Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers largely due to disseminated disease at the time of presentation. Here, we investigated the role and mechanism of action of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in all sporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, and metastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to the endoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumor cells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasome degradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases [cathepsin B (CTSB) and cathepsin D (CTSD)], in addition to promoting the secretion of the precursor form pro-CTSD. Importantly, the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functional downstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB, and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the results suggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permit new strategies for early detection as well as therapeutic management.

Abstract. In this paper we define the first "Regional Atmosphere and Land" (RAL) science configuration for kilometre scale modelling using the UM and JULES. "RAL1" defines the science configuration of the dynamics and physics schemes of the atmosphere and land. This configuration will provide a model baseline for any future weather or climate model developments to be described against and it is the intention that from this point forward significant changes to the system will be documented in literature. This is reproducing the process used for global configurations of the UM which was first documented as a science configuration in 2011. While it is our goal to have a single defined configuration of the model that performs effectively in all regions, this has not yet been possible. Currently we define two sub-releases, one for mid-latitudes (RAL1-M) and one for tropical regions (RAL1-T). The differences between RAL1-M and RAL1-T are documented and where appropriate, we define how the model configuration relates to the corresponding configuration of the global forecasting model.

Using an integrated approach incorporating proteomics, metabolomics and published mRNA data, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletion mutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysis based on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) is the foundation of the quantitative proteomics approach. This study identifies 260 differentially expressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity of the cellular response to oxidative stress and the dependency on the Sty1p stress-activated protein kinase. We show the relationship between these protein changes and mRNA expression levels identified in a parallel whole genome study, and discuss the regulatory mechanisms involved in protecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-activated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1 H NMR was also conducted alongside the protein analysis using the same sample sets, allowing examination of how the protein changes might affect the metabolic pathways and biological processes involved in the oxidative stress response. This combined analysis identifies a number of interlinked metabolic pathways that exhibit stress-and Sty1-dependent patterns of regulation.

Up-regulation of S100P, a member of the S100 calcium-binding protein family, is an early molecular event in the development of pancreatic cancer and it is expressed at high levels in both precursor lesions and invasive cancer. To gain more insight into the molecular mechanisms underlying the functional roles of this protein, we stably overexpressed S100P in the Panc1 pancreatic cancer cell line and identified the consequent changes in global protein expression by twodimensional difference in-gel electrophoresis. The observed changes in target proteins were confirmed by Western blot analysis and immunofluorescence, whereas their functional effect was investigated using motility and invasion assays. In this study, we have shown that overexpression of S100P led to changes in the expression levels of several cytoskeletal proteins, including cytokeratins 8, 18, and 19. We have also shown disorganization of the actin cytoskeleton network and changes in the phosphorylation status of the actin regulatory protein cofilin. Additionally, we have shown that overexpression of S100P leads to increased expression of another early pancreatic cancer marker, S100A6, as well as the aspartic protease cathepsin D, both of which are involved in cellular invasion. Functional studies showed that the increased invasive potential of S100P-overexpressing cells was at least partially due to the increase in cathepsin D expression. In summary, our data suggest that these changes could contribute to the metastatic spread of pancreatic cancer and may explain the devastating prognosis of this disease. [Cancer Res 2007;67(18):8633-42]

The evolution of pediatric solid tumors is poorly understood. There is conflicting evidence of intra-tumor genetic homogeneity vs. heterogeneity (ITGH) in a small number of studies in pediatric solid tumors. A number of copy number aberrations (CNA) are proposed as prognostic biomarkers to stratify patients, for example 1q + in Wilms tumor (WT); current clinical trials use only one sample per tumor to profile this genetic biomarker. We multisampled 20 WT cases and assessed genome-wide allele-specific CNA and loss of heterozygosity, and inferred tumor evolution, using Illumina CytoSNP12v2.1 arrays, a custom analysis pipeline, and the MEDICC algorithm. We found remarkable diversity of ITGH and evolutionary trajectories in WT. 1q + is heterogeneous in the majority of tumors with this change, with variable evolutionary timing. We estimate that at least three samples per tumor are needed to detect > 95% of cases with 1q +. In contrast, somatic 11p15 LOH is uniformly an early event in WT development. We find evidence of two separate tumor origins in unilateral disease with divergent histology, and in bilateral WT. We also show subclonal changes related to differential response to chemotherapy. Rational trial design to include biomarkers in risk stratification requires tumor multisampling and reliable delineation of ITGH and tumor evolution.

The lack of safe and efficient systemic gene delivery vectors has largely reduced the potential of gene therapy in the clinic. Previously, we have reported that polypropylenimine dendrimer PPIG3/DNA nanoparticles are capable of tumor transfection upon systemic administration in tumor-bearing mice. To be safely applicable in the clinic, it is crucial to investigate the colloidal stability of nanoparticles and to monitor the exact biodistribution of gene transfer in the whole body of the live subject. Our biophysical characterization shows that dendrimers, when complexed with DNA, are capable of forming spontaneously in solution a supramolecular assembly that possesses all the features required to diffuse in experimental tumors through the enhanced permeability and retention effect. We show that these nanoparticles are of sizes ranging from 33 to 286 nm depending on the DNA concentration, with a colloidal stable and well-organized fingerprint-like structure in which DNA molecules are condensed with an even periodicity of 2.8 nm. Whole-body nuclear imaging using small-animal nano-single-photon emission computed tomography/computer tomography scanner and the human Na

BackgroundKveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins.MethodsKv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay.ResultsWe observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4.ConclusionsStimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.

ObjectivesTo describe the rates for consulting a general practitioner (GP) for sequelae after acute covid-19 in patients admitted to hospital with covid-19 and those managed in the community, and to determine how the rates change over time for patients in the community and after vaccination for covid-19.DesignPopulation based study.Setting1392 general practices in England contributing to the Clinical Practice Research Datalink Aurum database.Participants456 002 patients with a diagnosis of covid-19 between 1 August 2020 and 14 February 2021 (44.7% men; median age 61 years), admitted to hospital within two weeks of diagnosis or managed in the community, and followed-up for a maximum of 9.2 months. A negative control group included individuals without covid-19 (n=38 511) and patients with influenza before the pandemic (n=21 803).Main outcome measuresComparison of rates for consulting a GP for new symptoms, diseases, prescriptions, and healthcare use in individuals admitted to hospital and those managed in the community, separately, before and after covid-19 infection, using Cox regression and negative binomial regression for healthcare use. The analysis was repeated for the negative control and influenza cohorts. In individuals in the community, outcomes were also described over time after a diagnosis of covid-19, and compared before and after vaccination for individuals who were symptomatic after covid-19 infection, using negative binomial regression.ResultsRelative to the negative control and influenza cohorts, patients in the community (n=437 943) had significantly higher GP consultation rates for multiple sequelae, and the most common were loss of smell or taste, or both (adjusted hazard ratio 5.28, 95% confidence interval 3.89 to 7.17, P<0.001); venous thromboembolism (3.35, 2.87 to 3.91, P<0.001); lung fibrosis (2.41, 1.37 to 4.25, P=0.002), and muscle pain (1.89, 1.63 to 2.20, P<0.001); and also for healthcare use after a diagnosis of covid-19 compared with 12 months before infection. For absolute proportions, the most common outcomes ≥4 weeks after a covid-19 diagnosis in patients in the community were joint pain (2.5%), anxiety (1.2%), and prescriptions for non-steroidal anti-inflammatory drugs (1.2%). Patients admitted to hospital (n=18 059) also had significantly higher GP consultation rates for multiple sequelae, most commonly for venous thromboembolism (16.21, 11.28 to 23.31, P<0.001), nausea (4.64, 2.24 to 9.21, P<0.001), prescriptions for paracetamol (3.68, 2.86 to 4.74, P<0.001), renal failure (3.42, 2.67 to 4.38, P<0.001), and healthcare use after a covid-19 diagnosis compared with 12 months before infection. For absolute proportions, the most common outcomes ≥4 weeks after a covid-19 diagnosis in patients admitted to hospital were venous thromboembolism (3.5%), joint pain (2.7%), and breathlessness (2.8%). In patients in the community, anxiety and depression, abdominal pain, diarrhoea, general pain, nausea, chest tightness, and tinnitus persisted throughout follow-up. GP consultation rates were reduced for all symptoms, prescriptions, and healthcare use, except for neuropathic pain, cognitive impairment, strong opiates, and paracetamol use in patients in the community after the first vaccination dose for covid-19 relative to before vaccination. GP consultation rates were also reduced for ischaemic heart disease, asthma, and gastro-oesophageal disease.ConclusionsGP consultation rates for sequelae after acute covid-19 infection differed between patients with covid-19 who were admitted to hospital and those managed in the community. For individuals in the community, rates of some sequelae decreased over time but those for others, such as anxiety and depression, persisted. Rates of some outcomes decreased after vaccination in this group.