Abciximab improves 6-month clinical outcome after rescue coronary angioplasty

Background Few data are available concerning the effects on clinical outcome and left ventricular function of abciximab administration in patients undergoing rescue percutaneous transluminal coronary angioplasty (PTCA) after failed thrombolysis for acute myocardial infarction. The aim of the study was to investigate such effects. Methods Eighty-nine consecutive patients referred to our laboratory from other hospitals for rescue PTCA within 24 hours from the onset of chest pain were prospectively randomized before the procedure to abciximab treatment (44 patients) or placebo (45 patients). No significant differences in baseline characteristics were observed between the 2 groups. Study end points were the occurrence of major adverse cardiac events (MACE) such as death, reinfarction, congestive heart failure, target lesion revascularization, or recurrent ischemia at 30-day and 6-month follow-up and the occurrence of periprocedural bleeding. Results Mean time from symptom onset to reperfusion was 8.5 ± 5.4 hours; rescue PTCA was successful in 96% of patients. The incidence of major, moderate, and minor bleeding was similar in the 2 groups. At 30-day follow-up, the echocardiographic left ventricular wall motion score index showed a significantly higher improvement in the abciximab group versus the placebo group (P < .001). At 6-month follow-up, the incidence of MACE was 11% in the abciximab group versus 38% in the placebo group (P = .004). Abciximab administration (P = .003) and cardiogenic shock (P = .005) were the only independent predictors of the occurrence of MACE at multivariable analysis. Conclusion Treatment with abciximab during rescue PTCA positively affects clinical outcome at 6-month follow-up without increasing periprocedural bleeding. (Am Heart J 2002;143:334-41.)     

Antiplatelet agents in acute coronary syndromes

Plaque disruption, platelet activation, and intracoronary artery thrombus formation are the key events in the pathogenesis of acute coronary syndromes. Antiplatelet therapies significantly reduce the risk of ischemic complications both during the acute phase and in the long term in patients with acute coronary syndromes. Aspirin remains the cornerstone of antiplatelet therapy, but there is incremental benefit when clopidogrel or ticlopidine is added to aspirin. Dual antiplatelet therapy with the combination of clopidogrel and aspirin is becoming the new standard of care for the management of patients with non-ST-segment elevation acute coronary syndrome and undergoing percutaneous coronary intervention and is currently being further evaluated in ST-segment elevation acute coronary syndrome.     

Genetic Polymorphisms of Functional Candidate Genes and Recurrent Acute Otitis Media With or Without Tympanic Membrane Perforation

Evaluation of the genetic contribution to the development of recurrent acute otitis media (rAOM) remains challenging. This study aimed to evaluate the potential association between single nucleotide polymorphisms (SNPs) in selected genes and rAOM and to analyze whether genetic variations might predispose to the development of complicated recurrent cases, such as those with tympanic membrane perforation (TMP).A total of 33 candidate genes and 47 SNPs were genotyped in 200 children with rAOM (116 with a history of TMP) and in 200 healthy controls.INFγ rs 12369470CT was significantly less common in the children with rAOM than in healthy controls (odds ratio [OR] 0.5, 95% confidence interval [CI] 0.25–1, P = 0.04). Although not significant, interleukin (IL)-1β rs 1143627G and toll-like receptor (TLR)-4 rs2737191AG were less frequently detected in the children with rAOM than in controls. The opposite was true for IL-8 rs2227306CT, which was found more frequently in the children with rAOM than in healthy controls. The IL-10 rs1800896TC SNP and the IL-1α rs6746923A and AG SNPs were significantly more and less common, respectively, among children without a history of TMP than among those who suffered from this complication (OR 2.17, 95% CI 1.09–4.41, P = 0.02, and OR 0.42, 95% CI 0.21–0.84, P = 0.01).This study is the first report suggesting an association between variants in genes encoding for factors of innate or adaptive immunity and the occurrence of rAOM with or without TMP, which confirms the role of genetics in conditioning susceptibility to AOM.     

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.