Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

Background:Bitter taste receptors(Tas2rs)are generally considered to sense various bitter compounds to escape the intake of toxic substances.Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo.Methods:To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues,multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique.A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes.Then,T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice.Quantitative real-time polymerase chain reaction(qRT-PCR)and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds.Perception to taste substance was also studied using twobottle preference tests.Results:We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique.Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice.But qRT-PCR results revealed the changed expression profile of m Tas2rs gene in taste buds of these mutant mice.With two-bottle preference tests,these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105.In addition,these mutant mice showed a loss of taste perception to quinine dihydrochloride,denatonium benzoate,and cucurbitacin B(CuB).Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception.Conclusions:These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

Comparing the difference in enhancement of kokumi-tastingγ-glutamyl peptides on basic taste via molecular modeling approaches and sensory evaluation

γ-Glutamyl peptides can enhance basic taste sensations such as saltiness,sweetness,and umaminess,while the molecular mechanism and the difference in taste enhancement remain elusive.Thus,two complex conformations:taste type 1 receptor 1(T1 R1)-MSG and taste type 1 receptor 2(T1 R2)-sucrose were constructed to form binding receptors.These peptides showed affinity for the two receptors,but a higher affi nity scores and more binding amino acid residues for the T1 R1-MSG receptor,implying that they may exhibit a higher umami-enhancing effect.Thereinto,γ-glutamyl alanine(γ-EA)displayed the highest affi nity for the two receptors through mobilizing multiple amino acid residues to form hydrophobic and hydrogen bonds,indicating it had the highest enhancement for umaminess and sweetness among these peptides.Sensory evaluation demonstrated the enhancement ofγ-EA on umaminess was superior to that of sweetness.Generally,γ-glutamyl peptides could enhance basic taste sensation via activating taste receptor,and exhibited a highest umami-enhancing effect.

Identification of the material basis of the medicinal properties in Curcuma Longa L. to enhance targeted clinical application

Objective:To identify the relationship between the active compounds in turmeric(Curcuma Longa L.,C.Longa)and their medicinal properties,and clarify the chemical material basis of the medicinal properties in C.Longa.Methods:High throughput screening models of the thermo-transient receptor potentials(thermo-TRPs)and human taste type 2 receptors(hTAS2Rs)were established to evaluate the activity of the active compounds in C.Longa.The biological processes and distributions of the related targets of the active compounds were acquired by data mining.The above results were then used as basic data to comprehensively analyze the material basis of the medicinal properties of turmeric.Results:Curcumin inhibited TRPV1,TRPV2,TRPV3,and TRPA1,while demethoxycurcumin(DMC)inhibited TRPV3,TRPA1,and TRPM8.In terms of the 21 hTAS2Rs,the response values of hTAS2R38,hTAS2R16,and hTAS2R44 intervened by curcumin were ranked top three;the response values of hTAS2R3,hTAS2R1,and hTAS2R9 by DMC were in top three;the response values of hTAS2R47,hTAS2R39,and hTAS2R43 by bisdemethoxycurcumin(BDMC)were in top three.The biological processes related to active compounds primarily involved blood circulation,ossification,and regulation of the inflammatory response.Conclusion:Curcumin,DMC,and BDMC were identified as the material basis of the property of Chinese materia medica of C.Longa.Among these,curcumin and DMC may contribute to its warming effect in the four qi.Curcumin,DMC,and BDMC may lead to its bitter flavor,and the three active compounds were consistent with the meridian entry of C.Longa.Although different compounds play diverse roles in the four qi,five flavors,and meridian entry,they all were relevant to the efficacy of C.Longa.It is helpful to further understand the important role of the property of Chinese materia medica for the clinical application of traditional Chinese medicine.