Search

SummaryT2R bitter receptors, encoded byTas2rgenes, are not only critical for bitter taste signal transduction but also important for defense against bacteria and parasites. However, little is known about whether and howTas2rgene expression are regulated. Here we show that, in an inflammation model mimicking bacterial infection, the expression of manyTas2rsare significantly up-regulated and mice displayed markedly increased neural and behavioral responses to bitter compounds. Using single-cell assays for transposase-accessible chromatin with sequencing (scATAC-seq), we found that the chromatin accessibility ofTas2rswas highly cell type specific and inflammation increased the accessibility of manyTas2rs. scATAC-seq also revealed substantial chromatin remodeling in immune response genes in taste tissue stem cells, suggesting potential long-term effects. Together, our results suggest an epigenetic mechanism connecting inflammation,Tas2rgene regulation, and altered bitter taste, which may explain heightened bitter taste that can occur with infections and cancer treatments.

Taste perception, initiated by activation of taste receptors in taste bud cells, is crucial for regulating nutrient intake. Genetic polymorphisms in taste receptor genes cannot fully explain the wide individual variations of taste sensitivity. Alternative splicing (AS) is a ubiquitous posttranscriptional mode of gene regulation that enriches the functional diversity of proteins. Here, we report the identification of a novel splicing variant of sweet taste receptor gene Tas1r2 (Tas1r2_∆e4) in mouse taste buds and the mechanism by which it diminishes sweet taste responses in vitro and in vivo. Skipping of Tas1r2 exon 4 in Tas1r2_∆e4 led to loss of amino acids in the extracellular Venus flytrap domain, and the truncated isoform reduced the response of sweet taste receptors (STRs) to all sweet compounds tested by generating nonfunctional T1R2/T1R3 STR heterodimers. The splicing factor PTBP1 (polypyrimidine tract-binding protein 1) promoted Tas1r2_∆e4 generation through binding to a polypyrimidine-rich splicing silencer in Tas1r2 exon 4, thus decreasing STR function and sweet taste perception in mice. Taken together, these data reveal the existence of a regulated AS event in Tas1r2 expression and its effect on sweet taste perception, providing a novel mechanism for modulating taste sensitivity at the posttranscriptional level.

Tissue engineering techniques have enabled to replicate the geometrical architecture of native tissues but usually fail to reproduce their exact cellular arrangements during the fabricating process, while it is critical for manufacturing physiologically relevant tissues. To address this problem, a "sewing-like" method of controlling cellular alignment during the fabricating process is reported here. By integrating the stretching step into the fabricating process, a static mechanical environment is created which, in turn, regulates the subsequent cellular alignment, elongation, and differentiation in the generated tissues. With this method, patterned cellular constructs can be fabricated with controlled cellular alignment. Moreover, this method shows a potent capability to fabricate physiologically relevant skeletal muscle constructs in vitro by mechanically inducing myoblast fusion and maturation. As a potential clinical application, aligned myofibers are directly fabricated onto injured muscles in vivo, which repair the damaged tissues effectively. This study shows that the "sewing-like" method can produce engineered tissues with precise control of cellular arrangements and more clinically viable functionalities. This study was supported by the Major International Cooperation Project of National Natural Science Foundation of China (No. 62120106004) and the Key R&D project of Ministry of Science and Technology of China (No. 2021YFB3200801).

Peripherally inserted central catheters (PICCs) are used for the administration of chemotherapy drugs, including vinorelbine. The present study aimed to construct a rabbit model with vinorelbine administration via PICC, and to dynamically monitor the formation of phlebitis and thrombosis. PICC was inserted into 48 rabbits following specific clinical procedures. The rabbits were randomly divided (n=6 per group) into the following eight groups: i) Control (PICC in place for 1 day); ii) 2nd day of PICC placement (received the first cycle of vinorelbine administration); iii) 3rd day of PICC placement; iv) 7th day of PICC placement; v) 14th day of PICC placement; vi) 21st day of PICC placement; vii) 23rd day of PICC placement (received the second cycle of vinorelbine administration); and viii) 24th day of PICC placement. Hematoxylin and eosin staining was performed on catheter, ear vein and anterior vena specimens. Prothrombin time was measured using an automatic coagulation analyzer, followed by routine blood tests. Serum levels of inflammation- and thrombosis-related factors, including C-reactive protein, D-dimer, interleukin-2, interleukin-6, P-selectin and E-selectin, were measured using ELISAs. X-ray examination confirmed that the rabbit model with vinorelbine administration via PICC was successfully constructed. On the 1st and 23rd day of PICC placement, thrombosis was observed in the catheter. Furthermore, on the 1st day of PICC placement, thrombosis was clearly observed in the ear vein and anterior vena samples. After vinorelbine administration, phlebitis occurred in the ear vein and anterior vena cava samples. With increasing time after vinorelbine administration via PICC, thrombosis and phlebitis were notably ameliorated. Moreover, on the day of vinorelbine administration, prothrombin time was significantly decreased and the serum levels of inflammation- and thrombosis-related factors were significantly increased compared with previous days. Collectively, the present study observed the formation and specific evolution of phlebitis and venous thrombosis after vinorelbine administration, providing a reference for the early prediction, timely prevention and treatment of PICC-related chemotherapy complications.

Purpose The aim of this study is to investigate the prevention and treatment patterns of deep vein thrombosis (DVT) in critically ill patients and to explore the risk factors for DVT in people from Zhejiang Province, China. Materials and methods This study prospectively enrolled patients admitted in intensive care units (ICUs) of 54 hospitals from 09/16/2019 to 01/16/2020. The risk of developing DVT and subsequent prophylaxis was evaluated. The primary outcome was DVT occurrence during ICU hospitalisation. Univariate and multivariate logistic regression were performed to determine the risk factors for DVT. Results A total of 940 patients were included in the study. Among 847 patients who received prophylaxis, 635 (75.0%) patients received physical prophylaxis and 199 (23.5%) patients received drug prophylaxis. Fifty-eight (6.2%) patients were diagnosed with DVT after admission to the ICU, and 36 patients were treated with anticoagulants (all patients received low molecular weight heparin [LMWH]). D-dimer levels (OR = 1.256, 95% CI: 1.132–1.990), basic prophylaxis (OR = 0.092, 95% CI: 0.016–0.536), and physical prophylaxis (OR = 0.159, 95% CI: 0.038–0.674) were independently associated with DVT in ICU patients. The short-term survival was similar between DVT and non-DVT patients. Conclusions DVT prophylaxis is widely performed in ICU patients. Prophylaxis is an independent protective factor for DVT occurrence. The most common treatment of DVT patients is LMWH, although it might increase the rate of bleeding.Key messagesThis is the only multicenter and prospective study of DVT in ICUs in China.d-dimer levels were independently associated with DVT in ICU patients.Prophylaxis was an independent protective factor for DVT occurrence in ICU.

Age-related macular degeneration (AMD), featured with dysfunction and loss of retinal pigment epithelium (RPE), is lacking efficient therapeutic approaches. According to our previous studies, human amniotic epithelial stem cells (hAESCs) may serve as a potential seed cell source of RPE cells for therapy because they have no ethical concerns, no tumorigenicity, and little immunogenicity. Herein, trichostatin A and nicotinamide can direct hAESCs differentiation into RPE like cells. The differentiated cells display the morphology, marker expression and cellular function of the native RPE cells, and noticeably express little MHC class II antigens and high level of HLA-G. Moreover, visual function and retinal structure of Royal College of Surgeon (RCS) rats, a classical animal model of retinal degeneration, were rescued after subretinal transplantation with the hAESCs-derived RPE like cells. Our study possibly makes some contribution to the resource of functional RPE cells for cell therapy. Subretinal transplantation of hAESCs-RPE could be an optional therapeutic strategy for retinal degeneration diseases.

We develop a novel identification method for a partially linear model with multiple endogenous variables of interest but a single instrumental variable, which could even be binary. We present an easy-to-implement consistent estimator for the parametric part. This estimator retains n -convergence rate and asymptotic normality even though we have a generated regressor in our setup. The nonparametric part of the model is also identified. We also outline how our identification strategy can be extended to a fully non-parametric model. Finally, we use our methods to assess the impact of smoking during pregnancy on birth weight.

To evaluate the therapeutic effect of electroacupuncture on acute gastrointestinal injury (AGI) in patients with severe traumatic brain injury (sTBI).A prospective randomized controlled trial was conducted. 126 consecutively hospitalized patients with AGI after sTBI admitted to intensive care unit (ICU) of the First Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine from January 2018 to December 2019 were enrolled. The patients were divided into observation group and control group by random number table. All the patients of two groups were given conventional treatment of western medicine for consecutive 7 days, including the treatments of primary diseases, indwelling nasogastric tube to extract gastric contents every 6 hours to determine gastric residual volume (GRV). When vital signs were basically stable, enteral nutrition (EN) was implemented and EN feeding amount and speed were adjusted according to GRV. On the basis of conventional western medicine treatment, the observation group was treated with electroacupuncture at Zusanli, Tianshu, Shangjuxu, Xiajuxu and Zhongwan, once in the morning and once in the evening, 30 minutes each time. The gastrointestinal function parameters including intra-abdominal pressure (IAP), serum diamine oxidase (DAO) and gastrointestinal failure (GIF) scores were observed before treatment and at day 3 and day 7 of treatment. The incidence of ICU hospital-acquired pneumonia (HAP-ICU), duration of mechanical ventilation (MV), length of ICU stay, 28-day mortality and adverse reactions of electroacupuncture were also observed in the two groups. Kaplan-Meier method was used for 28-day survival analysis.During the 7-day treatment and observation, 26 cases of 126 patients withdrew from the study, and 100 cases were actually enrolled, 50 cases in the observation group and 50 cases in the control group. IAP and DAO at day 3 of treatment in both groups were significantly lower than those before treatment [control group: IAP (cmHElectroacupuncture at corresponding acupoints can effectively improve gastrointestinal function in patients with AGI after sTBI, which is beneficial to shortening the length of ICU stay, promoting the recovery of the patients, and reducing the 28-day mortality.

Airway smooth muscle (ASM) contraction is a major pathophysiological characteristic of asthma. Although β2-adrenoceptor (β2-AR) agonists are currently used as bronchodilators, they cause rapid effect and long-term agonist-induced desensitization. Thus, it is necessary to search for more effective and safer relaxant agents for ASM cells. In this work, bitter taste receptors (TAS2Rs) were demonstrated to be expressed in primary mouse ASM cells endogenously, and they were considered as new drug targets for asthma treatment. Traditional Chinese medicines (TCMs) contained a wide range of TAS2R agonists and some of them had the efficacy of relieving cough and asthma with less toxic side effects. Then the electronic cell-substrate impedance sensor (ECIS) was used for the first time to establish a method to detect the contraction/relaxation effects of ASM cells. Therefore, we introduced a biomimetic in vitro respiratory system using ASM cells on ECIS chips to screen for potential TCMs against asthma. Quinine, nobiletin, and picfeltarraenin IA screened in this study could effectively inhibit the ASM contraction in a concentration-dependent manner, showing potential value as novel anti-asthma drugs. Furthermore, the effective screening of anti-asthma drugs was realized based on 3D ASM cell arrays and gel imaging system. Consistent results were found and the reliability of the biomimetic in vitro respiratory system for the screening of TCMs against asthma was further verified. The biomimetic system designed in this study has the advantages of operation simplicity, high throughput, non-invasive, real-time, and high sensitivity, and therefore provides a promising drug screening platform for asthma disease.

Quiescent stem cells have been found in multiple adult organs, and activation of these stem cells is critical to the restoration of damaged tissues in response to injury or stress. Existing evidence suggests that extrinsic cues from the extracellular matrix or supporting cells of various stem cell niches may interact with intrinsic components to initiate stem cell differentiation, but the molecular and cellular mechanisms regulating their activation are not fully understood. In the present study, we find that olfactory horizontal basal cells (HBCs) are stimulated by neural glial-related cell adhesion molecules (NrCAMs). NrCAM activation requires matrix metalloproteases (MMPs) and epidermal growth factor receptors (EGFRs). Inhibiting MMP activity or EGFR activation not only blocks HBC proliferation in the cultured olfactory organoids, but also severely suppresses HBC proliferation in the olfactory epithelium following methimazole-induced injury, resulting in a delay of olfactory mucosa reconstitution and functional recovery of the injured mice. Both NrCAMs and EGFR are expressed by the HBCs and their expression increases upon injury. Our data indicate that MMP-mediated cleavage of NrCAMs serves as an autocrine or paracrine signal that activates EGFRs on HBCs to trigger HBC proliferation and differentiation to reconstruct the entire olfactory epithelium following injury.

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.

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.

Detecting and modeling structural changes in time series models have attracted great attention. However, relatively little effort has been paid to the testing of structural changes in panel data models despite their increasing importance in economics and finance. In this paper, we propose a new approach to testing structural changes in panel data models. Unlike the bulk of the literature on structural changes, which focuses on detection of abrupt structural changes, we consider smooth structural changes for which model parameters are unknown deterministic smooth functions of time except for a finite number of time points. We use nonparametric local smoothing method to consistently estimate the smooth changing parameters and develop two consistent tests for smooth structural changes in panel data models. The first test is to check whether all model parameters are stable over time. The second test is to check potential time-varying interaction while allowing for a common trend. Both tests have an asymptotic N ( 0 , 1 ) distribution under the null hypothesis of parameter constancy and are consistent against a vast class of smooth structural changes as well as abrupt structural breaks with possibly unknown break points alternatives. Simulation studies show that the tests provide reliable inference in finite samples and two empirical examples with respect to a cross-country growth model and a capital structure model are discussed.

Salicin is a bitter compound with important pharmaceutical values. The detection of salicin in pharmaceutical industry usually employs standard methods of analytical chemistry such as high performance liquid chromatography, which is not suitable for primary detection due to the complicated operation process and expensive systems. In this study, we explore the feasibility of using a label-free bioengineered cell-based biosensor to detect salicin. A human bitter taste receptor, T2R16, which could specifically respond to salicin, was expressed in HEK-293 cells as sensing elements of the biosensor. The structural and functional expression of T2R16 was validated by immunofluorescent staining and calcium imaging. The cell-impedance sensor was utilized as secondary transducer to detected cellular responses to salicin. The cell-impedance response of the bioengineered cells to salicin was investigated by analyzing the response intensity under various concentrations. The cell-impedance results revealed that this bioengineered cells could respond to salicin in a dose-dependent relationship. Finally, the performance of this bioengineered cell-based biosensor was evaluated. In cell-impedance measurement, non-transfected HEK-293 cells were not responsive to salicin, which proved the specificity of the biosensor. The detection of salicin at two unmeasured concentrations in standard response curve also proved this biosensor was sensitive in salicin detection. With the advantage of label-free measurement, this bioengineered may provide a promising and valuable approach for primary and quick detection of salicin.

Hexadecanol (16OH) and hexadecyl acetate (16Ac) are two pheromones secreted in a large quantity by mouse preputial glands and act on male and female mice differentially. Yet the underlying molecular and cellular mechanisms remain to be elucidated. In this study, we examined the activation of vomeronasal sensory neurons (VSNs) by these two pheromones and mapped the downstream neural circuits that process and relay their chemosignals. Using the calcium imaging method and immunohistochemistry, we found that a small number of VSNs were activated by 16OH, 16AC, or both in the male and female mice, most of which were located apically in the vomeronasal epithelium, and their numbers did not increase when the concentrations of 16OH and 16Ac were raised by 10,000-fold except that of female VSNs in response to 16OH. In the accessory olfactory bulb (AOB), the two pheromones evoked more c-Fos+ neurons in the anterior AOB (aAOB) than in the posterior AOB (pAOB); and the increases in the number of c-Fos+ neurons in both aAOB and pAOB were dose-dependent; and between sexes, the female AOB responded more strongly to 16OH than to 16Ac whereas the male AOB had the opposite response pattern. This sexual dimorphism was largely retained in the downstream brain regions, including the bed nucleus of the stria terminalis (BNST), the medial amygdaloid nucleus (MeA), the posteromedial cortical amygdaloid nucleus (PMCo), the medial preoptic area (MPA), and the ventromedial hypothalamic nucleus (VmH). Taken together, out data indicate that there is one V1r receptor each for 16OH, 16Ac, or both, and that activation of these receptors evokes sexually dimorphic neural circuits, directing different behavioral outputs and possibly modulating other pheromone-induced responses.

In this study, we developed a novel bioelectronic taste sensor for the detection of specific bitter substances. A human bitter taste receptor, hT2R4, was efficiently expressed in Escherichia coli (E. coli), which was used as the primary recognition element. A simple and low-cost electrochemical device based on ITO-based electrolyte-semiconductor (ES) structure was innovatively employed as the transducer to assess bacterial metabolic consequences of receptor activation in real time. An apparent increase in extracellular acidification rate was observed, which was resulted from the triggering of hT2R4 receptors by their target ligand of denatonium. The sensor showed dose-dependent responses to denatonuim ranging from 50 nM to 500 nM, while non-bioengineered bacteria without hT2R4 receptors exhibited negligible responses to the same stimulus. In addition, the specificity of the proposed taste biosensor was verified using other typical bitter substances such as quinine and alpha-naphthylthiourea (ANTU). This research provides a simple and inexpensive approach for the construction of bioelectronic taste sensors.

Bitter detection has attracted extensive attention in industries and researches due to pharmacological and/or food safety issues. Common electronic tongues constructed with electrochemical, optical or mass sensors are often subjected to the drawback of low-specificity in detecting a wide range of bitter compounds with diverse chemical structures and functions. However, biological gustatory components present unique specificity to bitter compounds, which may provide a promising approach in bitter detection. The activation of G protein coupled receptors (GPCRs) by ligands will affect cell morphology in a specific manner, which can be monitored by cell-impedance sensor. Thus cells expressing bitter receptors (a subfamily of GPCRs) can be used as sensing material in specific bitter detection. In this study, we explored the feasibility of utilizing male mouse germ cells in testis and cell-based impedance sensors (CIS) to build a cell-based bitter biosensor. Male mouse germ cells express bitter receptor T2Rs, which can sensitively and specifically respond to bitter compounds. To verify bitter sensing capacity of the germ cells, calcium responses of germ cells to three bitter compounds were examined prior to cell impedance response measurement. Furthermore, a bitter receptor blocker, probenecid, was used to verify the specificity of T2R-mediated impedance responses in germ cells. Cell-impedance response profiles of germ cells to four bitter compounds were investigated by analyzing the response intensity under various concentrations. Finally, impedance responses to five basic tastes were examined to evaluate the performance of cell-based bitter biosensor in bitter detection. The results revealed that this hybrid bitter biosensor could respond to those bitter compounds in a dose-dependent manner. The detection threshold for quinine was 0.125 mM, lower than our previous cell-based bitter biosensor. The calcium imaging tests and use of probenecid confirmed that T2R activation contributed to the cell impedance responses of this cell-based biosensor. Moreover, this germ cell-based cell could specifically detect bitter compounds among five taste stimuli, which may provide a promising and valuable approach for detection of various bitter compounds.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit α-gustducin and other signaling proteins, have been found in the intestines. Here, we use α-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the α-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Serotonin or 5-hydroxytryptamine (5-HT) is an important neurotransmitter that is found in mammalian taste buds and can regulate the output of intragemmal signaling networks onto afferent nerve fibers. However, it is unclear how 5-HT is produced, synthesized locally inside taste buds or absorbed from outside sources. In this study, we attempt to address this question by delineating the process of possible 5-HT biosynthesis within taste buds. First, we verified that the rate-limiting enzyme tryptophan hydroxylase (TPH2) responsible for converting L-tryptophan into the intermediate 5-hydroxy-L-tryptophan (5-HTP) is expressed in a subset of type II taste bud cells (TBCs) whereas the enzyme aromatic L-aromatic amino acid decarboxylase (AADC) capable of converting 5-HTP into 5-HT is found in type III TBCs. And abolishment of TPH2 did not affect the production of intragemmal 5-HT or alter TBCs; the mutant mice did not show any changes in behavioral responses to all five primary taste qualities: sweet, umami, bitter, salty, and sour. Then we identified that 5-HTP as well as AADC are abundant in type III TBCs; and application of an AADC inhibitor significantly blocked the production of 5-HT in taste buds. In contrast, administration of an inhibitor on serotonin-reuptake transporters had minimal impact on the 5-HT amount in taste buds, indicating that exogenous 5-HT is not a major source for the intragemmal transmitter. Taken together, our data indicate that intragemmal serotonin is not biosynthesized de novo from tryptophan; instead, it is produced by AADC-mediated conversion of 5-HTP absorbed from the plasma and/or nerve fibers into 5-HT. Thus, our results suggest that the overall bodily 5-HTP level in the plasma and nervous system can regulate taste buds' physiological function, and provide an important molecular mechanism connecting these peripheral taste organs with the circulatory and nervous systems.

ObjectiveTo investigate the effects of postconditioning ischemia on the expressions of the hippocampus neuron autophagy-related proteins LC3-II and Beclin-1 in rats following cerebral ischemia reperfusion.MethodsA total of 128 male Sprague–Dawley rats were randomly divided into 4 groups: control, cerebral ischemia-reperfusion (IR), cerebral ischemia post-conditioning group (IP), and PI3K/Akt inhibitor (LY294002). The rat cerebral ischemia model was established by the improved Pulsinelli four vessel occlusion method. The durations across the platform and escape latent period were recorded using the water maze experiment. The changes in cell morphology and the number of surviving hippocampal neurons were detected by hematoxylin-eosin (HE) staining. The cells with Beclin-1 and LC3-II in the hippocampal region were detected by immunohistochemical staining and Western blotting.ResultsWhen compared with the IR at 48 and 72 h, the number of platform passes increased and the escape latency time was shortened. Consequently, the HE staining detected positive cells with LC3-II and Beclin-1 increased in number at each time point in immunohistochemistry and the expressions of the LC3-II and Beclin-1 proteins were improved in the IP (P < 0.05).ConclusionsCerebral ischemic post-conditioning promoted the expressions of autophagy-related proteins LC3-II and Beclin-1 while relieving the injuries caused by cerebral ischemia reperfusion.

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.

SummaryIn 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.

Background Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methods β2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization. Results Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization. Conclusion Amphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General Significance Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2’s kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.

The N-myc downstream regulated gene (Ndrg) family consists of four main members Ndrg1, 2, 3, and 4. The Ndrg genes are involved in many vital biological events including development. However, comprehensive expression patterns of this gene family during vertebrate embryogenesis remain largely unknown. Here, we analyzed the Ndrg family from the evolutionary perspective and examined the expression patterns of the Ndrg genes during Xenopus tropicalis embryogenesis. Different Ndrg family members of vertebrates are separated into different homology clusters which can be further classified into two groups and each Ndrg family member is well conserved during evolution. The temporal and spatial expression patterns of Ndrg1, 2, 3 and 4 are different during early Xenopus tropicalis development. Ndrg1, 2 and 4 are maternally expressed genes while Ndrg3 is a zygotically expressed gene. The Ndrg genes are differentially expressed in the developing central nervous system, the developing sensory organs, and the developing excretory organs. Moreover, they also show other specific expression domains. Our results indicate that the Ndrg genes exhibit specific expression patterns and may play different roles during vertebrate embryogenesis.