of the human progestin receptor and the human and rat glucocorticoid receptor proteins were covalently charged with the synthetic progestin, [3H]promegestone, by photoaffinity labeling. After labeling, the denaturated protein was cleaved and the mixture of peptides subjected to radiosequence analysis as previously described for the rat glucocorticoid receptor protein (Carlstedt-Duke, J., Strömstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and Jörnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). The radioactivity labels identified, corresponded to Met-759 and Met-909 after photoaffinity labeling of the human progestin receptor, and Met-622 and Cys-754 after labeling of the rat glucocorticoid receptor. The residues labeled in the glucocorticoid receptor are the same as those previously reported to bind triamcinolone actonide. The corresponding residues were also labeled in the human glucocorticoid receptor. Met-759 of the progestin receptor and Met-622 of the rat glucocorticoid receptor are positioned within a segment with an overall high degree of sequence similarity and are equivalent. However, Met-909 (progestin receptor) and Cys-754 (glucocorticoid receptor) do not occur within equivalent segments of the two proteins. Thus, although the two classes of steroid hormone share a common structure within the A-ring, there are subtle differences in their interaction with the two separate receptor proteins.