Odorant Receptor Inhibition Is Fundamental to Odor Encoding.

Most natural odors are complex mixtures of volatile components, competing to bind odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) of the nose. To date, surprisingly little is known about how OR antagonism shapes neuronal representations in the detection layer of the olfactory system. Here, we investigated its prevalence, the degree to which it disrupts OR ensemble activity, and its conservation across phylogenetically related ORs. Calcium imaging microscopy of dissociated OSNs revealed significant inhibition, often complete attenuation, of responses to indole-a commonly occurring volatile associated with both floral and fecal odors-by a set of 36 tested odorants. To confirm an OR mechanism for the observed inhibition, we performed single-cell transcriptomics on OSNs exhibiting specific response profiles to a diagnostic panel of odorants and identified three paralogous receptors-Olfr740, Olfr741, and Olfr743-which, when tested in vitro, recapitulated OSN responses. We screened ten ORs from the Olfr740 gene family with ∼800 perfumery-related odorants spanning a range of chemical scaffolds and functional groups. Over half of these compounds (430) antagonized at least one of the ten ORs. OR activity fitted a mathematical model of competitive receptor binding and suggests normalization of OSN ensemble responses to odorant mixtures is the rule rather than the exception. In summary, we observed OR antagonism occurred frequently and in a combinatorial manner. Thus, extensive receptor-mediated computation of mixture information appears to occur in the olfactory epithelium prior to transmission of odor information to the olfactory bulb.

An evaluation of the mid-ventilation method for the planning of stereotactic lung plans.

BACKGROUND AND PURPOSE: Stereotactic ablative body radiotherapy for lung plans requires 4DCT. Most radiotherapy centres use this to determine an internal target volume (ITV), despite studies suggesting that planning on a mid-ventilation (Mid-V) phase can reduce target volumes. The purpose of this study is two-fold: to determine whether the Mid-V approach provides adequate coverage and to discuss methods to enable the Mid-V approach to be applied more widely. METHOD: 4D scans of 79 patients were outlined on every phase. The mid-V phase was identified. Margins were determined from the range of motion, and plans generated with a 55 Gy prescription. A grid-based method was used to get the probability of tumour coverage in the presence of systematic and random uncertainties, with and without blurring for breathing motion. RESULTS: For the Mid-V plans with the margins calculated from the van-Herk formula, after blurring doses for breathing, the coverage (dose covering 95% of the CTV 95% of the time) was greater than for plans with isotropic 5 mm margins uncorrected for breathing (58.2 Gy v 57.3 Gy). Similar results were obtained for a linear margin chosen as 0.15 of the breathing range. Deformable contour propagation in a commercial outlining system (ProSoma) identified the same mid-V phase in the majority of cases. CONCLUSION: Our results confirm that a mid-V approach can be used to reduce the PTV size, with no loss of tumour coverage. We propose the use of a simplified margin formula equal to the margin ignoring breathing plus 0.15 of the range of motion.

The physician's role in perioperative management of older patients undergoing surgery.

Life-sustaining and life-improving surgical interventions are increasingly available to older, frailer patients, many of whom have multimorbidity. Physicians can help support perioperative multidisciplinary teams with assessment and preoperative optimisation of physiological reserve, comorbidities and associated geriatric syndromes. Similar structured support can be useful in the postoperative period where older patients are at increased risk of delirium, medical complications, increased functional dependency and where discharge planning can prove more difficult than in younger cohorts. Comprehensive geriatric assessment has been shown to improve outcomes and is now embedded in most UK-based services for traumatic hip fracture. Perioperative comprehensive geriatric assessment has been explored in other surgical disciplines and procedures and, where evaluated, has been associated with improved outcomes. The need to support older patients with frailty undergoing surgery exceeds the capacity of specialist geriatricians. Other groups of healthcare professionals need to nurture the core competencies to support this group perioperatively.

Heme oxygenase-1 gene promoter polymorphism is associated with the development of necrotizing acute pancreatitis.

OBJECTIVES: Acute pancreatitis is a severe and frequently life-threatening disease, which can lead to pancreatic necrosis, acute lung injury, systemic inflammatory response syndrome, and other complications. In this study, we hypothesized that the expression of heme oxygenase-1 determined by the number of guanidinium thiocyanate (GT) repeats can influence the occurrence of acute pancreatitis. METHODS: Patients with acute pancreatitis (n = 131) and age- and sex-matched healthy controls (n = 108) were studied. The polymerase chain reaction products were analyzed by ABI 3130 genetic analyzer and the exact size of the polymerase chain reaction products was determined by GeneMapper software. A short allele was defined as containing 27 GT repeats or fewer, whereas a long allele was more than 27 repeats. RESULTS: The subjects were categorized into 3 groups on the basis of the genotype results: 1 short and 1 long, 2 short, and 2 long alleles (L/L). Patients with necrotizing disease more frequently were carriers of LL genotype compared with those who had edematous acute pancreatitis. Furthermore, logistic regression analysis revealed that the presence of L/L allele type doubles the risk for developing pancreatic necrosis in patients with acute pancreatitis. CONCLUSIONS: The polymorphism of the GT repeats in the heme oxygenase-1 promoter region may be a risk factor for developing severe and necrotizing acute pancreatitis.

MicroRNA profiling in lung cancer reveals new molecular markers for diagnosis.

OBJECTIVE: To identify new molecular diagnostic markers for non-small cell lung carcinoma (NSCLC) by analyzing microRNA (miRNA) expression profile differences in samples from NSCLC patients and adults with nonneoplastic diseases. STUDY DESIGN: miRNA expression was studied in archival formalin-fixed, paraffin-embedded tissues by microarray and confirmed by real-time PCR analysis of NSCLC and normal lung tissues. An algorithm for discriminating normal, squamous cell carcinoma (SQCC), and adenocarcinoma (ADC) tissue was derived from miRNA expression studies and applied towards characterization of poorly differentiated NSCLC samples. RESULTS: Microarray data from a genome-wide scan revealed 34 differentially expressed miRNAs, 5 of which enabled algorithmic discrimination of normal tissue from carcinoma (SQCC or ADC), as well as SQCC from ADC. Expression of miR-21 was significantly increased in both tumor types, whereas levels of miR-451 and miR-486-5p were reduced. SQCC was distinguished from normal tissue and ADC by high-level miR-205 expression and decreased miR-26b. Comparison of miRNA profiles to histological and immunohistochemical findings in 19 poorly differentiated specimens demonstrated the potential clinical utility of miRNA profiling to provide important insights into the classification of SQCC and ADC. CONCLUSION: This study presents a novel algorithm for specimen classification in cases of poorly differentiated NSCLC.

Fluorescent imaging of high-grade bladder cancer using a specific antagonist for chemokine receptor CXCR4.

We previously reported that the expression of CXC chemokine receptor-4 (CXCR4) was upregulated in invasive bladder cancers and that the small peptide T140 was a highly sensitive antagonist for CXCR4. In this study, we identified that CXCR4 expression was induced in high-grade superficial bladder tumors, including carcinoma in situ and invasive bladder tumors. To visualize the bladder cancer cells using urinary sediments from the patients and chemically induced mouse bladder cancer model, a novel fluorescent CXCR4 antagonist TY14003 was developed, that is a T140 derivative. TY14003 could label bladder cancer cell lines expressing CXCR4, whereas negative-control fluorescent peptides did not label them. When labeling urinary sediments from patients with invasive bladder cancer, positive-stained cells were identified in all patients with bladder cancer and positive urine cytology but not in controls. Although white blood cells in urine were also labeled with TY14003, they could be easily discriminated from urothelial cells by their shape and size. Finally, intravesical instillation of TY14003 into mouse bladder, using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer model, demonstrated that fluorescent signals were detected in the focal areas of bladder of all mice examined at 12 weeks of BBN drinking by confocal microscopy and fluorescent endoscopy. On the contrary, all the normal bladders were found to be negative for TY14003 staining. In conclusion, these results indicate that TY14003 is a promising diagnostic tool to visualize small or flat high-grade superficial bladder cancer.

Structure-activity relationship study on artificial CXCR4 ligands possessing the cyclic pentapeptide scaffold: the exploration of amino acid residues of pentapeptides by substitutions of several aromatic amino acids.

Previously, downsizing of a 14-residue peptidic CXCR4 antagonist has led to the development of a highly potent CXCR4 antagonist [cyclo(-d-Tyr(1)-Arg(2)-Arg(3)-Nal(4)-Gly(5)-)]. In the present study, cyclic pentapeptide libraries that were designed by substitutions of several amino acids for d-Tyr(1) and Arg(2) in peptide were prepared and screened to evaluate binding activity for CXCR4. The above structure-activity relationship study led to the finding of several potent CXCR4 ligands.

Expression of CXCR4, a G-protein-coupled receptor for CXCL12 in yeast identification of new-generation inverse agonists.

G-protein-coupled receptors (GPCR) are prime targets for therapies with small molecule-antagonists. Since yeast have GPCR triggered signaling pathways analogous to those present in mammalian cells, it is possible to express human receptors in yeast coupled to the pheromone responsive signaling cascade in variants that contain mammalian-yeast Galpha subunit chimeras. CXCR4 and CXCR4(N119S), a constitutively active mutant were expressed in yeast coupled to pheromone responsive reporter genes, HIS3, lacZ, or FUI, and tested for signaling activity. Compounds derived from T140, an inverse agonist for CXCR4, were screened for activity using yeast cells expressing CXCR4(N119S) and containing a FUS1-lacZ reporter gene. Levels of inhibition of beta-galactosidase activities triggered by constitutive activation of the pheromone response pathway that were obtained in the presence of the T140 derived compounds correlated with affinities measured in radioligand binding inhibition experiments. The yeast signaling system may provide an effective approach for screening chemokine receptor antagonists.

Physical association of GPR54 C-terminal with protein phosphatase 2A.

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.