Heuristic results for the heterogeneous fleet instances.

<p>Heuristic results for the heterogeneous fleet instances.</p

Parameters and decision variables for heterogeneous fleet school bus scheduling problem.

<p>Parameters and decision variables for heterogeneous fleet school bus scheduling problem.</p

Exact and Metaheuristic Approaches for a Bi-Objective School Bus Scheduling Problem

<div><p>As a class of hard combinatorial optimization problems, the school bus routing problem has received considerable attention in the last decades. For a multi-school system, given the bus trips for each school, the school bus scheduling problem aims at optimizing bus schedules to serve all the trips within the school time windows. In this paper, we propose two approaches for solving the bi-objective school bus scheduling problem: an exact method of mixed integer programming (MIP) and a metaheuristic method which combines simulated annealing with local search. We develop MIP formulations for homogenous and heterogeneous fleet problems respectively and solve the models by MIP solver CPLEX. The bus type-based formulation for heterogeneous fleet problem reduces the model complexity in terms of the number of decision variables and constraints. The metaheuristic method is a two-stage framework for minimizing the number of buses to be used as well as the total travel distance of buses. We evaluate the proposed MIP and the metaheuristic method on two benchmark datasets, showing that on both instances, our metaheuristic method significantly outperforms the respective state-of-the-art methods.</p></div

Parameters and decision variables for homogeneous fleet bus scheduling problem.

<p>Parameters and decision variables for homogeneous fleet bus scheduling problem.</p

The school bus routing and scheduling instances RSRB and CSCB.

<p>The school bus routing and scheduling instances RSRB and CSCB.</p

Comparison of our algorithm with existing algorithm.

<p>Comparison of our algorithm with existing algorithm.</p

The Second Intracellular Loop of the Human Cannabinoid CB2 Receptor Governs G Protein Coupling in Coordination with the Carboxyl Terminal Domain

<div><p>The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the G_s protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both G_i- and G_s-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.</p></div

Functional characterization of cannabinoid receptor chimeras and mutants.

<p>The values are expressed as the mean ± SEM (<i>n = </i>3 experiments). % of maximal, the value of cAMP level percentage of the value obtained upon 10 µM forskolin treated only. Fold increase, the valve of cAMP level related to basal activity.</p><p>ND, not detectable.</p>α<p>The values were obtained in the absence of forskolin.</p>b<p>The values were obtained in the presence of 2 µM WIN55,212-2.</p

Comparison of effects of G_i inhibitor and PKA inhibitor on the activation of ERK in wild-type CB2 and P139L expressing cells.

<p>(<b>A</b>) Transiently transfected HEK293 cells were pretreated with or without 100 ng/mL <i>Pertussis toxin</i> (PTX) for 12 h or pretreated with or without 10 µM H89 for 30 min, and then stimulated with increasing concentrations of WIN55,212-2. (<b>B</b>) ERK signals were quantified by densitometry and expressed as a ratio of activated over total ERK. The maximal phosphorylation of ERK obtained in control cells at 5 min of stimulation with WIN55,212-2 in the absence of inhibitors were arbitrarily chosen as 100%. Each data point represents the mean ± SEM from three independent experiments. ***<i>P</i><0.001 compared with the entire control curve.</p

Effects of key residues in the ICL2 of the CB2 receptor on selectively G_i and G_s coupling.

<p>(<b>A</b>) Structures of CB2 mutations within the second intracellular loop as well as the C-terminal. (<b>B</b>) ELISA analysis of CB2 receptors expression. HEK293 cells were transiently transfected with Flag epitope-tagged receptors and the cell surface expression was measured by ELISA analysis, as described under <i>Methods</i>. The results represent the mean ± SEM of three independent experiments, each done in triplicate. (<b>C</b>) Dose response curve of cAMP accumulation for the CB2P139A and CB2P139L upon WIN55,212-2 stimulation. For cAMP measurements, cells were incubated with various concentrations of WIN55,212-2 for P139L and with various concentrations of WIN55,212-2 plus 10 µM forskolin for P139A and wild-type for 4 h. Values were expressed as percentage of forskolin stimulation for CB2P139A and CB2 wild-type, and as percentage of basal activity for CB2P139L. (<b>D</b>) Effects of substitutions of P139 with various kinds of amino acids in the CB2 receptor on WIN55,212-2-induced cAMP formation. HEK293 cells were treated with 2 µM WIN55,212-2, and cAMP production was measured as described in the <i>materials and methods</i>. The resulting increases in cAMP were expressed as fold increase above basal. Data are expressed as the mean ± SEM and are representative of three independent experiments.</p