Implementation of Enhanced Recovery After Surgery (ERAS) Across a Provincial Healthcare System: The ERAS Alberta Colorectal Surgery Experience.

BACKGROUND: Enhanced recovery after surgery (ERAS) colorectal guideline implementation has occurred primarily in standalone institutions worldwide. We implemented the guideline in a single provincial healthcare system, and our study examined the effect of the guideline on patient outcomes [length of stay (LOS), complications, and 30-day post-discharge readmissions] across a healthcare system. METHODS: We compared pre- and post-guideline implementation in consecutive elective colorectal patients, ≥ 18 years, from six Alberta hospitals between February 2013 and December 2014. Participants were followed up to 30 days post discharge. We used summary statistics, to assess the LOS and complications, and multivariate regression methods to assess readmissions and to estimate cost impacts. RESULTS: A total of 1333 patients (350 pre- and 983 post-ERAS) were analysed. Of this number, 55 % were males. Median overall guideline compliance was 39 % in pre- and 60 % in post-ERAS patients. Median LOS was 6 days for pre-ERAS compared to 4.5 days in post-ERAS patients with the longest implementation (p value <0.0001). Adjusted risk ratio (RR) was 1.71, 95 % CI 1.09-2.68 for 30-day readmission, comparing pre- to post-ERAS patients. The proportion of patients who developed at least one complication was significantly reduced, from pre- to post-ERAS, difference in proportions = 11.7 %, 95 % CI 2.5-21.0, p value: 0.0139. The net cost savings attributable to guideline implementation ranged between $2806 and $5898 USD per patient. CONCLUSION: The findings in our study have shown that ERAS colorectal guideline implementation within a healthcare system resulted in patient outcome improvements, similar to those obtained in smaller standalone implementations. There was a significant beneficial impact of ERAS on scarce health system resources.

A highly conserved cytoplasmic cysteine residue in the α4 nicotinic acetylcholine receptor is palmitoylated and regulates protein expression.

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4ß2 nAChR.

Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites.

• Inorganic arsenic (As(i) ) in rice (Oryza sativa) grains is a possible threat to human health, with risk being strongly linked to total dietary rice consumption and consumed rice As(i) content. This study aimed to identify the range and stability of genetic variation in grain arsenic (As) in rice. • Six field trials were conducted (one each in Bangladesh and China, two in Arkansas, USA over 2 yr, and two in Texas, USA comparing flooded and nonflood treatments) on a large number of common rice cultivars (c. 300) representing genetic diversity among international rice cultivars. • Within each field there was a 3-34 fold range in grain As concentration which varied between rice subpopulations. Importantly, As(i) correlated strongly with total As among a subset of 40 cultivars harvested in Bangladesh and China. • Genetic variation at all field sites was a large determining factor for grain As concentration, indicating that cultivars low in grain As could be developed through breeding. The temperate japonicas exhibited lower grain As compared with other subpopulations. Effects for year, location and flooding management were also statistically significant, suggesting that breeding strategies must take into account environmental factors.

Local entropy and structure in a two-dimensional frustrated system.

We calculate the local contributions to the Shannon entropy and excess entropy and use these information theoretic measures as quantitative probes of the order arising from quenched disorder in the diluted Ising antiferromagnet on a triangular lattice. When one sublattice is sufficiently diluted, the system undergoes a temperature-driven phase transition, with the other two sublattices developing magnetizations of equal magnitude and opposite sign as the system is cooled.(1) The diluted sublattice has no net magnetization but exhibits spin glass ordering. The distribution of local entropies shows a dramatic broadening at low temperatures; this indicates that the system's total entropy is not shared equally across the lattice. The entropy contributions from some regions exhibit local reentrance, although the entropy of the system decreases monotonically as expected. The average excess entropy shows a sharp peak at the critical temperature, showing that the excess entropy is sensitive to the structural changes that occur as a result of the spin glass ordering.

Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

The synthesis of 5-substituted ring E analogs of methyllycaconitine via the Suzuki-Miyaura cross-coupling reaction.

Novel 3,5-disubstituted ring E analogs of methyllycaconitine were prepared and evaluated in nicotinic acetylcholine receptor binding assays. The desired analogs were prepared through the Suzuki-Miyaura cross-coupling reaction of methyl 5-bromo-nicotinate. The Suzuki-Miyaura cross-coupling reactions of pyridines with electron withdrawing substituents have not been extensively described previously.

Pharmacological and immunological identification of native alpha7 nicotinic receptors: evidence for homomeric and heteromeric alpha7 receptors.

Controversy surrounds the expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, alpha7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [(125)I]alpha-bungarotoxin (alphaBGT) binding reaches equilibrium within 4 h and is saturable with a K(d) value of 4.2 nM. Using homologous competition experiments, the K(i) for binding of alphaBGT was 1.9 nM. These data are consistent with the expression of homomeric alpha7 nAChRs. Methyllycaconatine (MLA), which binds alpha7 nAChRs with high affinity, inhibits [(125)I]alphaBGT binding in a concentration-dependent manner with a K(i) of 30.6 nM; this value is approximately 10 fold higher than the reported affinity of MLA for alpha7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate alpha7 nAChRs, as has been previous demonstrated for bovine adrenal alpha3beta4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes alpha7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha3 and alpha5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. Together, these results support the expression of alpha7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha7 nAChRs.

Evidence for constitutive expression of bovine adrenal a3beta4* nicotinic acetylcholine receptors.

Many pathological conditions involve alterations in expression of nicotinic acetylcholine receptors (nAChRs). The following studies were designed to investigate cellular mechanisms involved with expression and turnover of alpha3beta4* nAChRs. These studies support constitutive turnover of adrenal alpha3beta4* nAChRs and the use of cultured adrenal chromaffin cells to study nAChR regulation.

Structure-activity studies with ring E analogues of methyllycaconitine on bovine adrenal alpha3beta4* nicotinic receptors.

The development of new agents that selectively interact with subtypes of neuronal nicotinic receptors (nAChRs) is of primary importance for the study of physiological processes and pathophysiological conditions involving these receptors. Our laboratory has evidence that simple ring E analogues of methyllycaconitine (MLA) act as antagonists to bovine adrenal alpha3beta4* nAChRs. The following studies were designed to characterize the concentration-response effects of several ring E analogues of MLA in order to assess structural requirements involved with their inhibitory activity on bovine adrenal alpha3beta4* nAChRs. Ring E analogues with various substitutions on the ring E nitrogen were tested for their ability to inhibit nicotinic stimulated adrenal catecholamine release and [3H]epibatidine binding to a bovine adrenal membrane preparation. Several N-alkyl derivatives inhibited secretion with IC50 values in the low micromolar range. The N-phenpropyl analogue was the most potent of the analogues tested (IC50, 11 microM) on adrenal secretion. Competition binding studies suggest a noncompetitive interaction of the analogues with bovine adrenal nAChRs. These studies identify several structural features of ring E analogues of MLA which significantly affect their inhibitory activity on bovine adrenal alpha3beta4* nAChRs.

[3H]Epibatidine binding to bovine adrenal medulla: evidence for alpha3beta4* nicotinic receptors.

In these studies, [3H]epibatidine is used as the radioligand to characterize nicotinic acetylcholine receptors (nAChRs) from bovine adrenal medulla. Specific binding reaches equilibrium within 30 min, and is saturable with a Kd value of 0.5 nM. The affinities of several cholinergic agents were determined, including nicotine (Ki, 0.2 microM), cytisine (Ki, 0.4 microM), carbachol (Ki, 4.7 microM), dihydro-beta-erythrodine (Ki, 33.6 microM), D-tubocurarine (Ki, 0.4 microM), 1,1-dimethyl-4-phenyl-piperazinium (Ki, 0.8 microM), decamethonium (Ki, 234 microM) and methyllycaconitine (Ki, 1.3 microM). These values are similar to reported values for recombinant alpha3beta4 nAChRs in transfected cell lines. These studies demonstrate [3H]epibatidine binding to an easily obtainable adrenal membrane preparation and support the characterization of adrenal nAChRs as alpha3beta4* nAChRs.

Position-space renormalization-group approach for driven diffusive systems applied to the asymmetric exclusion model.

This paper introduces a position-space renormalization-group approach for nonequilibrium systems and applies the method to a driven stochastic one-dimensional gas with open boundaries. The dynamics are characterized by three parameters: the probability alpha that a particle will flow into the chain to the leftmost site, the probability beta that a particle will flow out from the rightmost site, and the probability p that a particle will jump to the right if the site to the right is empty. The renormalization-group procedure is conducted within the space of these transition probabilities, which are relevant to the system's dynamics. The method yields a critical point at alpha(c)=beta(c)=1/2, in agreement with the exact values, and the critical exponent nu=2.71, as compared with the exact value nu=2.00.

Evidence for the involvement of adenomatous polyposis coli (APC) protein in maintaining cellular distributions of α3ß4 nicotinic receptors.

Evidence exists supporting the involvement of adenomatous polyposis coli (APC) protein in the assembly of neuronal nicotinic acetylcholine receptors (nAChRs) in the postsynaptic complex. In the following studies, the effects of APC protein on cellular distribution of recombinant α3ß4 nAChRs was investigated. RT-PCR and Western blotting techniques established the expression of APC protein both in bovine adrenal chromaffin cells, which express native α3ß4* nAChRs, and in a HEK293 cell line expressing recombinant bovine adrenal α3ß4 nAChRs (BMα3ß4 cells). Transfection of BMα3ß4 cells with siRNA to APC, reduced APC protein levels to 52.4% and 61.9% of control values at 24 and 48 h after transfection. To investigate the effects of APC on the cellular distribution of α3ß4 nAChRs, [(3)H]epibatidine binding approaches, coupled with APC siRNA treatment, were used. Twenty-four and 48 h after APC siRNA transfection, intracellular nAChRs were significantly reduced to 71% and 68% of control, respectively, while the total population of nAChRs were not significantly changed. Given that total cellular nAChRs represent the sum of surface and intracellular nAChRs, these studies support a re-distribution of nAChRs to the plasma membrane with APC siRNA treatment. Treatment of the cells with the protein synthesis inhibitor, puromycin, also caused a significant reduction (55%) in APC protein levels, and produced a similar re-distribution of cellular nAChRs. These studies support the involvement of APC protein in the maintenance of normal cellular distribution of α3ß4 nAChRs.

Presynaptic targeting of alpha4beta 2 nicotinic acetylcholine receptors is regulated by neurexin-1beta.

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between alpha4beta2 AChRs and the presynaptic cell adhesion molecule, neurexin-1beta. In non-neuronal tsA 201 cells, recombinant neurexin-1beta and mature alpha4beta2 AChRs form complexes. alpha4beta2 AChRs and neurexin-1beta also coimmunoprecipitate from rat brain lysates. When exogenous alpha4beta2 AChRs and neurexin-1beta are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1beta. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1beta lacking the extracellular domain. Additionally, when alpha4beta2 AChRs, alpha7 AChRs, and neurexin-1beta are coexpressed in the same neuron, only the alpha4beta2 AChR colocalizes with neurexin-1beta at presynaptic terminals. Collectively, these data suggest that neurexin-1beta targets alpha4beta2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on alpha4beta2 AChRs, may contribute to the etiology of these neurological disorders.

Analogs of methyllycaconitine as novel noncompetitive inhibitors of nicotinic receptors: pharmacological characterization, computational modeling, and pharmacophore development.

As a novel approach to drug discovery involving neuronal nicotinic acetylcholine receptors (nAChRs), our laboratory targeted nonagonist binding sites (i.e., noncompetitive binding sites, negative allosteric binding sites) located on nAChRs. Cultured bovine adrenal cells were used as neuronal models to investigate interactions of 67 analogs of methyllycaconitine (MLA) on native alpha3beta4* nAChRs. The availability of large numbers of structurally related molecules presents a unique opportunity for the development of pharmacophore models for noncompetitive binding sites. Our MLA analogs inhibited nicotine-mediated functional activation of both native and recombinant alpha3beta4* nAChRs with a wide range of IC(50) values (0.9-115 microM). These analogs had little or no inhibitory effects on agonist binding to native or recombinant nAChRs, supporting noncompetitive inhibitory activity. Based on these data, two highly predictive 3D quantitative structure-activity relationship (comparative molecular field analysis and comparative molecular similarity index analysis) models were generated. These computational models were successfully validated and provided insights into the molecular interactions of MLA analogs with nAChRs. In addition, a pharmacophore model was constructed to analyze and visualize the binding requirements to the analog binding site. The pharmacophore model was subsequently applied to search structurally diverse molecular databases to prospectively identify novel inhibitors. The rapid identification of eight molecules from database mining and our successful demonstration of in vitro inhibitory activity support the utility of these computational models as novel tools for the efficient retrieval of inhibitors. These results demonstrate the effectiveness of computational modeling and pharmacophore development, which may lead to the identification of new therapeutic drugs that target novel sites on nAChRs.

Expression of native alpha3beta4* neuronal nicotinic receptors: binding and functional studies investigating turnover of surface and intracellular receptor populations.

Several pathological conditions involve alterations in expression of neuronal nicotinic acetylcholine receptors (nAChRs). Although some studies have addressed processes involved with muscle nAChR expression, knowledge of the regulation of neuronal nAChRs is particularly sparse. The following studies were designed to investigate cellular mechanisms involved with expression of neuronal alpha3beta4* nAChRs. Catecholamine secretion assays and receptor binding studies coupled with receptor alkylation were used to study the nAChR regulation and turnover. Alkylation of adrenal nAChRs results in a rapid and complete loss of receptor-mediated neurosecretion and surface [(3)H]epibatidine binding sites. After alkylation, both neurosecretory function and nAChR binding slowly (24-48 h) return to prealkylation levels. When cells are treated with the protein synthesis inhibitor puromycin, after alkylation, receptor-mediated neurosecretion does not recover. Long-term treatment (24-48-h) with puromycin, in the absence of alkylation, results in a slow, time-dependent shift to the right, followed by a downward shift, in the nicotine concentration-response curve, documenting a disappearance of surface nAChRs. Puromycin treatment alone also results in a loss to both surface and intracellular [(3)H]epibatidine binding sites. nAChR beta4 subunit levels are significantly decreased after treatment with puromycin. These data support a constitutive turnover of adrenal alpha3beta4* nAChRs, requiring continual de novo synthesis of new receptor protein.