Inhibition of cytosolic phospholipase A2 mRNA expression: a novel mechanism for acetylsalicylic acid-mediated growth inhibition and apoptosis in colon cancer cells

Acetylsalicylic acid (ASA) has been confirmed to inhibit proliferation and to induce apoptosis in human colorectal cancer cells in vitro. However, the mechanism by which ASA exhibits antiproliferative and proapoptotic effects in cyclooxygenase 2 (COX-2)-negative cells remains to be further elucidated. In the present study, SW480, a COX-2-negative colon cancer cell line, was treated with various concentrations of ASA (0, 2.5, 5, and 10 mM). The antiproliferative and proapoptotic effects of ASA were confirmed by MTT assay, flow cytometry of propidium iodide (PI)-stained cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. After treatment with ASA, intracellular cyclic AMP (cAMP) levels were increased and the production of prostaglandin E2 (PGE2) was decreased. RT-PCR analysis revealed that treatment of ASA induced a concentration-dependent downregulation of cytosolic phospholipase A2 (cPLA2) mRNA expression in SW480 cells and also in two other colorectal cancer cell lines, Colo320 and HT-29 cells. Intracellular calcium levels were unaffected by ASA treatment. Our results indicate that the ASA-induced downregulation of cytosolic phospholipase A2 mRNA expression might be a novel mechanism for ASA-mediated growth inhibition and apoptosis in colon cancer cells.     

Serum Biomarkers in Patients with Relapsing Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss)

Introduction

Previous studies suggest a role for eotaxin-3, TARC/CCL17 and IgG4 in newly- diagnosed patients with eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss) with highly active disease. The role of these biomarkers in relapsing disease is unclear.

Methods

Serum levels of TARC/CCL17, eotaxin-3, IgG4, and IgG4/IgG ratio were determined in serum samples from a longitudinal cohort of patients with EGPA (105 visits of 25 patients). Epidemiological, clinical and laboratory data were available for all visits.

Results

At the first visit, 80% of patients were using glucocorticoids and 68% additional immunosuppressive drugs. Disease flares were seen at 18 visits. The median BVAS and BVAS/WG scores at time of relapse were 4 and 2, respectively. None of the biomarkers tested were useful to discriminate between active disease and remission. Patients treated with prednisone had lower eotaxin-3 and eosinophil levels compared to patients not taking glucocorticoids irrespective of disease activity. Use of immunosuppressive agents was not associated with biomarker levels.

Conclusions

Serum levels of TARC/CCL17, eotaxin-3, IgG4, and IgG4/IgG ratio do not clearly differentiate active and inactive disease in established EGPA. Defining biomarkers in EGPA remains a challenge especially during times of glucocorticoid use.     

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1−/− and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer''s disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1−/− and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1−/− mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.Cerebrospinal fluid (CSF)¹ consists of interstitial fluid that is in continuous exchange with the central nervous system and the peripheral blood system. It represents the only body fluid in humans that is in direct contact with brain tissue and accessible in a routine clinical setting. Thus, the easy accessibility from the periphery renders CSF perfectly suited to study pathologic neurological processes (1). Human CSF has a relatively low protein content (∼ 0.4 mg/ml), but features a highly diverse proteome. It is thus increasingly studied by modern mass spectrometry based proteomics (2). The proteomic analysis of human CSF typically involves various protein concentration and fractionation steps as well as the depletion of highly abundant proteins, such as serum albumin. This allows the identification of several hundred up to 2600 proteins from several milliliters of human CSF (3).Mice are the most popular animal model in preclinical research, because of their similarity to humans in genetics and physiology, their unlimited supply and their ease of genetic engineering. The study of their CSF can provide valuable insights into disease mechanisms and biomarker discovery and may allow the rapid translation of preclinical findings into human patients. However, the proteomic study of murine CSF has been limited because of several shortcomings. The low total CSF volume of ∼30 μl and an average yield of only ∼10 μl blood-free CSF pose a challenge for various protein concentration and depletion steps that are routinely applied to human CSF, where the sample volume is up to 1,000-fold more (4, 5). One study reported the identification of 289 proteins and the quantification of 103 proteins using pooled immunodepleted CSF from 10–12 mice per sample (6). A second study reported the identification of 566 proteins in murine CSF of individual mice, relying on time consuming fractionation by two dimensional liquid chromatography tandem MS (2D-LC-MS/MS) (7).Here we show that label-free quantitative proteomics in murine CSF can be achieved in unprecedented depth in individual animals using single ultra HPLC runs on the benchtop Q Exactive mass spectrometer. We demonstrate the feasibility of our approach by comparing the CSF of BACE1 (β-site amyloid precursor protein (APP) cleaving enzyme 1) −/− mice with their wild-type littermates.BACE1 is a membrane bound aspartyl protease that is essential in the pathogenesis of Alzheimer''s disease. It is the rate-limiting enzyme in a proteolytic cascade leading to the liberation of the neurotoxic Aβ peptide from the much larger amyloid precursor protein (APP) into the extracellular space (8, 9). Inhibition of BACE1 abolishes Aβ generation, rendering BACE1 a prime drug target for the therapy of Alzheimer''s disease (10). Besides APP, BACE1 processes numerous other substrates in vivo and in vitro, which raises concerns about mechanism based side effects on the therapeutic inhibition of this protease (11). Although BACE1 expression levels are the highest in the brain, it is currently unknown whether BACE1 substrate levels besides APP can be monitored in the CSF as a read-out of BACE1 activity. This would be desirable, as it would allow the longitudinal monitoring of BACE1 substrate levels on therapeutic inhibition of BACE1 in humans and thus an effective screening for possible adverse effects.Our approach allows the accurate identification and quantification of several hundred proteins in as little as 2 μl of murine CSF in ∼4.5 h per sample, at a much greater speed and proteomic depth than in previous studies, despite using lower sample amounts (6, 7). Overall, 715 proteins were identified with at least two unique peptides and 522 proteins were quantified in at least three biological replicates of both BACE1−/− and wild-type mice. We provide evidence that BACE1 activity is reflected in the composition of the CSF, as the secreted ectodomains of well-known BACE1 substrates were reduced in BACE1−/− animals. In addition, we identified and validated a previously unknown BACE1 substrate candidate and confirmed two recently described novel BACE1 substrates. The three proteins may represent novel prognostic or diagnostic biomarkers and may aid in the development of APP-specific BACE1 inhibitors.     

Sensitivity of Hyperdense Basilar Artery Sign on Non-Enhanced Computed Tomography

Purpose

The hyperdense basilar artery sign (HBAS) is an indicator of vessel occlusion on non contrast-enhanced computer tomography (NECT) in acute stroke patients. Since basilar artery occlusion (BAO) is associated with a high mortality and morbidity, its early detection is of great clinical value. We sought to analyze the influence of density measurement as well as a normalized ratio of Hounsfield unit/hematocrit (HU/Hct) ratio on the detection of BAO on NECT in patients with suspected BAO.

Materials and Methods

102 patients with clinically suspected BAO were examined with NECT followed immediately by Multidetector computed tomography Angiography. Two observers independently analyzed the images regarding the presence or absence of HBAS on NECT and performed HU measurements in the basilar artery. Receiver operating characteristic curve analysis was performed to determine the optimal density threshold for BAO using attenuation measurements or HU/Hct ratio.

Results

Sensitivity of visual detection of the HBAS on NECT was relatively low 81% (95%-CI, 54–95%) while specificity was high 91% (95%-CI, 82–96%). The highest sensitivity was achieved by the combination of visual assessment and additional quantitative attenuation measurements applying a cut-off value of 46.5 HU with 94% sensitivity and 81% specificity for BAO. A HU/Hct ratio >1.32 revealed sensitivity of 88% (95%-CI, 60–98%) and specificity of 84% (95%-CI, 74–90%).

Conclusion

In patients with clinically suspected acute BAO the combination of visual assessment and additional attenuation measurement with a cut-off value of 46.5 HU is a reliable approach with high sensitivity in the detection of BAO on NECT.     

Coupling GIS and LCA for biodiversity assessments of land use

Purpose  

Geospatial details about land use are necessary to assess its potential impacts on biodiversity. Geographic information systems (GIS) are adept at modeling land use in a spatially explicit manner, while life cycle assessment (LCA) does not conventionally utilize geospatial information. This study presents a proof-of-concept approach for coupling GIS and LCA for biodiversity assessments of land use and applies it to a case study of ethanol production from agricultural crops in California.     

An orally bioavailable positive allosteric modulator of the mGlu4 receptor with efficacy in an animal model of motor dysfunction

A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure–activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented.