Implausibility of the vibrational theory of olfaction

The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. A previous study reported that human subjects differentiated hydrogen/deuterium isotopomers (isomers with isotopic atoms) of the musk compound cyclopentadecanone as evidence supporting the theory. Here, we find no evidence for such differentiation at the molecular level. In fact, we find that the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR expression system and robustly responding to cyclopentadecanone and muscone, fails to distinguish isotopomers of these compounds in vitro. Furthermore, the mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other selected human and mouse ORs, responded similarly to normal, deuterated, and ¹³C isotopomers of their respective ligands, paralleling our results with the musk receptor OR5AN1. These findings suggest that the proposed vibration theory does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3, or other ORs examined. Also, contrary to the vibration theory predictions, muscone-d₃₀ lacks the 1,380- to 1,550-cm⁻¹ IR bands claimed to be essential for musk odor. Furthermore, our theoretical analysis shows that the proposed electron transfer mechanism of the vibrational frequencies of odorants could be easily suppressed by quantum effects of nonodorant molecular vibrational modes. These and other concerns about electron transfer at ORs, together with our extensive experimental data, argue against the plausibility of the vibration theory.In 1870, the British physician William Ogle wrote: “As in the eye and the ear the sensory impression is known to result not from the contact of material particles given off by the object seen or heard, but from waves or undulations of the ether or the air, one cannot but suspect that the same may be true in the remaining sense, and that the undulatory theory of smell… [may be] the true one” (1, 2). Of the 29 different “theories of odour” listed in the 1967 edition of The Chemical Senses (3), nine associate odor with vibrations, particularly those theories championed by Dyson (4, 5) and Wright (6–8). However, the premise that olfaction involves detection of vibrational frequencies of odorants remains highly speculative because neither the structures of the odorant receptors (ORs) nor the binding sites or the activation mechanisms triggered upon odorant binding to ORs have been established. In 1996–1997, Turin (9–12) elaborated on the undulatory theory of smell, as considered in more detail below, and suggested that a mechanism analogous to inelastic electron tunneling spectroscopy (13) may be involved, where tunneling electrons in the receptor probe the vibrational frequencies of odorants. In 2013, Gane et al. (14) commented that “whether olfaction recognizes odorants by their shape, their molecular vibrations, or both remains an open and controversial question” and that “a convenient way to address [this question] is to test for odor character differences between deuterated and nondeuterated odorant isotopomers since these have identical ground-state conformations but different vibrational modes.” Gane et al. (14) also stated that a particularly appropriate test case would involve odorants containing “more CH group… [such as] musks [which] are among the largest odorants and typically contain 15–18 carbons and 28 or more hydrogens.”In judging the plausibility of the vibration theory, we use a multipronged approach:

• i)We consider the concepts of shape vs. vibration theory and odorant perception vs. reception.
• ii)As a test of the vibration theory, we have prepared a series of isotopomers of musks and other compounds, containing up to 30 C–H or C–D bonds as test odorants, which are evaluated using in vitro activation of receptors identified by us and other groups as being highly responsive to these isotopomers.
• iii)We consider the confounding effects of impurities and isotope effects in interpreting odorant perception, as well as the validity of requirements for specific IR bands for recognition of musks by their receptors.
• iv)We examine the physical validity of the models developed to support the vibration theory.
• v)We consider the specific limitations of our in vitro approach using isotopomers to evaluate the vibration theory, based primarily on results obtained with a single identified human musk OR, in addition to other OR/ligand pairs.
• vi)We consider plausible nonvibration theory models for docking of musks to the human musk receptor, OR5AN1, where the musk carbonyl group functions as a hydrogen bond acceptor.

Gane et al. (14) have framed the argument for olfactory discrimination of hydrogen isotopomers as one of “shape” vs. “vibration.” However, neither the binding modes of isotopomers nor their activation mechanisms are known. ORs belong to the superfamily of class A G protein-coupled receptors (GPCRs), which are known to be activated through allosteric conformational changes induced upon ligand binding even without triggering any kind of electron transfer processes. Ligand–receptor interactions can be both attractive and repulsive, involving hydrogen bonding, van der Waals, cation–π, π–π, ion–ion, dipole–dipole, steric, and hydrophobic interactions with the receptor, with water channels and bridging water molecules mediating hydrogen bonds, as well as metal–ion coordination, as we have recently demonstrated in the latter case (15, 16). Therefore, molecular shape can be considered a “straw-man” alternative to the vibration theory when describing the differing affinities of ligands bound to GPCRs (17, 18), including isotopomers (19, 20). Some of these attractive and repulsive interactions were identified in 1940 by Pauling and Delbrück (21), who note that interacting biomolecules “must have complementary surfaces, like die and coin, and also a complementary distribution of active groups.” In addition, shape-related features are misrepresented by vibration theory proponents. For example, Franco et al. (17) stated: “Given that proteins are chiral, a shape-only theory cannot account for the identical odors of most enantiomeric pairs,” echoing similar comments by Turin (22): “One would therefore generally expect enantiomers to have completely different smells. This is emphatically not the case.” However, these assertions are clearly at odds with the highly developed ability of mice and other mammals to discriminate an array of nonpheromonal chiral odorant enantiomeric pairs (23–25), with the divergent in vitro responses to enantiomers by different combinations of ORs (26) and, in particular, with the highly selective response of the musk-sensitive mouse receptor, MOR215-1, to (R)-muscone (“l-muscone”) compared with (S)-muscone (“d-muscone”) (27).In addition to our concerns regarding shape, a second issue relates to describing how different smells are perceived, that is, the perception of an odorant. It is known that in vivo perception of odorants reflects the totality of perireceptor events as well as odorant–OR interactions (reception). Volatile odorants enter the nasal passage, where they dissolve in the nasal mucus overlying the olfactory epithelium and are then rapidly detected by ORs on the cilia of the olfactory sensory neurons, ultimately leading to signaling (28, 29). It is the mechanism of odorant–OR interactions, the reception of the odorant, that we seek to examine with isotopomers to determine whether the vibration theory is plausible, displaying isotope effects, because perception could be influenced by isotope effects due to the perireceptor events involving mucosal components, such as enzymes, mucopolysaccharides, salts, and antibodies.Whether deuterated and nondeuterated odorant isotopomers can be distinguished by smell and, even if they can, whether this distinction validates the vibration theory is a matter of contention. A 2001 paper by Haffenden et al. (30) reported that benzaldehyde-d₆ gave a statistically significant difference in odor perception relative to normal benzaldehyde, in support of the vibration theory. However, this study has been criticized for lacking double-blind controls to eliminate bias and because it used an anomalous version of the duo-trio test (31). Furthermore, the study failed to account for perireceptor events, namely, the enzyme-mediated conversion of odorants that has been shown to occur in nasal mucus. For example, benzaldehyde is converted to benzoic acid (32), a reaction potentially subject to significant primary isotope effects (2, 33, 34), which could explain the difference in odor perception for the benzaldehyde isotopomers. Earlier claims that human subjects can distinguish odors of acetophenone isotopomers (9, 35) have been shown to be untrue (14, 31). Recent studies indicate that Drosophila melanogaster can distinguish acetophenone isotopomers (36, 37) and that Apis mellifera L., the honey bee, can be trained to discriminate pairs of isotopomers (38). These studies differ from earlier insect studies in which isotopomer discrimination was not found. For example, systematic deuteration of 4-(p-hydroxyphenyl)-2-butanone acetate, a Dacus cucurbitae Coquillett (the male melon fly) attractant, did not affect the attractiveness of the compound to the fly (39); deuteration of alarm pheromones failed to alter the response toward these compounds by Pogonomyrmex badius worker ants (40); and honey bees could not distinguish between deuterated and nondeuterated nitrobenzene (41).Concerns have been raised (42) about aspects of the Drosophila study (36), which is “behavioural and not at the receptor level” (2) (also a concern with the Apis study). Also, given that the ORs and their downstream signaling in Drosophila and humans are completely unrelated, the Drosophila study should not be considered predictive of the ability of humans to distinguish isotopomers (2, 17). In view of the above discussion, it is interesting that in a blinded behavioral study, smell panelists distinguished between deuterated and nondeuterated isotopomers of cyclopentadecanone (1; Fig. 1A) and other musk odorants (14).Open in a separate windowFig. 1.(A) Preparation of deuterated 1–3. Deuterium could be selectively introduced, or selectively removed, adjacent to the carbonyl group using D₂O/K₂CO₃ or H₂O/K₂CO₃, respectively, at 130 °C; global replacement of all hydrogens could be achieved with Rh/C in D₂O at 150 °C. Repetition led to more complete deuteration as well as reduction of 1 to 3 and 2; oxidation of 2 gave 1 with ∼98% deuteration. Chromatography of deuterated 1 with freshly distilled pentane followed by repeated recrystallization from methanol/water to constant melting point gave samples showing no new peaks in their ¹H NMR spectra, other than very weak peaks corresponding to those peaks seen in undeuterated 1. (B) Deuterated (97%) muscone 4 was prepared via alcohol 5 as above. (C) 8-d₅ and 2,4,5,7-tetrathiaoctane-d_10, (9-d₁₀; 98% deuterium) were prepared as shown. Details of these syntheses are provided in SI Appendix.Here, we study the response of human musk-sensitive OR5AN1, identified through screening of heterologously expressed human ORs, to cyclopentadecanone (1) and muscone (4) isotopomers. We also present pharmacological data on the response of mouse ORs to deuterated and nondeuterated acetophenone and benzaldehyde, as well as selected ¹³C isotopomers. In addition, we present related studies on the response of various human and mouse ORs to other deuterated and nondeuterated odorants, including (methylthio)-methanethiol (MTMT, 8; Fig. 1C) and bis(methylthiomethyl) disulfide (9), studied in connection with our investigation of the role of copper coordination in the recognition of both sulfur-containing odorants by the mouse (methylthio)methanethiol receptor, MOR244-3 (15, 16). Insofar as the ability to distinguish odors of isotopomers directly tests the predictions of the vibration theory, the comparative response of human and mouse ORs to isotopomers of these selected ligands in the heterologous OR expression system constitutes a robust test of the vibration theory. Finally, we discuss the basis for recent vibration theories of olfaction and supporting computational evidence (37, 43–47) in light of well-established electron transfer theories (48). We point out that key assumptions underlying the vibration theory lack experimental support and are missing important physical features expected for biological systems.     

Hyperpolarized [1,3‐13C2]ethyl acetoacetate is a novel diagnostic metabolic marker of liver cancer

An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses ¹³C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized ¹³C‐MR. This work led to the identification of a class of substrates, low molecular weight ethyl‐esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3‐¹³C₂]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an ～4 times higher metabolic substrate‐to‐product ratio than in the surrounding healthy tissue, (p = 0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized ¹³C‐MR marker. This could be appreciated by the signal‐to‐noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state‐of‐the‐art hyperpolarized substrate, [1‐¹³C]pyruvate. Also, the contrast‐to‐noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1‐¹³C]pyruvate.     

Gene expression profile predictive of response to chemotherapy in metastatic colorectal cancer

Fluoropyrimidine-based chemotherapy (CT) has been the mainstay of care of metastatic colorectal cancer (mCRC) for years. Response rates are only observed, however, in about half of treated patients, and there are no reliable tools to prospectively identify patients more likely to benefit from therapy. The purpose of our study was to identify a gene expression profile predictive of CT response in mCRC. Whole genome expression analyses (Affymetrix GeneChip^® HG-U133 Plus 2.0) were performed in fresh frozen tumor samples of 37 mCRC patients (training cohort). Differential gene expression profiles among the two study conditions (responders versus non-responders) were assessed using supervised class prediction algorithms. A set of 161 differentially expressed genes in responders (23 patients; 62%) versus non-responders (14 patients; 38%) was selected for further assessment and validation by RT-qPCR (TaqMan^®Low Density Arrays (TLDA) 7900 HT Micro Fluidic Cards) in an independent multi-institutional cohort (53 mCRC patients). Seven of these genes were confirmed as significant predictors of response. Patients with a favorable predictive signature had significantly greater response rate (58% vs 13%, p = 0.024), progression-free survival (61% vs 13% at 1 year, HR = 0.32, p = 0.009) and overall survival (32 vs 16 months, HR = 0.21, p = 0.003) than patients with an unfavorable gene signature. This is the first study to validate a gene-expression profile predictive of response to CT in mCRC patients. Larger and prospective confirmatory studies are required, however, in order to successfully provide oncologists with adequate tools to optimize treatment selection in routine clinical practice.     

Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling

One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca²⁺). In the present study, we established conditions that allow the in vivo detection of Ca²⁺ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca²⁺ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.     

CD151-α3β1 integrin complexes are prognostic markers of glioblastoma and cooperate with EGFR to drive tumor cell motility and invasion

Glioblastoma, one of the most aggressive forms of brain cancer, is featured by high tumor cell motility and invasiveness, which not only fuel tumor infiltration, but also enable escape from surgical or other clinical interventions. Thus, better understanding of how these malignant traits are controlled will be key to the discovery of novel biomarkers and therapies against this deadly disease. Tetraspanin CD151 and its associated α3β1 integrin have been implicated in facilitating tumor progression across multiple cancer types. How these adhesion molecules are involved in the progression of glioblastoma, however, remains largely unclear. Here, we examined an in-house tissue microarray-based cohort of 96 patient biopsies and TCGA dataset to evaluate the clinical significance of CD151 and α3β1 integrin. Functional and signaling analyses were also conducted to understand how these molecules promote the aggressiveness of glioblastoma at molecular and cellular levels. Results from our analyses showed that CD151 and α3 integrin were significantly elevated in glioblastomas at both protein and mRNA levels, and exhibited strong inverse correlation with patient survival (p < 0.006). These adhesion molecules also formed tight protein complexes and synergized with EGF/EGFR to accelerate tumor cell motility and invasion. Furthermore, disruption of such complexes enhanced the survival of tumor-bearing mice in a xenograft model, and impaired activation of FAK and small GTPases. Also, knockdown- or pharmacological agent-based attenuation of EGFR, FAK or Graf (ARHGAP26)/small GTPase-mediated pathways markedly mitigated the aggressiveness of glioblastoma cells. Collectively, our findings provide clinical, molecular and cellular evidence of CD151-α3β1 integrin complexes as promising prognostic biomarkers and therapeutic targets for glioblastoma.