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In order to analyze the feasibility of recycled engine oil bottom (REOB) as a regenerant for old asphalt and to determine its improvement effect on the performance of recycled asphalt, this paper analyzes the functional groups of two kinds of old asphalt and matrix asphalt through infrared spectrum test, such as carbonyl group, butadiene double bond, styrene double bond, methyl group, methylene group, and aromatic ring in the molecules of REOB. Find out the compatibility characteristics between them from the microscopic point of view. The recycled asphalt with 4%, 6%, and 8% content was prepared, and the physical property test was conducted to analyze the regeneration effect of different contents of REOB, and the optimum amount of REOB for 10-year-old asphalt was determined to be 6%. The study found that REOB contains aromatic hydrocarbons and colloids, which can supplement the reduction of aromatic hydrocarbons and colloids caused by asphalt aging. Therefore, REOB helps to adjust the composition of aging asphalt, improve its performance, and achieve good regeneration.

Pervious concrete has good water permeability and, if used in construction, it can alleviate the heat island effect. However, its low strength and poor durability are major obstacles to its use. This study shows that nano-reinforced pervious concrete created by incorporating cellulose nanofibrils (CNFs) can improve the physical properties and increase the durability of pervious concrete. CNFs were added to the concrete mix in proportions ranging from 0.05% to 0.2% by weight of binder. The additions were found to alter matrix rheology. The hydration kinetics of matrix with differing CNF contents were compared and analyzed. The experimental results show the addition of CNFs delayed peak heat flow and maximum cumulative heat. The 28 d compressive strength of pervious concrete increased by up to 26.5% and 28 d flexural strength by up to 25.8% with the addition of 0.05–0.2% CNFs. Addition of 0.1% and 0.2% CNFs increased water permeability. Addition of 0.05–0.15% CNFs decreased mass loss by 73.2–83.7% after 150 freeze–thaw cycles, which corresponded to an increase in frost resistance. Denser matrices and stronger interfacial transition zones were observed using scanning electron microscopy when 0.05–0.2% CNFs were added.

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-lymphoma). The majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-lymphoma and how these mutations contribute to the development of lymphoma. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants and performed a range of in vitro assays. We found that the GPR34 Q340X truncation, but not the R84H and D151A mutants, conferred a significantly increased resistance to apoptosis and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization compared with the wild type after ligand (lysophosphatidylserine) stimulation. Among the 9 signaling pathways examined, the GPR34 Q340X truncation, and to a lesser extent the D151A mutant, significantly activated CRE, NF-κB, and AP1 reporter activities, particularly in the presence of ligand stimulation. We further described the enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, as well as their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B cells via GPR34, in which phospholipase A is released by LELs and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-lymphoma.

The genesis of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is driven by oncogenic co-operation among immunological stimulations and acquired genetic changes. We previously identified recurrent CCR6 mutations in MALT lymphoma, with majority predicted to result in truncated proteins lacking the phosphorylation motif important for receptor desensitization. Functional consequences of these mutational changes, the molecular mechanisms of CCR6 activation and how this receptor signaling contributes to MALT lymphoma development remain to be investigated. In the present study, we demonstrated that these mutations impaired CCR6 receptor internalization and were activating changes, being more potent in apoptosis resistance, malignant transformation, migration and intracellular signaling, particularly in the presence of the ligands CCL20, HBD2 (human beta defensin 2) and HD5 (human alpha defensin 5). CCR6 was highly expressed in malignant B cells irrespective of the lymphoma sites. HBD2 and CCL20 were constitutively expressed by the duct epithelial cells of salivary glands, and also those involved in lymphoepithelial lesions (LEL) in salivary gland MALT lymphoma. While in the gastric setting, HBD2, and HD5, to a less extent CCL20, were highly expressed in epithelial cells of pyloric and intestinal metaplasia respectively including those involved in LEL, which are adaptive responses to chronic Helicobacter pylori infection. These findings suggest that CCR6 signaling is most likely active in MALT lymphoma, independent of its mutation status. The observations explain why the emergence of malignant B cells and their clonal expansion in MALT lymphoma are typically around LEL, linking the innate immune responses to lymphoma genesis.

Summary Background Screening for Barrett's oesophagus relies on endoscopy, which is invasive and few who undergo the procedure are found to have the condition. We aimed to use machine learning techniques to develop and externally validate a simple risk prediction panel to screen individuals for Barrett's oesophagus. Methods In this prospective study, machine learning risk prediction in Barrett's oesophagus (MARK-BE), we used data from two case-control studies, BEST2 and BOOST, to compile training and validation datasets. From the BEST2 study, we analysed questionnaires from 1299 patients, of whom 880 (67·7%) had Barrett's oesophagus, including 40 with invasive oesophageal adenocarcinoma, and 419 (32·3%) were controls. We randomly split (6:4) the cohort using a computer algorithm into a training dataset of 776 patients and a testing dataset of 523 patients. We compiled an external validation cohort from the BOOST study, which included 398 patients, comprising 198 patients with Barrett's oesophagus (23 with oesophageal adenocarcinoma) and 200 controls. We identified independently important diagnostic features of Barrett's oesophagus using the machine learning techniques information gain and correlation-based feature selection. We assessed multiple classification tools to create a multivariable risk prediction model. Internal validation of the model using the BEST2 testing dataset was followed by external validation using the BOOST external validation dataset. From these data we created a prediction panel to identify at-risk individuals. Findings The BEST2 study included 40 diagnostic features. Of these, 19 added information gain but after correlation-based feature selection only eight showed independent diagnostic value including age, sex, cigarette smoking, waist circumference, frequency of stomach pain, duration of heartburn and acidic taste, and taking antireflux medication, of which all were associated with increased risk of Barrett's oesophagus, except frequency of stomach pain, with was inversely associated in a case-control population. Logistic regression offered the highest prediction quality with an area under the receiver-operator curve (AUC) of 0·87 (95% CI 0·84–0·90; sensitivity set at 90%; specificity of 68%). In the testing dataset, AUC was 0·86 (0·83–0·89; sensitivity set at 90%; specificity of 65%). In the external validation dataset, the AUC was 0·81 (0·74–0·84; sensitivity set at 90%; specificity of 58%). Interpretation Our diagnostic model offers valid predictions of diagnosis of Barrett's oesophagus in patients with symptomatic gastro-oesophageal reflux disease, assisting in identifying who should go forward to invasive confirmatory testing. Our predictive panel suggests that overweight men who have been taking antireflux medication for a long time might merit particular consideration for further testing. Our risk prediction panel is quick and simple to administer but will need further calibration and validation in a prospective study in primary care. Funding Charles Wolfson Charitable Trust and Guts UK.

Primary Aldosteronism (PA) is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60–80% of aldosterone-producing adenomas (APA). We undertook WES on 39 consecutive APAs in search of further variants. 1 APA revealed a somatic mutation (Val380Asp) within the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). An adjacent mutation (Gly379Asp) was discovered on WES from a PA patient in Munich. Both short and long isoforms (442 & 453 residues) of wild-type (WT) and both mutant CADM1 genes were cloned into lentivirus vectors and each transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion and other parameters. Previous studies in pancreatic islet cells suggested a role of CADM1 in regulating gap junction (GJ) communication. To assess this we microinjected single WT or mutant H295R cells with the GJ permeable dye calceinAM and counted the dye-positive cells after 1 hour. The effect of inhibiting or silencing GJs in H295R cells using peptide gap27 or a Dharmacon smartpool was assessed. H295R cells were also co-transfected with WT or mutant CADM1 and the GJ protein CX43, tagged with the mApple fluorophore. These were mixed with cells transfected with CX43-Venus, allowing confocal visualisation of GJ formation. Protein modelling was undertaken to determine whether Asp in the intramembranous domain changes angulation of CADM1. All mutant isoforms had consistently different effects, shown as a range compared to WT. Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (p Protein modelling showed mutations to increase the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2nm to 24.7 and 27.9nm. Mixing of Venus and mApple-tagged CX43 transfected cells showed fewer intact GJ channels in cells co-transfected with mutant compared to WT CADM1 [mutant 42/291 (14.4%) VS WT 68/212 (32.1%) p The CADM1 mutations shows the importance of membrane proteins in aldosterone regulation to extend beyond ion channels and transporters. A key role may be to bring opposing CX43 hemichannels close enough to form GJ channels, permitting the oscillating Ca2+ currents which regulate aldosterone in intact adrenal slices.

Supplemental Digital Content is available in the text., Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10−24) was ANO4, a putative Ca2+-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca2+-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10−7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca2+-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.

Heterogeneity among aldosterone-producing adenomas (APAs) has been highlighted by the discovery of somatic mutations. KCNJ5 mutations predominate in large zona fasciculata (ZF)–like APAs; mutations in CACNA1D , ATP1A1, ATP2B3 , and CTNNB1 are more likely to be found in small zona glomerulosa (ZG)–like APAs. Microarray comparison of KCNJ5 mutant versus wild-type APAs revealed significant differences in transcriptomes. NEFM , encoding a neurofilament subunit which is a D1R (dopamine D1 receptor)–interacting protein, was 4-fold upregulated in ZG-like versus ZF-like APAs and 14-fold more highly expressed in normal ZG versus ZF. Immunohistochemistry confirmed selective expression of NEFM (neurofilament medium) polypeptide in ZG and in ZG-like APAs. Silencing NEFM in adrenocortical H295R cells increased basal aldosterone secretion and cell proliferation; silencing also amplified aldosterone stimulation by the D1R agonist, fenoldopam, and inhibition by the D1R antagonist, SCH23390. NEFM coimmunoprecipitated with D1R, and its expression was stimulated by fenoldopam. Immunohistochemistry for D1R was mainly intracellular in ZG-like APAs but membranous in ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZF-like APAs was higher than in cells from ZG-like APAs. Transfection of mutant KCNJ5 caused a large reduction in NEFM expression in H295R cells. We conclude that NEFM is a negative regulator of aldosterone production and cell proliferation, in part by facilitating D1R internalization from the plasma membrane. Downregulation of NEFM in ZF-like APAs may contribute to a D1R/D2R imbalance underlying variable pharmacological responses to dopaminergic drugs among patients with APAs. Finally, taken together, our data point to the possibility that ZF-like APAs are in fact ZG in origin.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) CaV1.3. Using a novel antagonist of CaV1.3, compound 8, we investigated the role of CaV1.3 on steroidogenesis in the human adrenocortical cell line, H295R and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, CaV1.2, over CaV1.3. In H295R cells transfected with wild-type or mutant CaV1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 μM of compound 8. In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 μM). Selective CaV1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of CaV1.2-blockade and provides targeted treatment for ZG-like APAs with mutations of CaV1.3.

5081Background: The effect of novel drugs can be studied in primary prostate cancer (PC), if given prior to radical prostatectomy (RP). Altered PI3K/AKT/mTOR signalling is associated with aggressiv...

97 Background: Additional systemic therapy given to unselected patients (pts) around RP has yet to improved pt survival. Further trials should use appropriate pt selection, i.e. men at highest risk of relapse, ideally biomarker stratified. Using novel drugs prior to RP allows direct study of effects in primary prostate cancer (PC) that may guide design of future trials. Altered PI3K/AKT/mTOR pathway signalling is associated with aggressive PC hence we have undertaken a study vistusertib, an oral, dual mTORC1/mTORC2 inhibitor prior to RP. Methods: Primary endpoint was to measure mTOR1/2 inhibition by immunohistochemistry (IHC). Secondary endpoints were feasibility, safety, tolerability and determining plasma concentrations of vistusertib. Exploratory objectives included measurement of anti-cancer effects. Men, due for RP, with high volume or aggressive PC consented and received vistusertib, 50mg bd, for 15 days prior to RP. Diagnostic biopsy and RP tissue were collected for IHC. Adverse events (AE) were graded according to CTCAE v4. Plasma was collected to determine vistusertib concentrations. Results: 21 men, median age 62 (range 50 – 69) included 11 intermediate and 10 high risk PC. 18/21 were evaluable for primary endpoint. On day of RP mean plasma vistusertib concentration at RP was 648 ng/mL (range 386-852) and 6-8 hr post dose 484 ng/mL (range 211-660). Majority of related AEs were Grade (Gr) 1, the most common being rash, thrombocytopenia, oral mucositis, diarrhoea, lymphopenia and fatigue. Six pts experienced Gr3 AEs (3 pt lymphopenia, 1 pt each rash, mucositis and ALT rise). Two pts discontinued due to AEs (mucositis, thrombocytopenia) and one withdrew consent. Surgery was delayed in one pt (Gr 1 thrombocytopenia). IHC confirmed at RP: 4EBP1 was reduced in 100% pt (75% no staining post vistusertib) and pS6 was reduced in 67%, not changed in 25% and increased in 8%. A fall in PSA occurred in 4/18 pts. Conclusions: 2 weeks of AZD2014 can be given prior to RP. Active plasma concentrations were achieved. mTOR pathway inhibition was confirmed on IHC and a PSA fall occurred in 22% of pt. Analysis is ongoing, to be informed by concomitant studies in a PTEN null PC model. Clinical trial information: NCT02064608.

Clinical studies have shown the improved benefit of combining androgen deprivation therapy (ADT) with radiotherapy, indicating a potential interaction between the androgen signalling and DNA damage response (DDR) pathways in PCa. We have explored the underlying basis of this interaction by interrogating the mechanism of androgen regulation of the DDR in men with PCa, both untreated and treated with an androgen inhibitor. We show that androgen receptor (AR) signalling regulates the DDR and promotes DDR foci formation leading to ATM activation in PCa. Our data showing that AR regulates DDR provides an explanation for the improved outcome of combined ADT and radiotherapy. Thus, our work identifies the rationale for targeting both the AR and DDR pathways simultaneously to improve therapy outcome in advanced or high risk prostate cancer. Citation Format: Mohammad Asim, Heather Zecchini, Firas Tarish, Charlie Massie, Ajoeb Baridi, Anne Warren, Wanfeng Zhao, Leigh-Anne McDuffus, Patrice Mascalchi, Greg Shaw, Harveer Dev, Karan Wadhwa, Paul Wijnhoven, Josep Forment, Scott Lyons, Andy Lynch, Cormac O’Neill, Vincent Zecchini, Paul Rennie, Aria Baniahmad, Simon Tavare, Ian Mills, Niklas Schulz, Yaron Galanty, Thomas Helleday, David Neal. The human androgen receptor regulates DNA damage response (DDR) in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-176.

Resistance to androgen deprivation therapy (ADT) and the development of castration resistant prostate cancer (CRPC) remains a major challenge in the treatment of advanced prostate cancer (PCa). Elucidation of resistance mechanisms leading to CRPC is essential to develop novel therapeutic interventions. Using a systems biology approach, we interrogated the androgen-regulated proteome and identified a mitotic Kinase as a novel androgen regulated kinase in PCa. The kinase was shown to be androgen-regulated in PCa and its nuclear localisation significantly increased in CRPC and was associated with biochemical relapse. Interestingly, the Kinase interacted directly with the amino terminus transactivation and carboxy terminus ligand binding domain of androgen receptor (AR), resulting in stabilisation of AR protein and its activity. The AR-driven global transcriptional program was regulated by this coactivator kinase including pathways involved in tumour invasion and metastasis. The inhibition of the kinase function decreased AR levels and activity, and slowed growth of PCa cell lines and human PCa explants. Thus the mitotic kinase is a novel AR-Stablised kinase with potential for clinical applications as both a candidate biomarker of PCa progression and a therapeutic target in advanced disease. Citation Format: Mohammad Asim, Charlie Massie, Anne Warren, Katarina Luko, Brinder Chohan, Suraj Menon, Ajoeb Baridi, Folake Orafidiya, Wanfeng Zhao, Carlos Escriu, Hisham Mohammed, Clive D’Santos, Xiaoping Yang, Chris Taylor, Arham Qureshi, Kate Watt, Vincent Zecchini, Luke Selth, Scott Dehm, Ian Mills, Jason Carroll, Wayne Tilley, Iain McEwan, Aria Baniahmad, David Neal. Androgen-regulated proteome reveals a therapeutically relevant androgen receptor coactivator target in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-003.