effects-taste-signaling-protein-abolishment-on-gut-inflammation-an

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Zhejiang University

Monell Chemical Senses Center

We characterized the genomic structure and developmental expression of the Dleu7 (deleted in lymphocytic leukemia, 7) gene in Xenopus tropicalis and the evolution of the gene across species. Within the protein-coding sequence (CDS) region, X. tropicalis Dleu7 consists of two exons and one intron. However, bioinformatic analysis indicates that this 211-amino-acid protein contains no obvious functional domains or known motifs. Reverse-transcription polymerase chain reaction and whole-mount in situ hybridization results revealed that, in addition to its expression in the blood island region, some regions of the central nervous system, and subdomains of the neural tube, X. tropicalis Dleu7 is zygotically expressed primarily in mesoderm tissues such as notochord and muscles during early embryogenesis. Expression in notochord is consistent with results from genome-wide association studies suggesting that DLEU7 is related to human adult height. Expression in the blood island region, where blood cell precursors (including B cells) are generated, implies a potential conserved role for Dleu7 in B-cell development between amphibians and mammals. Expression of Dleu7 in some regions of the central nervous system and subdomains of the neural tube also suggests other functions in development. Phylogenetic analysis indicated that Dleu7 is a vertebrate-specific gene and undergoes strong selective pressure in lower vertebrates but is functionally constrained in higher mammals. When subcellular localization was examined by overexpression of enhanced green fluorescent protein fusion protein, Dleu7 showed centrosome localization with main distribution in cytoplasm. Treating gastrula embryos with SU5402, a small molecular inhibitor of the fibroblast growth factor (FGF) receptor, confirmed that Dleu7 expression in mesoderm is regulated by FGF signaling. Our data provide important clues for pathogenesis and physiology during development from the perspective of evolutionary conservation.

Characterization and expressional analysis of Dleu7 during Xenopus tropicalis embryogenesis.

It is highly desirable to obtain functional cells with specific olfactory receptors (ORs) for the development of cell-based biosensors towards odorant detection. In this study, we explored the feasibility of bioengineered primary olfactory sensory neurons (OSNs) as sensing elements of biomimetic olfactory-based biosensors, in which light addressable potentiometric sensor (LAPS) was used to monitor bioengineered OSNs membrane potential responses to odorant molecules. An olfactory receptor of C. elegances, ODR-10, as a model receptor, was expressed on the plasma membrane of OSNs by transient transfection. The response profile of bioengineered OSNs to odorant molecules was investigated by analyzing extracellular potential firings features in frequency and time domains. The results indicated that bioengineered OSNs can specifically respond to diacetyl, the natural ligand of ODR-10. In addition, bioengineered OSNs showed different temporal firing patterns in responding to different concentrations of diacetyl. All the results demonstrate that bioengineered OSNs are useful and promising to serve as novel sensing elements of biosensors for specific odorant molecule detection. It is suggested that bioengineering techniques could provide novel approaches for preparing sensitive elements as well as promoting the development of practical applicable olfactory-based biosensors.

Bioengineered olfactory sensory neuron-based biosensor for specific odorant detection.

The biological olfactory system can recognize and discriminate thousands of volatile organic compounds (VOCs) with extremely high sensitivity and specificity. The most fundamental elements are olfactory receptors (ORs) in the cilia of olfactory sensory neurons (OSNs), which contribute greatly to the high-performance olfactory system. The excellent properties of ORs are generally recognized in the development of biomimetic OR-based biosensors. Over the past two decades, much work has been done in developing OR-based biosensors due to their promising potential in many applications. In this article, we will outline the latest advances of OR-based biosensors. Two current crucial issues in this field will be discussed, namely, the production methods and immobilization techniques of ORs. We will also elaborate on various OR-based biosensors and their latest developments. Finally, current research trends and future challenges in this field will be discussed.

Recent advances in olfactory receptor-based biosensors.

It is of substantial interest to mimic mechanisms of biological sensing systems for the development of novel biosensors. This paper presents a novel biomimetic bitter receptor-based biosensor for the detection of specific bitter substances, in which bitter receptors were used as sensitive elements for the first time. A simple and practical self-assembled aptamer-based strategy was proposed for functional immobilization and purification of bitter receptors. A human bitter receptor, T2R4, was expressed on the plasma membrane of HEK-293 cells and fused with a His6-tag on its C-terminal. The membrane fractions containing the expressed T2R4 were extracted and immobilized on the gold surface of a quartz crystal microbalance (QCM) pretreated with a monolayer of self-assembled aptamers that can specifically recognize and capture biomolecules labeled with His6-tags. The QCM device was used to monitor the responses of T2R4 to various bitter stimuli. The results indicate that this biosensor can detect denatonium with high sensitivity and specificity, which is the specific target of T2R4. In addition, this biosensor shows dose-dependent responses to a certain concentration range of denatonium. The sensitivity of bitter receptor-based biosensors prepared by an aptamer-based method is 1.21 kHz mM(-1), which is 2 times higher than that prepared by a SAM-based method. The major advances on bitter receptor immobilization and purification presented in this work could substantially be very useful for developing other membrane receptor-based biosensors and molecular sensor arrays. This bitter receptor-based biosensor has great potential to be used as a valuable tool for bitter detection as well as for the research of taste signal transduction. 

A biomimetic bitter receptor-based biosensor with high efficiency immobilization and purification using self-assembled aptamers.

New methods for functional assays of chemical receptors are highly essential for the research of chemical signal transduction mechanisms and for the development of chemical biosensors. This study described a novel bioengineered cell-based biosensor for label-free functional assays of chemical receptors by localized extracellular acidification measurement with a light-addressable potentiometric sensor (LAPS). A human taste receptor, hT2R4, and an olfactory receptor of Caenorhabditis elegans (C. elegans), ODR-10, were selected as models of chemical receptors, which were expressed on the plasma membrane of human embryonic kidney (HEK)-293 cells. The specific ligand binding function of expressed chemical receptors was monitored by localized extracellular acidification measurement using LAPS chip with a movable focused laser illuminating on the desired single cell. The function of expressed olfactory receptors was further validated using MDL12330A, which can specifically inhibit the activity of adenylyl cyclase. The obtained results indicate that both of chemical receptors were successfully expressed in HEK-293 cells and can be functionally assayed by this bioengineered cell-based biosensor that shows dose-dependent responses to the target ligands of chemical receptors. This bioengineered cell-based biosensor exhibits the sensitivity of 1.0 mV/s for hT2R4 assays, and 9.8 mV/s for ODR-10 assays. The negative control cells without any chemical receptor expression show no response to all the chemical stimuli tested. All the results demonstrate this bioengineered cell-based biosensor can be used to detect the interactions between chemical receptors and their ligands. This provides a valuable and promising approach for label-free functional assays of chemical receptors as well as for the research of other GPCRs.

Label-free functional assays of chemical receptors using a bioengineered cell-based biosensor with localized extracellular acidification measurement.

5-hydroxytryptamine (5-HT) is an important neurotransmitter in regulating emotions and related behaviors in mammals. To detect and monitor the 5-HT, effective and convenient methods are demanded in investigation of neuronal network. In this study, hippocampal neuronal networks (HNNs) endogenously expressing 5-HT receptors were employed as sensing elements to build an in vitro neuronal network-based biosensor. The electrophysiological characteristics were analyzed in both neuron and network levels. The firing rates and amplitudes were derived from signal to determine the biosensor response characteristics. The experimental results demonstrate a dose-dependent inhibitory effect of 5-HT on hippocampal neuron activities, indicating the effectiveness of this hybrid biosensor in detecting 5-HT with a response range from 0.01μmol/L to 10μmol/L. In addition, the cross-correlation analysis of HNNs activities suggests 5-HT could weaken HNN connectivity reversibly, providing more specificity of this biosensor in detecting 5-HT. Moreover, 5-HT induced spatiotemporal firing pattern alterations could be monitored in neuron and network levels simultaneously by this hybrid biosensor in a convenient and direct way. With those merits, this neuronal network-based biosensor will be promising to be a valuable and utility platform for the study of neurotransmitter in vitro. 

Detection of 5-hydroxytryptamine (5-HT) in vitro using a hippocampal neuronal network-based biosensor with extracellular potential analysis of neurons.

In many sensory organs, specialized receptors are strategically arranged to enhance detection sensitivity and acuity. It is unclear whether the olfactory system utilizes a similar organizational scheme to facilitate odor detection. Curiously, olfactory sensory neurons (OSNs) in the mouse nose are differentially stimulated depending on the cell location. We therefore asked whether OSNs in different locations evolve unique structural and/or functional features to optimize odor detection and discrimination. Using immunohistochemistry, computational fluid dynamics modeling, and patch clamp recording, we discovered that OSNs situated in highly stimulated regions have much longer cilia and are more sensitive to odorants than those in weakly stimulated regions. Surprisingly, reduction in neuronal excitability or ablation of the olfactory G protein in OSNs does not alter the cilia length pattern, indicating that neither spontaneous nor odor-evoked activity is required for its establishment. Furthermore, the pattern is evident at birth, maintained into adulthood, and restored following pharmacologically induced degeneration of the olfactory epithelium, suggesting that it is intrinsically programmed. Intriguingly, type III adenylyl cyclase (ACIII), a key protein in olfactory signal transduction and ubiquitous marker for primary cilia, exhibits location-dependent gene expression levels, and genetic ablation of ACIII dramatically alters the cilia pattern. These findings reveal an intrinsically programmed configuration in the nose to ensure high sensitivity to odors.

An Olfactory Cilia Pattern in the Mammalian Nose Ensures High Sensitivity to Odors.

The olfactory system is a natural biosensor since its peripheral olfactory sensory neurons (OSNs) respond to the external stimuli and transmit the signals to the olfactory bulb (OB) where they are integrated and processed. The axonal connections from the OSNs expressing about 1000 different types of odorant receptors are precisely organized and sorted out onto 1800 glomeruli in the OB, from which the olfactory information is delivered to and perceived by the central nervous system. This process is carried out with particularly high sensitivity, specificity and rapidity, which can be used for explosive detection. Biomimetic olfactory biosensors use various biological components from the olfactory system as sensing elements, possessing great commercial prospects. In this study, we utilized the genetically labeled murine M72 olfactory sensory neurons with the green fluorescent protein (GFP) as sensing components and obtained long-term in vivo electrophysiological recordings from the M72 OSNs by implanting the microelectrode arrays (MEAs) into the behaving mouse's OB. The electrophysiological responses showed high reliability, reproducibility and specificity for odor detection, and particularly, the high sensitivity for the detection of odorants that contain benzene rings. Furthermore, our results indicated that it can detect trinitrotoluene (TNT) in liquid at a concentration as low as 10M and can distinguish TNT from other chemicals with a similar structure. Thus our study demonstrated that the in vivo biomimetic olfactory system could provide novel approaches to enhancing the specificity and increasing working lifespan of olfactory biosensors capable of detecting explosives.

In vivo bioelectronic nose using transgenic mice for specific odor detection.

Taste and smell are very important chemical senses that provide indispensable information on food quality, potential mates and potential danger. In recent decades, much progress has been achieved regarding the underlying molecular and cellular mechanisms of taste and odor senses. Recently, biosensors have been developed for detecting odorants and tastants as well as for studying ligand-receptor interactions. This review summarizes the currently available biosensing approaches, which can be classified into two main categories: in vitro and in vivo approaches. The former is based on utilizing biological components such as taste and olfactory tissues, cells and receptors, as sensitive elements. The latter is dependent on signals recorded from animals' signaling pathways using implanted microelectrodes into living animals. Advantages and disadvantages of these two approaches, as well as differences in terms of sensing principles and applications are highlighted. The main current challenges, future trends and prospects of research in biomimetic taste and odor sensors are discussed.

Biomimetic Sensors for the Senses: Towards Better Understanding of Taste and Odor Sensation.

Inner ear hair cells are mechanoreceptors responsible for hearing. Pathogenic defects of hair cell-specific genes are one of the major causes of deafness. The BarH class homeobox gene Barhl1 is a deafness gene expressed in developing hair cells, yet the role of Barhl1 during hair cell development remains poorly understood. In the present study, we first established an in vitro differentiation system to efficiently obtain mouse embryonic stem cell (mESC)-derived hair cell-like cells. Subsequently, a mESC line carrying a targeted disruption of Barhl1 was generated using CRISPR/Cas9 technology and subjected to the established in vitro hair cell differentiation protocol. Targeted disruption of Barhl1 does not affect the induction of mESCs toward early primitive ectoderm-like (EPL) cells and otic progenitors but strongly inhibits the differentiation of hair cell-like cells. Using RNA-sequencing and bioinformatics, we further unravel the molecular mechanism underlying Barhl1-mediated hair cell development. Our data demonstrate the essential role of Barhl1 during hair cell development and provide a basis for the treatment of Barhl1 mutation-based deafness.

Barhl1 is required for the differentiation of inner ear hair cell-like cells from mouse embryonic stem cells.

Corrigendum to "Barhl1 is required for the differentiation of inner ear hair cell-like cells from mouse embryonic stem cells" [Int. J. Biochem. Cell Biol. 96 (2018) 79-89].

Inflammatory bowel disease (IBD) is a debilitating immune-related condition that affects over 1.4 million Americans. Recent studies indicate that taste receptor signaling is involved in much more than sensing food flavor, and taste receptors have been localized in a variety of extra-oral tissues. One of the newly revealed functions of taste receptors and downstream signaling proteins is modulation of immune responses to microbes and parasites. We previously found that components of the taste receptor signaling pathway are expressed in subsets of the intestinal epithelial cells. α-Gustducin, a key G-protein α subunit involved in sweet, umami, and bitter taste receptor signaling, is expressed in the intestinal mucosa. In this study, we investigated the role of α-gustducin in regulation of gut mucosal immunity and inflammation using α-gustducin knockout mice in the dextran sulfate sodium (DSS)-induced IBD model. DSS is a chemical colitogen that can cause intestinal epithelial damage and inflammation. We analyzed DSS-induced colitis in α-gustducin knockout versus wild-type control mice after administration of DSS in drinking water. Our results show that the knockout mice had aggravated weight loss, diarrhea, intestinal bleeding, and inflammation over the experimental period compared to wild-type mice, concurrent with augmented immune cell infiltration and increased expression of TNF and IFN-γ but decreased expression of IL-13 and IL-5 in the colon. These results suggest that the taste receptor signaling pathway may play critical roles in regulating gut immune balance and inflammation.

Aggravated gut inflammation in mice lacking the taste signaling protein α-gustducin.

Most olfactory receptors in vertebrates are G protein-coupled receptors, whose activation by odorants initiates intracellular signaling cascades through heterotrimeric G proteins consisting of α, β, and γ subunits. Abolishment of the α subunits such as Gαolf in the main olfactory epithelium and Gαi2 and Gαo in the vomeronasal organ resulted in anosmia and/or impaired behavioral responses. In this study, we report that a G protein γ subunit, Gγ13, is expressed in a spatiotemporal manner similar to those of Gαolf and Gαi2 in the olfactory system and vomeronasal organ, respectively. In addition, Gγ13 was found in the glomeruli of the main olfactory bulb but was largely absent in the glomeruli of the accessory olfactory bulb. Using the Cre-loxP system, the Gγ13's gene Gng13 was nullified in the mature olfactory sensory neurons and apical vomeronasal sensory neurons where the Cre recombinase was expressed under the promoter of the Omp gene for the olfactory marker protein. Immunohistochemistry indicated much reduced expression of Gγ13 in the apical vomeronasal epithelium of the mutant mice. Behavioral experiments showed that the frequency and duration of aggressive encounters in the male mutant mice were significantly lower than in WT male mice. Taken together, these data suggest that the Gγ13 subunit is a critical signaling component in both the main olfactory epithelium and apical vomeronasal epithelium, and it plays an essential role in odor-triggered social behaviors including male-male aggression.

Article	Year
Characterization and expressional analysis of Dleu7 during Xenopus tropicalis embryogenesis. Gene\| PubMed ID: 22939871	2012
Bioengineered olfactory sensory neuron-based biosensor for specific odorant detection. Biosensors & bioelectronics\| PubMed ID: 23036770	2013
Recent advances in olfactory receptor-based biosensors. Biosensors & bioelectronics\| PubMed ID: 23261691	2013
A biomimetic bitter receptor-based biosensor with high efficiency immobilization and purification using self-assembled aptamers. The Analyst\| PubMed ID: 23951570	2013
Label-free functional assays of chemical receptors using a bioengineered cell-based biosensor with localized extracellular acidification measurement. Biosensors & bioelectronics\| PubMed ID: 24333934	2014
Detection of 5-hydroxytryptamine (5-HT) in vitro using a hippocampal neuronal network-based biosensor with extracellular potential analysis of neurons. Biosensors & bioelectronics\| PubMed ID: 25530536	2015
An Olfactory Cilia Pattern in the Mammalian Nose Ensures High Sensitivity to Odors. Current biology : CB\| PubMed ID: 26365258	2015
In vivo bioelectronic nose using transgenic mice for specific odor detection. Biosensors & bioelectronics\| PubMed ID: 29128717	2018
Biomimetic Sensors for the Senses: Towards Better Understanding of Taste and Odor Sensation. Sensors (Basel, Switzerland)\| PubMed ID: 29232897	2017
Barhl1 is required for the differentiation of inner ear hair cell-like cells from mouse embryonic stem cells. The international journal of biochemistry & cell biology\| PubMed ID: 29413750	2018
Corrigendum to "Barhl1 is required for the differentiation of inner ear hair cell-like cells from mouse embryonic stem cells" [Int. J. Biochem. Cell Biol. 96 (2018) 79-89]. The international journal of biochemistry & cell biology\| PubMed ID: 29477628	2018
Aggravated gut inflammation in mice lacking the taste signaling protein α-gustducin. Brain, behavior, and immunity\| PubMed ID: 29678794	2018
G protein γ subunit Gγ13 is essential for olfactory function and aggressive behavior in mice. Neuroreport\| PubMed ID: 30157062	2018

Liquan Huang

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