Spherical-supported membranes as platforms for screening against membrane protein targets.

Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.

Obstetric bleeding among women with inherited bleeding disorders: a retrospective study.

INTRODUCTION: Women with inherited bleeding disorders are at increased risk for bleeding complications during pregnancy and the postpartum period, particularly postpartum haemorrhage (PPH). AIM: This retrospective study evaluates pregnancy management through the Inherited Bleeding Disorders Clinic of Southeastern Ontario, the clinical factors associated with pregnancy-related abnormal bleeding and assesses tranexamic acid use in the postpartum treatment of bleeding disorder patients. METHODS: A chart review of 62 pregnancies, from 33 women, evaluated patient characteristics (age, haemostatic factor levels) and delivery conditions (mode of delivery, postpartum treatment) in relation to abnormal postpartum bleeding. RESULTS: This cohort revealed increased risk of immediate PPH with increased age at delivery (mean age: 30.1 years with PPH, 26.5 years without PPH, P < 0.013), and birth by vaginal delivery (P < 0.042). Low von Willebrand factor (VWF) antigen or factor VIII (FVIII) in the third trimester was not associated with an increased risk of PPH; however, low VWF:RCo was associated with increased immediate PPH despite treatment with continuous factor infusion (P < 0.042). Women treated with tranexamic acid postpartum had less severe bleeding in the 6-week postpartum (P < 0.049) with no thrombotic complications. CONCLUSIONS: This study contributes to the growing body of work aimed at optimizing management of bleeding disorder patients through pregnancy and the postpartum period, showing patients are at a higher risk of PPH as they age. Risk factors such as low third trimester VWF:RCo have been identified. Treatment with tranexamic acid in the postpartum period is associated with a reduced incidence of abnormal postpartum bleeding.

Electronic parameters for charge transfer along DNA.

We systematically examine all the tight-binding parameters pertinent to charge transfer along DNA. The pi molecular structure of the four DNA bases (adenine, thymine, cytosine, and guanine) is investigated by using the linear combination of atomic orbitals method with a recently introduced parametrization. The HOMO and LUMO wave functions and energies of DNA bases are discussed and then used for calculating the corresponding wave functions of the two B-DNA base-pairs (adenine-thymine and guanine-cytosine). The obtained HOMO and LUMO energies of the bases are in good agreement with available experimental values. Our results are then used for estimating the complete set of charge transfer parameters between neighboring bases and also between successive base-pairs, considering all possible combinations between them, for both electrons and holes. The calculated microscopic quantities can be used in mesoscopic theoretical models of electron or hole transfer along the DNA double helix, as they provide the necessary parameters for a tight-binding phenomenological description based on the pi molecular overlap. We find that usually the hopping parameters for holes are higher in magnitude compared to the ones for electrons. Our findings are also compared with existing calculations from first principles.

Topical anaesthesia before nasendoscopy: a randomized controlled trial of co-phenylcaine compared with lignocaine.

OBJECTIVE: To evaluate the relative effectiveness of co-phenylcaine (lignocaine 5% with phenylephrine) and lignocaine 5% sprays when administered prior to rigid nasendoscopy. DESIGN: Randomized, double blind controlled study. SETTING: Teaching hospital otolaryngology unit. PARTICIPANTS: Thirty patients requiring routine outpatient rigid nasendoscopy were administered five puffs of either co-phenylcaine or lignocaine 5% spray which had been randomly assigned to either the first or the second visit. Ten minutes later nasendoscopy was performed. Immediately after nasendoscopy the ease of performance of the procedure and the quality of the view achieved was rated on a visual analogue scale by the endoscopist and the patients recorded the level of pain experienced on a visual analogue scale. Two weeks later, the patients returned for a repeat nasendoscopy, receiving the alternate spray. MAIN OUTCOMES MEASURES: Ease of performance and quality of view of achieved by endoscopists and pain experienced by patients, both measured with visual analogue scales. RESULTS: The ease of passage of the endoscope and quality of the view obtained was found to be greater after the administration of co-phenylcaine [visual analogue scores 84 (95% CI: 80-89) than after lignocaine and 77 (95% CI: 73-81) (P < 0.01)]. The two sprays produced similar levels of topical anaesthesia. CONCLUSIONS: Nasendoscopy can be performed with minimal discomfort after the administration of either co-phenylcaine or lignocaine 5% sprays. The vasoconstricting action of co-phenylcaine increases the ease of passage of the endoscope and quality of the view obtained by the endoscopist.

Functional evidence for a colorectal cancer tumor suppressor gene at chromosome 8p22-23 by monochromosome transfer.

Chromosome 8p is considered, from loss of heterozygosity analysis, to be a strong candidate for the location of a tumor suppressor gene inactivated in colorectal cancer. We have found a 53% (27 of 51) rate of allelic loss at the LPL locus on 8p22, with the smallest region of overlap of deletions including the region D8S258 to D8S277. Using microcell-mediated monochromosome 8 transfer into three colorectal cancer cell lines, SW480, SW620 and HT29, we have demonstrated a reduction of tumorigenicity in SW620 hybrids. Partial deletions of chromosome 8 in some SW620/8 hybrids further delineate the critical region(s) to 8p22-23. Hybrids of the colorectal cancer cell lines SW480 and HT29 containing chromosome 8 did not show suppression of tumorigenesis, but the H29/8 hybrid showed total suppression of soft agar clonicity. This indicates an alternate pathway of mutational progression in these three lines, despite the fact that SW480 was derived from the same patient as SW620.

Increased binding of benzo[a]pyrene metabolites to lymphocytes from patients with lung cancer.

This study was performed to determine whether lymphocytes from individuals with lung cancer were more likely to bind benzo[a]pyrene (BP) metabolites in an in vitro test. The in vitro system consisted of peripheral blood lymphocytes incubated with increasing concentrations of [3H] benzo[a]pyrene in the presence of beta-naphthoflavone-induced rat liver microsomes and a NADPH-generating system. The radioactivity bound to a TCA-precipitate of the lymphocytes was determined. The apparent affinity (Km) and maximal binding (Vmax) of this binding were calculated from double reciprocal plots of the data. Seven patients with primary lung cancer (squamous cell carcinoma and undifferentiated small cell carcinoma), none of whom had smoked for at least 2 months, were studied as were 10 lung cancer free smokers, and the results compared with 13 age- and sex-matched controls. In lymphocytes from patients with primary lung cancer, Vmax of radiolabel bound to TCA-precipitable material was almost double that of non-smoking controls (205 +/- 19.2 pmol X 2h X 10(6) cells vs. 121 +/- 10.8 pmol X 2h X 10(6) cells, mean +/- S.E.M., P less than 0.01). Among individuals without lung cancer, smokers did not differ from non-smokers. In addition, there was no difference in Km of radiolabel binding to lymphocytes from patients in all 3 groups. It is concluded that binding of carcinogenic metabolites to cell components is increased in patients with lung cancer. Further studies are required to determine whether this increased binding is related to individual susceptibility of some smokers to lung cancer.