Emergency department visits and unplanned hospitalizations in the treatment period for head and neck cancer patients treated with curative intent: A population-based analysis.

BACKGROUND: Mucosal head and neck squamous cell cancers are often managed with multimodality treatment which can be associated with significant toxicity. The objective of this study was to assess emergency department visits and unplanned hospitalizations for these patients during and immediately after their treatment. METHODS: A cohort of patients treated for head and neck squamous cell carcinoma was developed using administrative data. Emergency department visits and hospitalizations in the 90-day post-treatment period was determined. If a second treatment was initiated prior to the completion of 90 days, the attributable risk period was changed to the second treatment. RESULTS: Cohort of 3898 patients (1312 larynx/hypopharynx; 2586 oral cavity/oropharynx) from 2008 to 2012. The number of unplanned hospitalizations or ED visits (per 100 patient days) were 0.69 for surgery, 0.78 for surgery followed by concurrent chemoradiotherapy (CCRT), 0.55 for surgery followed by radiotherapy, 0.86 for CCRT, and 0.50 for radiation. Patients receiving CCRT had a statistically higher likelihood of treatment period events. The larynx/hypopharynx cancer subsite, higher comorbidity and more advanced stage of disease were all independent predictors of events. CONCLUSIONS: Patients undergoing treatment for head and neck cancer have significant unplanned hospitalizations and visits to the emergency department in the treatment period. Rates are higher in patients receiving CCRT. Quality improvement interventions should be used to improve these rates.

Characterization and Identification of Methods for Phenotyping Soybean Populations With Tolerance to the Soybean Aphid (Hemiptera: Aphididae).

The development of soybeans tolerant to the soybean aphid [Aphis glycines Matsumura (Hemiptera: Aphididae)] remains unexplored. The objectives of this research were to determine the susceptibility of two high-yielding soybean [Glycine max (L.) Merrill (Fabales: Fabaceae)] genotypes involved in a breeding platform to develop aphid-tolerant recombinant inbred lines (RILs); characterize the peroxidase activity and relative expression of peroxidase transcripts in the parents of RILs; and identify an assay to phenotype aphid-tolerant RILs. Enzyme kinetic assays documented the total peroxidase activity for tolerant (KS4202), susceptible (SD76R), and two high-yielding (U09-105007 and U11-611112) soybeans during two vegetative stages (V1 and V3) at three sampling days (D4, D6, and D8 after aphid introduction). Enzyme kinetic assays showed that V3 infested tolerant and U11-611112 plants had significantly higher peroxidase activity than their respective control plants at D4, and infested tolerant plants were also higher than control plants at D6. There were no apparent trends when comparing the expression of peroxidase-specific transcripts in the absence of aphids (basal levels) in both V1 and V3. Relative expression analyses of two peroxidase transcripts (PRX52 and PRX2) performed to compare differences among the soybean genotypes indicated that, despite basal levels being similar for the treatments analyzed, tolerant soybeans had a tendency for a higher expression of PRX52 in the presence of aphids. Based on the different patterns observed and the feasibility of analyses performed in this study, enzyme kinetics using V3 infested plants may be a marker for screening RILs in a breeding program targeting the development of aphid-tolerant soybeans.

Documenting Resistance and Physiological Changes in Soybean Challenged by Aphis glycines Matsumura (Hemiptera: Aphididae).

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a limiting factor in soybean production in the North Central region of the USA. The objectives of this work were to identify sources of resistance to A. glycines in 14 soybean genotypes, and also document changes in total protein, peroxidase, and chlorophyll in response to aphid feeding. A reduced number of A. glycines was observed on the genotypes UX 2569-159-2-01 and UX 2570-171- 04, indicating the presence of antixenosis and/or antibiosis. UX 2569-159-2-01 expressed the highest level of resistance; whereas, UX 2570-171-04 had moderate levels of resistance to A. glycines. Chlorophyll content was relatively unaffected by A. glycines, except for a reduction in UX 2569-159-2-01 infested plants at 5 and 15 days after infestation (DAI). No changes were detected in total protein content between infested and control plants for the genotypes analyzed; however, peroxidase activity was higher in infested UX 2570-171-04 at both 5 and 10 DAI. This improvement in peroxidase content in infested UX 2570-171-04 may be playing multiple roles in the plant tolerance.

Patterns and Drivers of Costs for Neuroendocrine Tumor Care: A Comparative Population-Based Analysis.

BACKGROUND: Little is known about resource use in the care of neuroendocrine tumors (NETs). This study defined patterns of costs in NET management and compared them with those of a more common malignancy, colon cancer (CC). METHODS: Using a provincial cancer registry (2004-2012), NET patients were identified and matched at a ratio of 1-3 with CC patients. Four phases of care were examined: pre-diagnostic (PreDx: -2 years to -181 days), diagnostic (Dx: -180 days to +180 days), postdiagnostic (PostDx: +181 days to +3 years), and prolonged post-diagnostic (PPostDx: +181 days to +9 years). The mean costs per patient were compared, and cost predictors were analyzed with quintile regression. RESULTS: Of 3827 NETs, 3355 were matched with 9320 CCs. The PreDx mean NET costs were higher than the CC costs ($5877 vs $5368; p = 0.06), driven by nondrug costs. They were lower in the Dx and PostDx phases (both p < 0.01). For PPostDx, the drug costs were higher for NETs ($26,788 vs $7827; p < 0.01), representing 41% of the costs versus 16% of the costs for CC. Older age and comorbidities predicted higher NET costs in all phases. Lower socioeconomic status (SES) predicted higher costs in the initial phases and higher SES costs in the PPost-Dx phase. Gastroenteric NETs were associated with lower costs in the Dx phase [parameter estimate (PE), -$13,644] and pancreatic NETs with higher costs in PostDx phase (PE, $3348). CONCLUSION: Currently, NETs represent a potential important health care burden. The NET cost patterns differed from those for CC, with the highest costs during the PPostDx phase. The SES and primary NET site affected costs differently at different time points. These data can inform resource allocation tailored to the needs for NETs.

A population-based study of ethnicity and breast cancer stage at diagnosis in Ontario.

BACKGROUND: Breast cancer stage at diagnosis is an important predictor of survival. Our goal was to compare breast cancer stage at diagnosis (by American Joint Committee on Cancer criteria) in Chinese and South Asian women with stage at diagnosis in the remaining general population in Ontario. METHODS: We used the Ontario population-based cancer registry to identify all women diagnosed with breast cancer during 2005-2010, and we applied a validated surname algorithm to identify South Asian and Chinese women. We used logistic regression to compare, for Chinese or South Asian women and for the remaining general population, the frequency of diagnoses at stage ii compared with stage i and stages ii-iv compared with stage i. RESULTS: The registry search identified 1304 Chinese women, 705 South Asian women, and 39,287 women in the remaining general population. The Chinese and South Asian populations were younger than the remaining population (mean: 54, 57, and 61 years respectively). Adjusted for age, South Asian women were more often diagnosed with breast cancer at stage ii than at stage i [odds ratio (or): 1.28; 95% confidence interval (ci): 1.08 to 1.51] or at stages ii-iv than at stage i (or: 1.27; 95% ci: 1.08 to 1.48); Chinese women were less likely to be diagnosed at stage ii than at stage i (or: 0.82; 95% ci: 0.72 to 0.92) or at stages ii-iv than at stage i (or: 0.73; 95% ci: 0.65 to 0.82). CONCLUSIONS: Breast cancers were diagnosed at a later stage in South Asian women and at an earlier stage in Chinese women than in the remaining population. A more detailed analysis of ethnocultural factors influencing breast screening uptake, retention, and care-seeking behavior might be needed to help inform and evaluate tailored health promotion activities.

Large scale refolding and purification of the catalytic domain of human BACE-2 produced in E. coli.

Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.

Published randomized controlled trials of drug therapy for dementia often lack complete data on harm.

OBJECTIVE: The objective of the study was to determine the extent to which published randomized controlled trials (RCTs) report data on harm. STUDY DESIGN AND SETTING: A systematic search strategy was used to identify RCTs published between 1996 and 2005 on the use of cholinesterase inhibitors or atypical antipsychotics in patients with dementia. A structured abstraction form was used to determine if data on mortality or serious adverse events were reported and if the articles followed Consolidated Standards of Reporting Trials format for reporting harm. RESULTS: Thirty-three RCTs were identified (27 on cholinesterase inhibitors and 6 on atypical antipsychotics). Nineteen trials (58%) had explicit data on mortality and only four (12%) reported regulatory-agency-defined serious adverse events. Most abstracts (31, 94%) stated that harm was studied but few studies (9, 27%) provided a clear definition of the measures of harm. CONCLUSIONS: Although most published RCTs state that they examine harm, many failed to provide data on mortality and most lacked clear definitions or detailed analyses of harm. Better reporting of harm would provide timely and important information that could help physicians and the public to make more informed decisions.

Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.

BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

Expression of a structural domain of the beta 2 subunit essential for alpha M beta 2 ligand recognition.

The beta2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of beta2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the alpha and beta subunit. As an approach to identify and characterize regulatory domains of the beta2 subunit, we have generated two different subdomains of the beta2 subunit for expression on the surface of mammalian cells through a phosphatidyl-inositol glycan anchor. The first subdomain contains the putative beta2 MIDAS motif implicated in ligand binding [beta2(LB)], whereas the second beta2 subdomain contains the cysteine-rich region [beta2(CR)]. Cells expressing alphaM and beta2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant alphaMbeta2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete alphaMbeta2 heterodimer. In contrast, cells expressing alphaMbeta2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected alpha or beta chains alone failed to adhere to immobilized iC3b. These results indicate that along with alphaM, the beta2(LB) subdomain contains the sufficient components within the beta2 subunit essential for ligand recognition. These findings support the hypothesis that the beta2 subunit cooperates with site(s) within the alphaM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction.

A peptide inhibitor of cholesteryl ester transfer protein identified by screening a bacteriophage display library.

We screened a bacteriophage display library of random decapeptides to identify peptide inhibitors of cholesteryl ester transfer protein (CETP). After affinity selection against CETP, bacteriophage-infected Escherichia coli were plated at clonal density and 36 random clones were isolated. Analysis of the relevant portion of the bacteriophage DNA from a group of 12 clones that had a relatively high affinity for CETP revealed that the corresponding amino acid sequences of the displayed peptides exhibited an ... Xaa-Arg-Met-Arg-Tyr-Xaa ... composite motif. Based on those results, decapeptides from this group were synthesized and one of them, DP1 (NH2-VTWRMWYVPA-COOH), inhibited CETP-catalyzed transfer of cholesteryl esters and triglycerides. Amino- and carboxy-terminal truncations of DP1 demonstrated that the original decapeptide could be reduced to a pentapeptide without loss of either its ability to bind to CETP or its ability to inhibit CETP-mediated lipid transfer. That pentapeptide, NH2-WRMWY-COOH (WRMWY, PNU-107368E), binds directly to CETP and its inhibition is consistent with that of a competitive inhibitor of CETP with a Ki of 164 microM. WRMWY or modified versions of this peptide may be useful in studying the interactions between CETP and plasma lipoproteins.

Restriction and functional changes of dopamine release in rat striatum from young adult and old rats.

In order to investigate the age-related changes in dopaminergic activity in rats, we have utilized the K(+)- and veratridine-stimulated [14C]dopamine release from striatum in vitro as a functional index of responsiveness to these stimuli in aging. We found that the K(+)-stimulated dopamine release from old (12 months) rats decreased by more than 50% compared to that from young adult rats (3 months). Reserpine (5 mg/kg) led to a pronounced decrease of the K(+)-stimulated dopamine release of young adult as well as old rats. Whereas ouabain (10 mumol/l) decreased the K(+)-stimulated dopamine release from young adult rats, in old rats the K(+)-induced dopamine release was increased up to 250%. However, in old rats which were reserpine pretreated, ouabain was unable to stimulate the K(+)-induced dopamine release. In contrast, the veratridine-stimulated dopamine release of old rats was increased up to 200% compared to that of young adult rats and was highly sensitive to reserpine pretreatment but not to ouabain. However, reserpine did not alter this veratridine-stimulated dopamine release from young adult rats. The present data indicate that the age-related reduction of exocytosis-related, Ca(2+)-dependent release mechanisms (K+) are probably compensated via an increase in Ca(2+)-independent, uptake carrier-mediated release processes (veratridine).

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells.

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2 micrograms/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band of M(r) ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.

Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition.

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.

Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope.

Libraries of random peptides can be screened to identify species which interact with antibodies or receptors. Similarly, maps of native molecular interactions can frequently be deduced by screening a limited set of peptide fragments derived from sequences within a native antigen or ligand. However, the existence of cross-reactive sequences that mimic original epitopes and the limited replaceability of amino acid residues suggest that the sequence space accessible by a receptor can be much broader. Definition of this space is of particular importance where structural information is required for peptidomimetic or drug design. We have used a two-stage selection scheme to expand the sequence space accessible by a phage display library and to define peptide epitopes of the anti-FLAG octapeptide monoclonal M2 antibody. Affinity selection of a primary library of 2 x 10(6) random decapeptides identified a non-contiguous core of three residues in the binding motif Tyr-Lys-Xaa-Xaa-Asp. A second stage library with 2 x 10(7) individual clones bearing the core motif but with the remaining flanking and internal residues re-randomized permitted access to a broader sequence space represented in a library equivalent to several orders of magnitude larger. Data here demonstrate that extended access to binding sequence space permitted by multi-stage screening of phage display libraries can reveal not only essential residues required for ligand binding, but also the ligand structural range permitted within the receptor binding pocket.

Long-term effects of postnatal hypoxia and flunarizine on the dopaminergic system.

Long-term changes of learning behavior and of the striatal dopaminergic system were observed in a rat model of early postnatal hypoxia. Striatal dopamine (DA) concentration, K(+)-stimulated DA release from slices, and DA uptake into crude synaptosomal preparations (S1 fractions) were used as markers of the striatal DAergic system. Active avoidance learning was tested as behavioral criterion. Cyclodextrin and flunarizine were found to produce long-term effects on the DAergic system in control animals. While cyclodextrin normalized hypoxia-induced effects in DA release, flunarizine prevented those in DA uptake and improved avoidance learning.

Effects of potassium channel openers and their antagonists on rat locus coeruleus neurones.

1. Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP-sensitive potassium (K(ATP)) channels, RO 31-6930 (10 microM) and cromakalim (100 microM) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential. 2. Of two K(ATP) channel blockers, tolbutamide (300 microM) increased the firing rate, while glibenclamide (3 microM) only tended to do so. In addition, both compounds antagonized the effect of RO 31-6930 (10 microM). Neither glibenclamide (3 microM) nor tolbutamide (300 microM) altered the resting membrane potential. 3. Tetrodotoxin (0.5 microM) depressed the firing, but did not influence the inhibitory action of RO 31-6930 (10 microM). The excitatory amino acid antagonist, kynurenic acid (500 microM), did not change the spontaneous discharge of action potentials. 4. Small shifts (2-4 mV) of the membrane potential by hyper- or depolarizing current injections markedly decreased and increased the firing rate, respectively. 5. Noradrenaline (100 microM) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 microM) or tolbutamide (300 microM). Ba2+ (2 mM), a blocker of both ATP-sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31-6930 (10 microM) and noradrenaline (100 microM). 6. These data suggest that K(ATP) channels are present on the noradrenergic LC neurones, but are not coupled to alpha 2-adrenoceptors.