Map-independent representation of an aggression-promoting social cue in the main olfactory pathway

While the olfactory system is required for proper social behaviors, the molecular basis for how social cues are detected via the main olfactory pathway of mammals is not well-characterized. Trimethylamine is a volatile, sex-specific odor found in adult male mouse urine that selectively activates main olfactory sensory neurons that express trace amine-associated receptor 5 (TAAR5). Here we show that trimethylamine, acting via TAAR5, elicits state-dependent attraction or aversion in male mice and drives inter-male aggression. Genetic knockout of TAAR5 significantly reduces aggression-related behaviors, while adding trimethylamine augments aggressive behavior towards juvenile males. We further show that transgenic expression of TAAR5 specifically in olfactory sensory neurons rescues aggressive behaviors in knockout mice, despite extensive remapping of TAAR5 projections to the olfactory bulb. Our results identify a specific main olfactory input that detects a prominent male-specific odor to induce inter-male aggression in a mammalian species and reveal that apparently innate behavioral responses are independent of patterned glomerular input to the olfactory bulb.

A Physiologically Based Pharmacokinetic (PBPK) Model of Vitamin B-6 Metabolism in the Mouse Incorporated With Visualization of Complex Compartmental Models

Objectives To develop a compartmental model which describes vitamin B-6 metabolism in the blood and tissues of the mouse under a variety of circumstances and to develop a computer application which can utilize the data to display the movement of tracer through the various compartments. Methods A circulation model was developed. Literature data from vitamin B-6 feeding studies and tracer experiments were used to develop an appropriate compartmental model using the SAAM II program (The Epsilon Group, Charlottesville, VA). A web-based application transforms the mass and flux data into intuitive and interactive graphical illustrations. Results Describing the interconversions between pyridoxine, pyridoxine 5’-phosphate, pyridoxal, pyridoxal 5’-phosphate, pyridoxamine, pyridoxamine 5’-phosphate and 4-pyridoxic acid in multiple tissues required 231 compartments. The largest amount of data deals with liver and brain. The model includes less detailed information on plasma, erythrocytes, gut, bone, muscle, heart, kidney, skin, adipose tissue and lung. The model includes adjustments to food intake, water intake, cardiac output, binding sites and Vmax values for enzymes based on the specified body weight of experimental animals. We did not include growth curves at this time. The model uses two parallel systems to monitor the steady state of endogenous metabolites as well as following tracer administration. Binding mechanisms are included to provide conservation of vitamin B-6 when intake is reduced. The model provides reasonable agreement with literature data on various vitamin B-6 intakes as well as oral and intravenous administration of tracer. It also reveals some areas which need clarification. For example, we have not found any detailed analysis of vitamin B-6 metabolites in mouse urine. There is little pyridoxic acid in plasma or urine suggesting that pyridoxic acid may not be the primary end product for vitamin B-6 in the mouse. The visualization application shows changes in the content of all 231 compartments over time illustrating the value of such computer applications in the interpretation of large, complex models. Conclusions This model facilitates the simulation of various dietary and physiological conditions on vitamin B-6 metabolism in mice. We hope to adapt it to rats, pigs and humans. Funding Sources Purdue University Fort Wayne.

Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion

The diversity and near universal expression of G protein-coupled receptors (GPCR) reflects their involvement in most physiological processes. The GPCR superfamily is the largest in the human genome, and GPCRs are common pharmaceutical targets. Therefore, uncovering the function of understudied GPCRs provides a wealth of untapped therapeutic potential. We previously identified an adhesion-class GPCR, Gpr116, as one of the most abundant GPCRs in the kidney. Here, we show that Gpr116 is highly expressed in specialized acid-secreting A-intercalated cells (A-ICs) in the kidney using both imaging and functional studies, and we demonstrate in situ receptor activation using a synthetic agonist peptide unique to Gpr116. Kidney-specific knockout (KO) of Gpr116 caused a significant reduction in urine pH (i.e., acidification) accompanied by an increase in blood pH and a decrease in pCO2compared to WT littermates. Additionally, immunogold electron microscopy shows a greater accumulation of V-ATPase proton pumps at the apical surface of A-ICs in KO mice compared to controls. Furthermore, pretreatment of split-open collecting ducts with the synthetic agonist peptide significantly inhibits proton flux in ICs. These data suggest a tonic inhibitory role for Gpr116 in the regulation of V-ATPase trafficking and urinary acidification. Thus, the absence of Gpr116 results in a primary excretion of acid in KO mouse urine, leading to mild metabolic alkalosis (“renal tubular alkalosis”). In conclusion, we have uncovered a significant role for Gpr116 in kidney physiology, which may further inform studies in other organ systems that express this GPCR, such as the lung, testes, and small intestine.

BCG-induced neutrophil extracellular traps formation and its regulatory mechanism

Background: Intravesical BCG is one of the most effective immunotherapies for bladder cancer. Our previous study showed that BCG induces the formation of neutrophil extracellular traps (NETs), which play an important role in bladder tumor treatment. To identify how BCG induced NETs formation, we examined NETs formation induced by BCG in vitro and in a mouse model, then analyzed the effects of NETs on BCG and the relevant regulatory mechanism. Results: We observed the NETs at different time points by Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscopy (SEM), the results found that BCG induced in vitro NETs formation in a time-dependent fashion, which was inhibited by pretreatment with DNase I and protease. FITC-labeled BCG was used to observe capturing by NETs. Interestingly, BCG was trapped but not killed in vitro by NETs, which was different from the effect on Staphylococcus aureus. Moreover, ROS was required for BCG-induced NETs formation, which was regulated by MEK, p38, PI3K, and PKC pathways. Finally, NETs formation was observed in mouse urine and subcutaneous tumors after BCG perfusion. Conclusions: By exploring how BCG induced the formation of NETs and the regulatory mechanism, we conclude that a novel immune reaction involving neutrophils exists in the early stages of BCG treatment.

SO039BETA3-ADRENORECEPTOR AS A NEW PLAYER IN SYMPATHETIC REGULATION OF THE ACID-BASE HOMEOSTASIS IN THE KIDNEY

Background and Aims Sympathetic nervous system has a key role in the regulation of renal function. We previously showed that, in mouse, the β3-adrenoreceptor (β3-AR) localizes in most of the nephron segments and its selective agonism promotes a potent antidiuretic effect. Here, we evaluate the expression of β3-AR in kidney intercalated cells (ICs) which are associated with the regulation of acid-base homeostasis in distal segments of the kidney tubule. The aim of this study is to investigate whether the β3-AR might play a role in the acid-base homeostasis operated by the kidney. Method The β3-AR expression in mouse kidney ICs was investigated by confocal microscopy. Wild type (wt) and β3-AR knock-out (ko) mice were used to study the role of β3-AR in the renal acid-base homeostasis. Mouse urine were collected using metabolic cages and pH was measured. Immunofluorescence and Western blotting experiments were performed to evaluate the localization and the abundance of renal H+-ATPase in wt and β3-AR ko mice. Kidney cells expressing human β3-AR (M1-β3-AR) and endogenous H+-ATPase were used to investigate the possible effects of β3-AR stimulation on the activity/localization of H+-ATPase. Live imaging experiments using the pH-sensitive dye BCECF were carried out to determine intracellular pH and assess H+-ATPase activity. Immunofluorescence experiments were performed to reveal the effects of β3-AR activation on H+-ATPase localization. Results Co-localization study of β3-AR with either H+-ATPase or Cl−/HCO3− exchanger pendrin in mouse kidney showed that β3-AR is expressed in H+-secreting type A, in HCO3— secreting type B, and in non-A non-B ICs. The urine pH of β3-AR ko mice was significantly higher compared with wt mice. In line with these results, localization and expression analysis showed that renal H+-ATPase significantly decreased in β3-AR ko mice compared to wt mice, supporting the idea that H+ secretion is partially blunted in these animals. Of note, exposure of M1-β3-AR cells to a selective β3-AR agonist induced a 2.5 fold increase of H+-ATPase activity compared to resting cells and this effect was prevented by a selective β3-AR antagonist or by the H+-ATPase inhibitor bafilomycin. Moreover, β3-AR agonism enhanced H+-ATPase apical expression in M1-β3-AR cells. In addition, the PKA inhibitor H89 abolished the stimulatory effect of β3-AR agonism, demonstrating the involvement of the cAMP/PKA pathway. Conclusion The present data suggest that modulation of H+-ATPase activity in renal ICs by endogenous β3-AR agonists may play an addition physiological role in hormonal control of renal acid-base homeostasis.

The Microbial Biotransformation of Soy Genistein Significantly Enhances Its Trapping Capacities of Reactive Carbonyl Metabolites

Objectives Carbonyl stress is the abnormal accumulation of carbonyl metabolites, such as methylglyoxal (MGO) and acrolein (ACR) that leads to increased modification of protein, lipids and DNA, and contributes to cell and tissue dysfunction resulting in aging and diseases, such as diabetes, cardiovascular diseases and neurodegenerative diseases. Carbonyl stress is caused by an imbalance in the formation and metabolism of carbonyl metabolites and also by increased exposure to exogenous carbonyl species. In vitro studies have shown that dietary flavonoids have the capacities to detoxify reactive carbonyl metabolites. However, whether flavonoids can trap carbonyl metabolites in vivo and whether biotransformation especially microbial metabolism limits the trapping capacities of flavonoids remain virtually unknown. The objective of this study is to use soy genistein as an example to test the impacts of bioavailability and biotransformation on the in vivo trapping capacities of RCS by genistein. Methods Chemically, we synthesized the MGO and ACR conjugates of genistein as authentic standards. In mice, we oral gavaged 200 mg/kg genistein or vehicle to mice. Urine and feces were collected in metabolic cages for 24 h. The urine samples from genistein treated mice were also used to prepare the RCS conjugates of genistein metabolites. Using LC tandem mass and the high-resolution accurate mass, we searched and identified the formation of genistein metabolites and their corresponding RCS conjugates. The RCS conjugates of genistein and its metabolites were also quantified using the synthetic standards. Results We found that 1) absorbed genistein trapped endogenous MGO and ACR by forming mono-RCS adducts and eventually be excreted into mouse urine; 2) absorbed genistein could produce active phase I metabolite, orobol, to scavenge endogenous MGO and ACR; and 3) considerable amounts of microbial metabolites of genistein displayed enhanced anti-RCS capacity both in the body and in the gut, compared to genistein. Conclusions Our findings demonstrate that in vivo anti-RCS ability of dietary polyphenols cannot be reflected solely based on their in vitro ability. The bioavailability and biotransformation of individual polyphenols especially gut microbiome contribute to in vivo anti-RCS ability of dietary polyphenols. Funding Sources N/A.

Analysis of Total Thiols in the Urine of a Cystathionine β-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography

Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine β-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.

BCG induced neutrophil extracellular traps formation and its regulatory mechanism

Background Intravesical BCG is one of the most effective immunotherapies for bladder cancer. Our previous study showed that BCG induces the formation of neutrophil extracellular traps (NETs), which play an important role in bladder tumor treatment. To identify how BCG-induced NETs formation, we examined NETs formation induced by BCG in vitro and in a mouse model, then analyzed the effects of NETs on BCG and the relevant regulatory mechanism. Methods The formation of NETs was visualized using Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscopy (SEM). NETs quantitation was evaluated by the strength of extracellular DNA. Reactive oxygen species (ROS) and NETs formation were determined by co-culturing with inhibitors of ROS, NADPH oxidase, and relevant pathways. FITC–labeled BCG was used to observe capturing by NETs. Finally, NETs formation was observed in mouse urine and subcutaneous tumors after BCG perfusion. Results BCG induced in vitro NETs formation in a time-dependent fashion, as well as urine and subcutaneous tumors in mice, which was inhibited by pretreatment with DNase I and protease. Interestingly, BCG was trapped but not killed in vitro by NETs, which was different from the effect on Staphylococcus aureus. Moreover, ROS was required for BCG-induced NETs formation, which was regulated by star pathways, such as the MEK, p38, PI3K, and PKC pathways. Conclusions By exploring how BCG induced the formation of NETs and the regulatory mechanism, we conclude that a novel immune reaction involving neutrophils exists in the early stages of BCG treatment.

BCG induced neutrophil extracellular traps formation and its regulatory mechanism

Background Intravesical BCG is one of the most effective immunotherapies for bladder cancer. Our previous study showed that BCG could induce the formation of neutrophil extracellular traps (NETs), which play an important role in bladder tumor treatment. To identify how BCG induced NETs formation, in this study, we examined NETs formation induced by BCG in vitro and in mouse model, then analyzed the effects of NETs on BCG, and the relevant regulatory mechanism.Methods The formation of NETs was visualized by Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscopy (SEM). NETs quantitation was evaluated by the strength of extracellular DNA. ROS and NETs formation were examined by co-culturing with the inhibitors of ROS, NADPH oxidase and relevant pathways respectively. FITC–labeled BCG was used to observe the capturing by NETs. Finally, NETs formation was observed in mouse urine and subcutaneous tumor after BCG perfusion.Results BCG time-dependently induced in vitro NETs formation, as well as in urine and subcutaneous tumor of mouse, which could be inhibited by pretreatment with DNase I and protease. Interestingly, BCG was trapped but not killed in vitro by NETs, which was different from the effect on S. aureus. Moreover, ROS was required for BCG-induced NETs formation, which was regulated by star pathways, such as MEK, p38, PI3K and PKC pathways.Conclusions By exploring how BCG induce the formation of NETs and regulatory mechanism, we concluded a novel immune reaction of neutrophils in the early stages of BCG treatment.