Targeting of the P2X7 receptor in pancreatic cancer and stellate cells

The ATP‐gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu‐1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu‐1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7(‐/‐) animals. PancTu‐1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120‐treated mice showed reduced bioluminescence compared to saline‐treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120‐treated tumours

From aptamer-based biomarker discovery to diagnostic and clinical applications: an aptamer-based, streamlined multiplex proteomic assay

Recently, we reported an aptamer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community

NK Cell Levels Correlate with Disease Activity in Patients with Multiple Sclerosis on Ocrelizumab/Rituximab Therapy

Background: Recently, research on the pathogenesis of multiple sclerosis (MS) has focused on the role of B lymphocytes and the possibility of using specific drugs, such as Ocrelizumab and Rituximab, directed toward these cells to reduce inflammation and to slow disease progression. Objective: We aimed to evaluate the effect of Ocrelizumab/Rituximab on laboratory immune parameters and identify the predictors of treatment responses. Methods: A retrospective single-center study was conducted among patients who received infusion therapy with an anti-CD20 drug to treat MS. Results: A total of 64 patients met the inclusion criteria, with 277 total cycles of therapy studied. Compared with the baseline values, anti-CD20 infusions resulted in absolute-value and percentage decreases in B lymphocyte levels and increased the absolute and percentage levels of NK cells 3 and 5 months after therapy (p < 0.001). After multivariate logistic regression analysis, a reduced percentage level of NK cells 3 months after infusion could predict disease activity 6 months after Ocrelizumab/Rituximab administration (p = 0.041). Conclusions: Lower percentage levels of NK cells 3 months after anti-CD20 infusion correlate with the presence of disease activity 6 months after therapy, confirming a possible protective role of NK cells in MS

From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community

A New Conceptual Model for Musical Sources and Musicological Studies

We present a new multi-layered, conceptual model for associating musical source materials to musicological arguments. We describe our proposal for operationalizing these concepts through a framework for musical annotation which we have implemented using RDF. Briefly stated, this model shows how portions of digitized data in various files and formats can be identified, selected, labelled, and compared

Estimadores robustos de autocorrelación espacial basados en la varianza muestral

La Geoestadística se ha convertido en una herramienta muy útil en las ciencias de la tierra. La aplicaremos para estudiar las características espaciales de las rocas reservorios de petróleo, desde la perspectiva de la ingeniería de reservorios. El comportamiento espacial de la permeabilidad y de la porosidad se describe en este trabajo mediante la autocorrelación. Una medida tradicional de la autocorrelación es el semivariograma. En este trabajo se proponen dos nuevos estimadores de la autocorrelación espacial. El primero es LV (Varianza Local), basado en la varianza de la distribución. Se deduce que LV es también un estimador del valor medio del semivariograma dentro de una región. LV es muy robusto y resistente a los valores extremos. Es también muy suave y por eso fácil de ser representado por un modelo teórico. El segundo, SLV (Semivariograma desde la Varianza Local), está relacionado con las derivadas del primero. Es un estimador del semivariograma propiamente dicho. LV y SLV se aplican a conjuntos de datos reales y sintéticos. Sus resultados se comparan con los de otros estimadores clásicos e integrales. LV y SLV se comportan mejor que el estimador clásico punto a punto de Matheron, que es impreciso a distancias de separación moderadas a grandes. Además se comportan de manera similar a los estimadores integrales de Li y Lake, siendo su principal ventaja el tiempo de cómputo, que es tres veces menor

Connexin-43 channels are a novel pathway for discharging lactate from glycolytic pancreatic ductal adenocarcinoma cells

Glycolytic cancer cells produce large quantities of lactate which must be removed in order to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H+ -coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility of other venting mechanisms becoming important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was high between Colo357 cells, lower yet hypoxiainducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with expression of Cx43, a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H+ ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150µm). MiaPaCa2 cells, which are Cx43-negative in monolayer culture, showed markedly increased Cx43-immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma) which can explain the advantage of junctional transmission over MCT in vivo. We propose that connexin43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Additionally, lactate entry into the better-perfused recipient cells has a favourable alkalinizing effect and supplies substrate for oxidative phosphorylation. Connexin43 is thus a novel target for influencing metabolite handling in junctionally-coupled tumours