Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs

Highly variable response to cytotoxic chemotherapy in carcinoma-associated fibroblasts (CAFs) from lung and breast

<p>Abstract</p> <p>Background</p> <p>Carcinoma-associated fibroblasts (CAFs) can promote carcinogenesis and tumor progression. Only limited data on the response of CAFs to chemotherapy and their potential impact on therapy outcome are available. This study was undertaken to analyze the influence of chemotherapy on carcinoma-associated fibroblasts (CAFs) <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>The <it>in vivo </it>response of stromal cells to chemotherapy was investigated in 22 neoadjuvant treated breast tumors on tissue sections before and after chemotherapy. Response to chemotherapy was analyzed <it>in vitro </it>in primary cultures of isolated CAFs from 28 human lung and 9 breast cancer tissues. The response was correlated to <it>Mdm2</it>, <it>ERCC1 </it>and <it>TP53 </it>polymorphisms and <it>TP53 </it>mutation status. Additionally, the cytotoxic effects were evaluated in an <it>ex vivo </it>experiment using cultured tissue slices from 16 lung and 17 breast cancer specimens.</p> <p>Results</p> <p>Nine of 22 tumors showed a therapy-dependent reduction of stromal activity. Pathological response of tumor or stroma cells did not correlate with clinical response. Isolated CAFs showed little sensitivity to paclitaxel. In contrast, sensitivity of CAFs to cisplatinum was highly variable with a GI50 ranging from 2.8 to 29.0 μM which is comparable to the range observed in tumor cell lines. No somatic <it>TP53 </it>mutation was detected in any of the 28 CAFs from lung cancer tissue. In addition, response to cisplatinum was not significantly associated with the genotype of <it>TP53 </it>nor <it>Mdm2 </it>and <it>ERCC1 </it>polymorphisms. However, we observed a non-significant trend towards decreased sensitivity in the presence of <it>TP53 </it>variant genotype. In contrast to the results obtained in isolated cell culture, in tissue slice culture breast cancer CAFs responded to paclitaxel within their microenvironment in the majority of cases (9/14). The opposite was observed in lung cancer tissues: only few CAFs were sensitive to cisplatinum within their microenvironment (2/15) whereas a higher proportion responded to cisplatinum in isolated culture.</p> <p>Conclusion</p> <p>Similar to cancer cells, CAF response to chemotherapy is highly variable. Beside significant individual/intrinsic differences the sensitivity of CAFs seems to depend also on the cancer type as well as the microenvironment.</p

Systematic review on the recurrence of postoperative nausea and vomiting after a first episode in the recovery room – implications for the treatment of PONV and related clinical trials

BACKGROUND: Despite the presence of a plethora of publications on the prevention of postoperative nausea and vomiting (PONV) only little is known how to treat established symptoms. Besides the high effort of performing these efficacy trials (much more patients must give their consent than are actually included in a study) and ethical concerns, little is known about the rate of re-occurring PONV/vomiting after placebo. As a consequence investigators will have difficulties defining a clinically relevant effect for the new treatment which is crucial for any planning. A quantitative systematic review was performed in order to provide more reliable estimates of the incidence of re-occurring PONV/vomiting after placebo and to help investigators defining a clinically relevant treatment effect. METHODS: A systematic search of the literature was performed using an extended search strategy of a previous review. Data on the recurrence of PONV (any nausea or emetic symptom) and vomiting (retching or vomiting) was extracted from published reports treating PONV with placebo and unpublished results from two observational trials where no treatment was given. A nonlinear random effects model was used to calculate estimates of the recurrence of symptoms and their 95%-confidence intervals (95%-CI). RESULTS: A total of 29 trials (including the unpublished data) were eligible for the calculations. Depending on the length of observation after administering placebo or no treatment the recurrence rate of PONV was between 65% (95%-CI: 53%...75%) and 84% (95%-CI: 73%...91%) and that of vomiting was between 65% (95%-CI: 44%...81%) and 78% (95%-CI: 59%...90%). CONCLUSION: Almost all trials showed a considerable and consistently high rate of recurrence of emetic symptoms after placebo highlighting the need for a consequent antiemetic treatment. Future (placebo) controlled efficacy trials may use the presented empirical estimates for defining clinically relevant effects and for statistical power considerations

+Fièr : une application mobile pour aider les jeunes issus de la communauté LGBTQ+ et leur famille

Les personnes LGBTQ+ sont 1,5 à 4 fois plus à risque que les personnes hétérosexuelles de souffrir de troubles de santé mentale (p. ex. dépression, anxiété, comportements suicidaires).Objectif L’objectif est de décrire le processus de développement d’une application mobile pour les jeunes LGBTQ+ et leur famille. L’article fait partie d’un programme de recherche ayant comme but général de doter les jeunes LGBTQ+ et leur famille d’outils technologiques leur permettant de développer et soutenir des stratégies d’adaptation face à la stigmatisation. En effet, ceux-ci sont confrontés à des stresseurs uniques, à la fois dans la sphère publique (p. ex. victimisation) et personnelle (p. ex. développement identitaire et le processus de coming out).Méthode Nous adapterons l’application mobile +Fort © développée par l’équipe dirigée par de la docteure Isabelle Ouellet-Morin, conçue pour soutenir les jeunes vivant de l’intimidation à réduire ces expériences, et nous créerons +Fièr/+ Proud, qui fera l’objet d’un codesign et d’une étude pilote auprès de participants LGBTQ+ âgés de 13 à 25 ans.Retombées À terme, notre espoir est que les jeunes LGBTQ+, à l’échelle nationale et internationale, puissent explorer et développer des stratégies d’adaptation soutenant leur mieux-être, apprendre à l’aide d’outils personnalisés, partager leurs expériences uniques et informer leurs proches des défis auxquels ils font face et se battent en silence.LGBTQ+ people are anywhere from 1.5 to 4 times more likely than heterosexual people to report depression, anxiety, suicidal behaviors, substance abuse, eating disorders, risky sexual behaviors, homelessness, and victimization.Objective The purpose is to describe the development of a mobile application for LGBTQ youth and their family. This article is part of a research program intended to equip LGBTQ+ youth and their families with technological tools to help them foster adaptive strategies in the face of stigma. LGBTQ+ youth face unique stressors both publicly (e.g. victimization) as well as personally (e.g. identity development and “coming out” process).Method We build upon Isabelle Ouellet-Morin’s team +Fort: Stronger than Bullying © mobile application designed to reduce victimization among youth. We will create a new app called +Fièr/+Proud, to be designed and piloted in collaboration with LGBTQ+ participants ages 13-25 and their families.Impact Our hope is to bring LGBTQ+ youth together nationally and internationally to explore health promoting coping strategies, learn from custom training modules, share their unique experiences, and help inform parents of the experiences that LGBTQ+ people often face and fight in silence

Inverse agonistic action of 3-iodothyronamine at the human trace amine-associated receptor 5.

Application of 3-iodothyronamine (3-T1AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T1AM. However, 3-T1AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T1AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct Gs-, Gi/o-, G12/13-, Gq/11- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T1AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the Gq/11 pathway but show differences in the basal activity in Gs and MAP kinase signaling. In contrast to mTaar5, 3-T1AM application at hTAAR5 resulted in significant reduction in basal IP3 formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T1AM, exhibiting inhibitory effects on IP3 formation and MAP kinase signaling pathways, but does not mediate Gs signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T1AM-mediated effects may differ between rodents and humans

MAP kinase activation and G_s signaling parameters of wild type TAAR5 and chimeric receptors.

<p><b>(A)</b> HEK293 cells expressing mouse or human TAAR5 or chimeric receptors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117774#pone.0117774.t002" target="_blank">Table 2</a> for details) were stimulated with 100 μM DMEA. The cAMP accumulation was measured by competitive cAMP assay based on AlphaScreen technology. Results are depicted as either fold over basal mock or fold over DMEA stimulated mock transfection. Data are shown as mean ± SEM from n ≥ 3 independent experiments with three or more replicates. Statistical analyses were carried out with an unpaired two-tailed Welch-corrected t-test; ***p ≤ 0.001, compared to the respective basal activity. <b>(B)</b> MAP kinase activation was reported by luciferase activity in a luciferase reporter gene assay (SRE-luc). HEK293 cells were co-transfected with a reporter construct containing a serum response element and the firefly luciferase reporter gene, and the different receptor constructs. Cells were stimulated with 10 μM 3-T₁AM and SRE-luc levels were determined. Results are presented as mean ± SEM as either fold over basal mock transfection for basal value or fold over 3-T₁AM-stimulated mock. An unpaired two-tailed Welsh-corrected t-test was performed for statistical analyses; *p ≤ 0.05.</p

Signaling pathways, basal activity and stimulation with 3-T₁AM at mouse and human TAAR5.

<p>Signaling pathways, basal activity and stimulation with 3-T₁AM at mouse and human TAAR5.</p

Signaling parameters of human and murine TAAR5 after treatment with 3-T₁AM.

<p><b>(A)</b> HEK293 cells expressing human TAAR1 were stimulated with 10 μM 3-T₁AM. For G_s signal determination cAMP accumulation was measured. Results are depicted as fold over basal mock or fold over 3-T₁AM-stimulated mock. Data is shown as mean ± SEM from n ≥ 3 independent experiments with 3 or more replicates. 3-T₁AM is a potent agonist for hTAAR1 (**p < 0.01). Statistical analysis was carried out with an unpaired two-tailed Welch-corrected t-test. <b>(B)</b> HEK293 cells transiently expressing hTAAR5 or mTaar5 were stimulated with 10 μM 3-T₁AM and IP3-luc levels were determined. Results are presented as either fold over basal mock transfection for basal value or fold over 3-T₁AM stimulated mock. An unpaired two-tailed Welsh-corrected t-test was used for statistical analyses, **p ≤ 0.01. Data are obtained from 3 to 6 independent experiments measured in at least triplicates and are shown as mean ± SEM. <b>(C)</b> Human TAAR5 was stimulated with 3-T₁AM concentrations ranging from 1 nM to 100 μM. The concentration-dependent IP3-luc signaling curve indicated the inverse agonism of 3-T₁AM at hTAAR5 with an EC₅₀ value of 4.4 ± 0.9 μM. <b>(D)</b> MAP kinase activation was determined by luciferase activity in a luciferase reporter gene assay (SRE-luc). HEK293 cells were co-transfected with a reporter construct containing a serum response element linked to the firefly luciferase reporter gene and in combination with the different receptor constructs, respectively. Cells were stimulated with 10 μM 3-T₁AM and SRE-luc levels were determined. Results are presented as mean ± SEM as either fold over basal mock transfection for basal value or fold over 3-T₁AM-stimulated mock. An unpaired two-tailed Welsh-corrected t-test was performed for statistical analyses; *p ≤ 0.05. <b>(E)</b> Cell surface expression studies of hTAAR5 were conducted in COS-7 cells for 6 hours after stimulation with or without 10 μM 3-T₁AM using an ELISA. Results are depicted as mean ± SEM obtained from 3 independent assays measured in 4 replicates. Data are presented as fold over basal hTAAR5. An unpaired two-tailed t-test with Welch-correction was performed.</p

Expression of mTaar1 and mTaar5 in various murine brain regions.

<p>Transcript expression studies were analyzed by <i>in situ</i> hybridization using a LNA (locked nucleic acid) probe. C57BL/6 mouse brains were sectioned and treated with the corresponding LNA probes. Signals were visualized by an avidin-biotin complex using DY-light 488 streptavidin <b>(A-F)</b> or DAB (3,3’-diaminobenzidin) staining <b>(G-M)</b>. III = third ventricle; OT = optical tract; DY-light 488 streptavidin labeled samples are shown with a 40-fold, DAB stained sections are depicted with a 20-fold magnification. Bar scale in (B) equals 100 μm, bar scale in (K) equals 200 μm. <b>(A-F)</b>: mTaar5 expression; <b>(A), (C)</b> and <b>(E)</b> represent negative controls using a scrambled LNA probe showing homogenous staining. <b>(B), (D)</b> and <b>(F)</b> show expression of mTaar5 in arcuate nucleus (ARC), in ventromedial hypothalamus (VMH) and amygdala, respectively. <b>(G-M)</b>: mTaar1 expression, the brain regions of interest are highlighted by circles. <b>(G)</b>, <b>(I)</b> and (<b>L)</b> represent negative controls using a scrambled LNA probe. <b>(K)</b> and <b>(M)</b> show expression of mTaar1 in mice brains in VMH and amygdala, respectively. <b>(H)</b> No expression could be detected in the ARC.</p