Olfactory marker protein contains a leucine-rich domain in the Ω-loop important for nuclear export.

Olfactory marker protein (OMP) is a cytosolic protein expressed in mature olfactory receptor neurons (ORNs). OMP modulates cAMP signalling and regulates olfactory sensation and axonal targeting. OMP is a small soluble protein, and passive diffusion between nucleus and cytoplasm is expected. However, OMP is mostly situated in the cytosol and is only sparsely detected in the nuclei of a subset of ORNs, hypothalamic neurons and heterologously OMP-expressing cultured cells. OMP can enter the nucleus in association with transcription factors. However, how OMP is retained in the cytosol at rest is unclear. Because OMP is proposed to affect cell differentiation, it is important to understand how OMP is distributed between cytoplasm and nucleus. To elucidate the structural profile of OMP, we applied several bioinformatics methods to a multiple sequence alignment (MSA) of OMP protein sequences and ranked the evolutionarily conserved residues. In addition to the previously reported cAMP-binding domain, we identified a leucine-rich domain in the Ω-loop of OMP. We introduced mutations into the leucine-rich region and heterologously expressed the mutant OMP in HEK293T cells. Mutations into alanine increased the nuclear distribution of OMP quantified by immunocytochemistry and western blotting. Therefore, we concluded that OMP contains a leucine-rich domain important for nuclear transport.

Olfactory marker protein is unlikely to be cleaved by calpain 5.

Olfactory maturation marker protein (OMP) is expressed in olfactory receptor neurons and hypothalamic neurons. OMP is a nested gene located in the intron of calpain 5 (CAPN5), a Ca2+-dependent cysteine protease. Despite being located at the same genomic locus, genetic regulation of the reciprocal expression of OMP and CAPN5 has been suggested. By performing a motif search, we detected possible calpain cleavage sites in OMP. However, the direct proteolytic regulation of OMP by CAPN5 is unclear. Here, we generated OMP fused with Myc-tag and His-tag at its N- and C-termini and examined whether CAPN5 cleaves OMP into fragments by detecting immunoreactivity against Myc, OMP and His. Western blotting demonstrated that OMP was unlikely to be cleaved even in the presence of Ca2+ in vitro. We expressed OMP and CAPN5 in HEK293T cells and applied a calcium ionophore under physiological conditions in cellulo, which resulted in no apparent fragmentation of OMP. We also applied liquid chromatography/mass spectrometry to the electrophoresed fractions smaller than the uncut Myc-OMP-His signals, which demonstrated no significant fragmentation of OMP. These results collectively indicate that OMP is unlikely to be cleaved by CAPN5.

Olfactory receptor 78 is expressed in hypothalamic vasopressin/oxytocin neurons, parenchymal microglia and choroidal macrophages in mice.

Olfactory receptors have been detected in extraolfactory organs. Olfactory receptor 78 (Olfr78), proposed to respond to small organic acids, is widely expressed in the kidney, arterioles, colon, and prostate. However, its expression patterns in the brain remain largely unknown. Using immunohistochemistry, we revealed that Olfr78 was densely expressed in the hypothalamus and choroid plexus and sparsely expressed throughout the parenchyma. By costaining with cellular markers, we further found that Olfr78 was expressed in the somata and axons of vasopressin/oxytocin neurons in the hypothalamic paraventricular/supraoptic nuclei. Olfr78 was also strongly expressed in macrophages in the choroid plexus and moderately expressed in microglia near the parenchymal vasculature. Considering that these brain regions should communicate with cerebral blood flow, Olfr78 could contribute to sensing the humoral conditions surrounding the cerebrovascular system.

Olfactory marker protein interacts with adenosine nucleotide derivatives.

Olfactory marker protein (OMP) is a genetic signature for mature olfactory receptor neurons (ORNs). Recently, it has been proposed that OMP directly captures odour-induced cAMP to swiftly terminate the olfactory signal transduction to maintain neuronal sensitivity. In the present study, we show that OMP can also interact with other adenosine nucleotides as ATP, ADP and AMP with different affinities. We performed bioluminescent resonant energy transfer (BRET) assay to measure the binding actions of the adenosine nucleotide derivatives in competition to cAMP. Amongst all, ATP showed the bell-shape affinity to OMP in the presence of cAMP; ADP and AMP showed fewer affinities to OMP than ATP. In the absence of cAMP analogues, ATP alone bound to OMP in a dose dependent manner with a lower affinity than to cAMP. Thus, OMP possessed different affinities to ATP in the presence or absence of cAMP. OMP may interact differentially with ATP and cAMP depending on its supply and demand along the cAMP-associated signalling in the limited spaces of cilia of ORNs.

Olfactory marker protein contributes to the evaluation of odour values by olfactory glomerular processing.

Olfaction starts from olfactory receptor neurons (ORNs) that express olfactory marker protein (OMP). OMP deficit results in various behavioural phenotypes indicating olfactory dysfunction due to the impaired responses of ORNs. Recently, OMP was demonstrated to maintain strong olfaction by buffering olfactory cAMP signalling. However, the impact of OMP on olfaction behaviours, the assessment of which requires time to evaluate odour values, remains largely unexplained. Here, we examined the behaviour of heterozygous OMP+/GFP (HET) mice vs. homologous GFP-knock-in OMP-deficient OMP GFP/ GFP (KI) mice during the olfactory investigation of odours with different values. When a swab containing an organic odour was presented, both HET and KI mice swiftly approached and investigated the swab with gradual habituation over test sessions. However, when another similar odour was presented, KI mice investigated the new swab much less intensively than HET mice. Next, mice were placed in a chamber with an aversive odour source in one corner of a test chamber. KI mice more frequently approached the compartment containing the aversive odour source than HET mice. Finally, we trained mice to associate two odours with solutions by utilizing reward-penalty values. HET mice stayed close to the reward-associated odour, while KI mice initially approached the reward-associated odour, occasionally turned towards the penalty-associated odour source and eventually stayed in the reward-odour compartment. Histologically, c-Fos-expressing juxtaglomerular cells were fewer and more broadly distributed around glomeruli in KI mice than HET mice. In conclusion, OMP contributes to the evaluation of odour values by glomerular processing during an olfactory investigation task.

Virus database annotations assist in tracing information on patients infected with emerging pathogens.

The global pandemic of SARS-CoV-2 has disrupted human social activities. In restarting economic activities, successive outbreaks by new variants are concerning. Here, we evaluated the applicability of public database annotations to estimate the virulence, transmission trends and origins of emerging SARS-CoV-2 variants. Among the detectable multiple mutations, we retraced the mutation in the spike protein. With the aid of the protein database, structural modelling yielded a testable scientific hypothesis on viral entry to host cells. Simultaneously, annotations for locations and collection dates suggested that the variant virus emerged somewhere in the world in approximately February 2020, entered the USA and propagated nationwide with periodic sampling fluctuation likely due to an approximately 5-day incubation delay. Thus, public database annotations are useful for automated elucidation of the early spreading patterns in relation to human behaviours, which should provide objective reference for local governments for social decision making to contain emerging substrains. We propose that additional annotations for past paths and symptoms of the patients should further assist in characterizing the exact virulence and origins of emerging pathogens.

Olfactory marker protein elevates basal cAMP concentration.

Olfactory marker protein (OMP), which is expressed abundantly in mature olfactory receptor neurons, operates as a cAMP-binding protein. OMP captures phasic cAMP surges induced by sensory stimuli and punctuates the downstream signalling in the cilia. On the other hand, OMP is also abundant in the soma. At equilibrium, OMP should exhibit association/dissociation reactions with cAMP. To examine the steady-state function of OMP, we expressed OMP in an HEK293 heterologous expression system and measured the activity of cAMP-dependent protein kinase (PKA) using a cAMP response element/luciferase reporter assay. In the presence of OMP, the basal activity level of PKA was elevated to approximately twice as much as that in the absence of OMP. Upon tonic stimulation by membrane-permeable cAMP, the PKA activity increased in a dose-dependent manner and was greater in the presence of OMP at all doses until saturation. These results indicate that OMP, a cytosolic cAMP-binding protein, operates as a cAMP reservoir by increases the basal cAMP concentration and enhances tonic cAMP actions. Together with the previous finding that OMP acutely sequesters cAMP-related responses, these results indicate that OMP can buffer acute surges in cAMP and tonic production, which stabilizes the basal cAMP pool in the long run.

Olfactory marker protein captures cAMP produced via Gαs-protein-coupled receptor activation.

Olfactory marker protein (OMP) labels the matured stage of olfactory receptor neurons (ORN) and has promoted the investigation on the physiology of olfaction. OMP regulates olfactory sensitivity and axonal projection of ORNs, both of which are under the control of the olfactory signaling mediator cAMP. Recently, it has been reported that OMP contains cAMP-binding sites. OMP directly captures the photo-uncaged cAMP in the cytosol and rapidly terminates the olfactory cyclic nucleotide-gated (CNG) channels activity to sharpen the olfactory responses. Here, we investigate the contribution of OMP to cAMP acutely produced via activation of Gαs-protein coupled receptors (GPCR). We expressed OMP and non-desensitizing CNGA2 channels in HEK293T cells together with ß1-adrenergic receptors (ADRB1) or photo-sensitive ß2-adrenergic receptors (opto-ß2). Continuous puff of adrenergic agonist isoproterenol to HEK29T cells with ADRB1 induced the lasting CNGA2 currents in the absence of OMP, while OMP rapidly deactivated the CNGA2 channel activity with residual currents. Photo-activation of opto-ß2 in the absence of OMP induced the CNGA2 currents with a prolonged increase, while OMP swiftly deactivated the CNGA2 channels after the initial surge. Therefore, cytosolic OMP rapidly uncouples CNGA2 channels and cAMP-signaling produced via GPCRs in the submembrane compartment.

Olfactory marker protein directly buffers cAMP to avoid depolarization-induced silencing of olfactory receptor neurons.

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.

Olfactory receptor neurons express olfactory marker protein but not calpain 5 from the same genomic locus.

Gene expression is highly regulated to functionally diversify cells. Genes that cooperate in the same physiological processes occasionally reside within nearby regions in a chromosome. Olfactory marker protein (OMP) is highly expressed in mature olfactory receptor neurons (ORNs), but its physiological roles are not fully understood. According to the genomic map, the OMP gene is located within an intron of the calcium-dependent protease, calpain 5 (CAPN5); in other words, the OMP gene is a nested intronic gene. Thus, we attempted to investigate the gene expression and protein distribution of CAPN5 in the olfactory epithelium compared with that in the central nervous system (CNS). By performing reverse-transcriptase PCR and in situ hybridization, we confirmed that CAPN5 mRNA was expressed in the olfactory epithelium. We then performed immunohistological investigations using sliced preparations obtained from mice expressing GFP under OMP promoter activity. The detected GFP fluorescence was restricted to the knob, soma and axon bundles of the ORNs, while CAPN5 immunoreactivity (CAPN5-IR) was ubiquitously detected in the olfactory epithelial layer and lamina propria; signals were strongly detected in the supporting cells within the epithelium. In the CNS, CAPN5 signals were widely detected and were especially strong in the hippocampal formation and the piriform cortex as previously indicated. Therefore, these data indicate that ORNs express OMP but not CAPN5 from CAPN5 gene expression even though they are localized in the same genomic locus. The mechanisms by which the OMP promoter is regulated require detailed investigations.

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis).

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Orbital magnetic resonance imaging combined with clinical activity score can improve the sensitivity of detection of disease activity and prediction of response to immunosuppressive therapy for Graves' ophthalmopathy.

The aim of this study was to demonstrate that the addition of orbital magnetic resonance (MR) imaging can provide improvement in sensitivity of detection of active disease and the prediction of the response to intravenous glucocorticoid therapy (ivGC), over clinical activity score (CAS) alone. A prospective case series was studied at our institution. Forty eight patients were examined by CAS and orbital MR imaging. The maximum of T2 relaxation times of extraocular muscles (maxT2RT) and other parameters were evaluated by MR imaging. Thirty five of 48 patients underwent ivGC. Twenty of 35 patients, whose CAS was 2 points or less, were evaluated for the response to ivGC. The correlation between CAS and maxT2RT was evaluated. Differentiation of active and inactive GO was performed by CAS and orbital MR imaging. The response to ivGC was evaluated by CAS, orbital MR imaging and ophthalmic parameters. As a result, CAS and maxT2RT showed significant positive correlation (r=0.58, p<0.0001), and 15 patients were positive by CAS and orbital MR imaging. However, 20 patients were positive by only MR imaging. In those 20 patients, there was significant improvement after ivGC. We concluded that orbital MR imaging combined with CAS could improve the sensitivity of detection of active disease and the prediction of the response to ivGC. In addition, even if only one parameter of CAS is positive, further examination with orbital MR imaging is advised.

Is death-feigning adaptive? Heritable variation in fitness difference of death-feigning behaviour.

The adaptation of death-feigning (thanatosis), a subject that has been overlooked in evolutionary biology, was inferred in a model prey-and-predator system. We studied phenotypic variation among individuals, fitness differences, and the inheritance of death-feigning behaviour in the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Two-way artificial selections for the duration of death-feigning, over 10 generations, showed a clear direct response in the trait and a correlated response in the frequency of death-feigning, thus indicating variation and inheritance of death-feigning behaviour. A comparison of the two selected strains with divergent frequencies of death-feigning showed a significant difference in the fitness for survival when a model predator, a female Adanson jumper spider, Hasarius adansoni Audouin (Araneomophae: Salticidae), was presented to the beetles. The frequency of predation was lower among beetles from strains selected for long-duration than among those for short-duration death-feigning. The results indicate the possibility of the evolution of death-feigning under natural selection.