Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.National Institutes of Health (U.S.) (grant R01 AI087879

Un enfoque fisiológico para los procesos oceánicos y los cambios glaciares-interglaciares del CO2 atmosférico

18 pages, 6 figures, 1 table[EN] One possible path for exploring the Earth’s far-from-equilibrium homeostasis is to assume that it results from the organisation of optimal pulsating systems, analogous to that in complex living beings. Under this premise it becomes natural to examine the Earth’s organisation using physiological-like variables. Here we identify some of these main variables for the ocean’s circulatory system: pump rate, stroke volume, carbon and nutrient arterial-venous differences, inorganic nutrients and carbon supply, and metabolic rate. The stroke volume is proportional to the water transported into the thermocline and deep oceans, and the arterial-venous differences occur between recently-upwelled deep waters and very productive high-latitudes waters, with atmospheric CO2 being an indicator of the arterial-venous inorganic carbon difference. The metabolic rate is the internal-energy flux (here expressed as flux of inorganic carbon in the upper ocean) required by the system’s machinery, i.e. community respiration. We propose that the pump rate is set externally by the annual cycle, at one beat per year per hemisphere, and that the autotrophic ocean adjusts its stroke volume and arterial-venous differences to modify the internal-energy demand, triggered by long-period astronomical insolation cycles (external-energy supply). With this perspective we may conceive that the Earth’s interglacial-glacial cycle responds to an internal organisation analogous to that occurring in living beings during an exercise-recovery cycle. We use an idealised double-state metabolic model of the upper ocean (with the inorganic carbon/nutrients supply specified through the overturning rate and the steady-state inorganic carbon/nutrients concentrations) to obtain the temporal evolution of its inorganic carbon concentration, which mimics the glacial-interglacial atmospheric CO2 pattern[ES] Un posible camino para el estudio de la homeóstasis fuera-de-equilibrio de la tierra es suponer que resulta de la organización de sistemas pulsátiles optimizados, análoga a aquélla en seres vivos complejos. Bajo esta premisa parece natural examinar la organización de la tierra utilizando variables de tipo fisiológico. Aquí identificamos algunas de las principales variables del sistema circulatorio oceánico: tasa de bombeo del corazón, volumen de latido, diferencias arteriovenosas de carbono y nutrientes, suministro de carbono y nutrientes inorgánicos, y tasa metabólica. El volumen de latido es proporcional al transporte de agua hacia la termoclina y océano profundo, y las diferencias arterio-venosas ocurren entre las aguas profundas recientemente afloradas y aquellas altamente productivas de altas latitudes, con el CO2 atmosférico siendo un indicador de la diferencia arterio-venosa de carbono inorgánico. La tasa metabólica es el flujo de energía interna (aquí expresado como flujo de carbono inorgánico en el océano superior) requerido por la maquinaria que sostiene el sistema, i.e. respiración total de la comunidad. Se propone que la tasa de latido está impuesta externamente, un latido por año por hemisferio, y que el océano autotrófico ajusta su volumen de latido y las diferencias arteriovenosas a cambios en la demanda de energía interna, inducido por ciclos de insolación astronómica de largo período (suministro de energía externa). Bajo esta perspectiva podemos concebir que el ciclo interglacial-glacial de la tierra responde a una organización interna análoga a la que ocurre en seres vivos durante un ciclo de ejercicio-recuperación. Se utiliza un modelo metabólico idealizado de dos estados para el océano superior (con el suministro de carbono/nutrientes inorgánicos especificado mediante la tasa de recirculación de aguas profundas y las concentraciones de carbono/nutrientes inorgánicos en estado estacionario) para obtener la evolución temporal de su concentración de carbono inorgánico, la cual mimetiza el patrón glacial-interglacial del CO2 atmosféricoThis work was supported by the Spanish government through the CANOA project (CTM2005-00444/MAR)Peer reviewe

The potential of marginal coastal nursery habitats for the conservation of a culturally important Caribbean marine species

Aim: Identifying the potential of marginal habitats for species conservation is of key importance when their core high-quality habitats are under substantial disturbances and threats. However, there is currently a knowledge gap on how useful marine marginal habitats may be for conserving endangered marine species. Here, we investigate the potential of groundwater-fed coastal areas for the conservation of the queen conch, an economically and culturally important marine gastropod. Location: The inlet of Xel-Ha, typical of groundwater-fed coastal areas widely distributed along the Yucatan Peninsula coast in Mexico and partially protected by a network of marine protected areas. Methods: We tracked 66 queen conchs (Lobatus gigas) using acoustic telemetry over a period of 3.5 years. We investigated for ontogenetic niche shift using a network analysis and by modelling their growth. Results: The queen conchs exhibited the same ontogenetic niche shift required to complete their life cycle in this marginal habitat as they do in offshore core habitats. A total of 33 individuals departed the inlet and migrated from shallow groundwater-affected nursery grounds to deeper marine habitats more suitable for breeding aggregation. Main conclusions: As the broad-scale movement behaviour of queen conch in this inlet is similar to that observed on the overfished core habitats, our findings suggest that groundwater-fed coastal areas should be included in conservation planning for an effective management of this species within a network of marine protected areas

Downregulation of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular carcinoma and their prognostic significance

<p>Abstract</p> <p>Background</p> <p>Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates and detoxification of xenobiotics and chemotherapeutics. The transporters became pharmaceutically interesting, because OCTs are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. No data exist about the relevance of OCTs in hepatocellular carcinoma (HCC).</p> <p>Methods</p> <p>OCT1 (<it>SLC22A1</it>) and OCT3 (<it>SLC22A3</it>) mRNA expression was measured in primary human HCC and corresponding non neoplastic tumor surrounding tissue (TST) by real time PCR (n = 53). Protein expression was determined by western blot analysis and immunofluorescence. Data were correlated with the clinicopathological parameters of HCCs.</p> <p>Results</p> <p>Real time PCR showed a downregulation of <it>SLC22A1 </it>and <it>SLC22A3 </it>in HCC compared to TST (p ≤ 0.001). A low <it>SLC22A1 </it>expression was associated with a worse patient survival (p < 0.05). Downregulation was significantly associated with advanced HCC stages, indicated by a higher number of T3 tumors (p = 0.025) with a larger tumor diameter (p = 0.035), a worse differentiation (p = 0.001) and higher AFP-levels (p = 0.019). In accordance, <it>SLC22A1 </it>was less frequently downregulated in tumors with lower stages who underwent transarterial chemoembolization (p < 0.001) and liver transplantation (p = 0.001). Tumors with a low <it>SLC22A1 </it>expression (< median) showed a higher <it>SLC22A3 </it>expression compared to HCC with high <it>SLC22A1 </it>expression (p < 0.001). However, there was no significant difference in tumor characteristics according to the level of the <it>SLC22A3 </it>expression.</p> <p>In the western blot analysis we found a different protein expression pattern in tumor samples with a more diffuse staining in the immunofluorescence suggesting that especially OCT1 is not functional in advanced HCC.</p> <p>Conclusion</p> <p>The downregulation of OCT1 is associated with tumor progression and a worse patient survival.</p