Quantitative changes in intracellular calcium and extracellular-regulated kinase activation measured in parallel in CHO cells stably expressing serotonin (5-HT) 5-HT2A or 5-HT2C receptors

<p>Abstract</p> <p>Background</p> <p>The serotonin (5-HT) 2A and 2C receptors (5-HT_2AR and 5-HT_2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (<it>Ca_i</it>⁺⁺) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.</p> <p>Results</p> <p>We optimized the <it>Ca_i</it>⁺⁺assay for stable cell lines expressing either 5-HT_2AR or 5-HT_2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT_2AR- and 5-HT_2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT_2AR-expressing cells showed significant release of <it>Ca_i</it>⁺⁺in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT_2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.</p> <p>Conclusions</p> <p>These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT_2AR and 5-HT_2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.</p

Differential Expression of Alpha 4 Integrins on Effector Memory T Helper Cells during Bordetella Infections. Delayed Responses in Bordetella pertussis

Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, a respiratory disease that is reemerging worldwide. Mechanisms of selective lymphocyte trafficking to the airways are likely to be critical in the immune response to this pathogen. We compared murine infection by B. pertussis, B. parapertussis, and a pertussis toxin-deleted B. pertussis mutant (BpΔPTX) to test the hypothesis that effector memory T-helper cells (emTh) display an altered pattern of trafficking receptor expression in B. pertussis infection due to a defect in imprinting. Increased cell recruitment to the lungs at 5 days post infection (p.i.) with B. parapertussis, and to a lesser extent with BpΔPTX, coincided with an increased frequency of circulating emTh cells expressing the mucosal-associated trafficking receptors α4β7 and α4β1 while a reduced population of these cells was observed in B. pertussis infection. These cells were highly evident in the blood and lungs in B. pertussis infection only at 25 days p.i. when B. parapertussis and BpΔPTX infections were resolved. Although at 5 days p.i., an equally high percentage of lung dendritic cells (DCs) from all infections expressed maturation markers, this expression persisted only in B. pertussis infection at 25 days p.i. Furthermore, at 5 days p.i with B. pertussis, lung DCs migration to draining lymph nodes may be compromised as evidenced by decreased frequency of CCR7+ DCs, inhibited CCR7-mediated in vitro migration, and fewer DCs in lung draining lymph nodes. Lastly, a reduced frequency of allogeneic CD4+ cells expressing α4β1 was detected following co-culture with lung DCs from B. pertussis-infected mice, suggesting a defect in DC imprinting in comparison to the other infection groups. The findings in this study suggest that B. pertussis may interfere with imprinting of lung-associated trafficking receptors on T lymphocytes leading to extended survival in the host and a prolonged course of disease

Bioavailability of Macro and Micronutrients Across Global Topsoils: Main Drivers and Global Change Impacts

Understanding the chemical composition of our planet\u27s crust was one of the biggest questions of the 20th century. More than 100 years later, we are still far from understanding the global patterns in the bioavailability and spatial coupling of elements in topsoils worldwide, despite their importance for the productivity and functioning of terrestrial ecosystems. Here, we measured the bioavailability and coupling of thirteen macro- and micronutrients and phytotoxic elements in topsoils (3–8 cm) from a range of terrestrial ecosystems across all continents (∼10,000 observations) and in response to global change manipulations (∼5,000 observations). For this, we incubated between 1 and 4 pairs of anionic and cationic exchange membranes per site for a mean period of 53 days. The most bioavailable elements (Ca, Mg, and K) were also amongst the most abundant in the crust. Patterns of bioavailability were biome-dependent and controlled by soil properties such as pH, organic matter content and texture, plant cover, and climate. However, global change simulations resulted in important alterations in the bioavailability of elements. Elements were highly coupled, and coupling was predictable by the atomic properties of elements, particularly mass, mass to charge ratio, and second ionization energy. Deviations from the predictable coupling-atomic mass relationship were attributed to global change and agriculture. Our work illustrates the tight links between the bioavailability and coupling of topsoil elements and environmental context, human activities, and atomic properties of elements, thus deeply enhancing our integrated understanding of the biogeochemical connections that underlie the productivity and functioning of terrestrial ecosystems in a changing world

Exploration of Synthetic Approaches and Pharmacological Evaluation of PNU-69176E and Its Stereoisomer as 5-HT_2C Receptor Allosteric Modulators

Allosteric modulators of the serotonin (5-HT) 5-HT_2C receptor (5-HT_2CR) present a unique drug design strategy to augment the response to endogenous 5-HT in a site- and event-specific manner with great potential as novel central nervous system probes and therapeutics. To date, PNU-69176E is the only reported selective positive allosteric modulator for the 5-HT_2CR. For the first time, an optimized synthetic route to readily access PNU-69176E (<b>1</b>) and its diastereomer <b>2</b> has been established in moderate to good overall yields over 10 steps starting from commercially available picolinic acid. This synthetic approach not only enables a feasible preparation of a sufficient amount of <b>1</b> for use as a reference compound for secondary pharmacological studies, but also provides an efficient synthesis of key intermediates to develop novel and simplified 5-HT_2CR allosteric modulators. Compound <b>1</b> and its diastereomer <b>2</b> were functionally characterized in Chinese hamster ovary (CHO) cells stably transfected with the 5-HT_2CR using an intracellular calcium (Ca_<i>i</i>²⁺) release assay. Compound <b>1</b> demonstrated efficacy and potency as an allosteric modulator for the 5-HT_2CR with no intrinsic agonist activity. Compound <b>1</b> did not alter 5-HT-evoked Ca_<i>i</i>²⁺ in CHO cells stably transfected with the highly homologous 5-HT_2AR. In contrast, the diastereomer <b>2</b> did not alter 5-HT-evoked Ca_<i>i</i>²⁺ release in 5-HT_2AR-CHO or 5-HT_2CR-CHO cells or exhibit intrinsic agonist activity

Functional analyses of lung DCs derived from <i>Bordetella</i> infections.

<p>(<b>A</b>) <b>Transwell CCR7 dependent migration of lung DCs</b>. 5×10⁵ isolated lung DCs from <i>Bordetella</i> infections were placed in the transwell upper chamber and allowed to migrate through a 5 µm polycarbonate membrane in the presence or absence of chemokines. Migrated cells were harvested, stained for CD11c & MHC-II, and enumerated by flow cytometry. Chemotaxis index data are calculated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052903#s2" target="_blank">Materials and Methods</a>. Depicted are combined data of three experiments. In each experiment, five mice were used per treatment group. *: p<0.05 as analyzed by a two-tailed student's <i>t</i>-test. (<b>B</b>) <b>Enumeration of CD11c⁺ cells in the mediastinal lymph nodes.</b> The mediastinal lymph nodes were harvested, stained with CD11c and CD45, and enumerated with Trubeads by flow cytometry. The absolute number of CD11c⁺ cells from each treatment was normalized to that from <i>B. pertussis</i> (1.0 by definition) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052903#s2" target="_blank">Materials and Methods</a>. The bar chart represents compiled data with error bars showing the SEM of three independent experiments. In each experiment, three mice were used per treatment group. *: p<0.05, and no asterisk indicates p<0.05 as analyzed by the one sample two-tailed student's <i>t</i>-test.</p

Characterization of lung CD11c⁺ cells during infection with <i>B. pertussis</i> and <i>B. parapertussis</i>.

<p>(<b>A</b>) Enumeration of lung CD11c⁺ cells during the early phase of infection with <i>Bordetella sp</i>. Mononuclear cells were isolated from lungs and enriched for DCs (CD11c⁺) as detailed. After column enrichment, the total number of CD11c⁺ cells per mouse was estimated using a hemocytometer. (<b>B</b>) CD11c⁺ enriched lung mononuclear cells were gated on singlets and then on CD11c⁺ cells (DCs). The frequency of CD11c⁺ cells expressing maturation markers was assessed at 5 days p.i. (<b>C</b>) and at 25 days p.i. (<b>D</b>), and normalized to the respective cells in uninfected control mice (1.0 by definition). In addition, the frequency of lung CD11c⁺ cells expressing CCR7 and CXCR4 at 5 days p.i. was measured (<b>E</b>). The bar charts (<b>C, D, E</b>) represent compiled data with error bars showing the SEM of four independent experiments. In each experiment, six mice were used per treatment group. *: p<0.05, and no asterisk indicates p<0.05 as analyzed by a two-tailed student's <i>t</i>-test.</p

Antibodies used to stain T cells.

<p>Antibodies used to stain T cells.</p

α4β7 and α4β1 imprinting on allogeneic Thy1.1⁺/CD4⁺/CD38⁺⁺ cells.

<p>Lung DCs from 5 days p.i. with one of four infection types—<i>B. pertussis</i>, <i>B. parapertussis</i>, <i>BpΔPTX</i>, or uninfected controls—were co-cultured for 4 days with naïve allogeneic Thy1.1⁺/CD4⁺ splenocytes at a 1∶5 ratio. (<b>A</b>) Lung DCs from <i>Bordetella</i> infections were used to stimulate purified naïve Thy1.1⁺/CD4⁺ splenocytes labeled with CFSE. Histograms depict CFSE dilution profiles. Approximately four cell cycles of division are observed in each system as estimated by FlowJo proliferation analysis. Low mRMS values obtained in every allogeneic system confirmed the accuracy of the analysis. (<b>B</b>) Co-cultured cells were gated on lymphocytes, followed by gating on singlets, and then on Thy1.1⁺/CD4⁺/CD38⁺⁺ cells. (<b>C</b>) Proportion of Thy1.1⁺/CD4⁺/CD38⁺⁺ cells expressing α4β7 or α4β1 following co-culture with lung DCs derived from <i>Bordetella</i> infections was measured and normalized to the respective cells from the uninfected control co-culture (1.0 by definition). The bar charts represent compiled data with error bars showing the SEM of three independent experiments. In each experiment, five mice were used per treatment group. *: p≤0.05, no asterisk: p<0.05.</p

<i>B. pertussis</i> induces a longer course of infection than <i>B. parapertussis</i>.

<p>BALB/c mice were infected intranasally with 5×10⁶ CFU of <i>Bordetella</i> strains in 20 µl of PBS, or mock infected (as detailed in the Methods section). Bacterial load in the lungs was assessed at the time points shown above. 3 mice were used at each time point. This graph represents data from two independent experiments.</p

Antibodies used to stain dendritic cells.

<p>Antibodies used to stain dendritic cells.</p