Single-nucleotide polymorphisms in a short basic motif in the ABC transporter ABCG2 disable its trafficking out of endoplasmic reticulum and reduce cell resistance to anticancer drugs

ATP-binding cassette (ABC) subfamily G member 2 (ABCG2) belongs to the ABC transporter superfamily and has been implicated in multidrug resistance of cancers. Although the structure and function of ABCG2 have been extensively studied, little is known about its biogenesis and the regulation thereof. In this study, using mutagenesis and several biochemical analyses, we show that the positive charges in the vicinity of the RKR motif downstream of the ABC signature drive trafficking of nascent ABCG2 out of the endoplasmic reticulum (ER) onto plasma membranes. Substitutions of and naturally occurring single-nucleotide polymorphisms within these positively charged residues disabled the trafficking of ABCG2 out of the ER. A representative ABCG2 variant in which the RKR motif had been altered underwent increased ER stress-associated degradation. We also found that unlike WT ABCG2, genetic ABCG2 RKR variants have disrupted normal maturation and do not reduce accumulation of the anticancer drug mitoxantrone and no longer confer resistance to the drug. We conclude that the positive charges downstream of the ABC signature motif critically regulate ABCG2 trafficking and maturation. We propose that single-nucleotide polymorphisms of these residues reduce ABCG2 expression via ER stress-associated degradation pathway and may contribute to reduced cancer drug resistance, improving the success of cancer chemotherapy

Task Offloading and Resource Allocation for Tasks with Varied Requirements in Mobile Edge Computing Networks

Edge computing enables devices with insufficient computing resources to offload their tasks to the edge for computing, to improve the service experience. Some existing work has noticed that the data size of offloaded tasks played a role in resource allocation shares but has not delved further into how the data size of an offloaded task affects resource allocation. Among offloaded tasks, those with larger data sizes often consume a larger share of system resources, potentially even monopolizing system resources if the data size is large enough. As a result, tasks with small or regular sizes lose the opportunity to be offloaded to the edge due to their limited data size. To address this issue, we introduce the concept of an emergency factor to penalize tasks with immense sizes for monopolizing system resources, while supporting tasks with small sizes to contend for system resources. The joint offloading decision and resource allocation problem is formulated as a mixed-integer nonlinear programming (MINLP) problem and further decomposed into an offloading decision subproblem and a resource allocation subproblem. Using the KKT conditions, we design a bisection search-based algorithm to find the optimal resource allocation scheme. Additionally, we propose a linear-search-based coordinate descent (CD) algorithm to identify the optimal offloading decision. Numerical results show that our proposed algorithm converges to the optimal scheme (for the minimal delay) when tasks are of regular size. Moreover, when tasks of immense, small and regular sizes coexist in the system, our scheme can exclude tasks of immense size from edge resource allocation, while still enabling tasks of small size to be offloaded

The Cardiac Ca2+-Sensitive Regulatory Switch, a System in Dynamic Equilibrium

The Ca 2+ -sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca 2+ , and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements suggest that the switch activates in two steps: a TnI-independent Ca 2+ -priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation after rapid addition of a lacking component (Ca 2+ or TnI) and deactivation after rapid chelation of Ca 2+ . Time-resolved measurements, stopped-flow measurements, and Ca 2+ -titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal that 1), the Ca 2+ -induced priming step, which precedes opening, is the rate-limiting step in activation; 2), closing is the rate-limiting step in de-activation; 3), TnI induces opening; 4), there is an incompletely deactivated population when regulatory Ca 2+ is not bound, which generates an accessory pathway of activation; and 5), there is incomplete activation by Ca 2+ —when regulatory Ca 2+ is bound, a 3:2 mixture of dynamically interconverting open (active) and primed-closed (partially active) conformers is observed (15°C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermokinetic parameterization of opening: the enthalpy change (Δ H = −33.4 kJ/mol), entropy change (Δ S = −0.110 kJ/mol/K), heat capacity change (Δ C p = −7.6 kJ/mol/K), the enthalpy of activation ( δ ‡ = 10.6 kJ/mol) and the effective barrier crossing attempt frequency ( ν adj = 1.8 × 10 4 s −1 )

A Spatiotemporal Hierarchical Analysis Method for Urban Traffic Congestion Optimization Based on Calculation of Road Carrying Capacity in Spatial Grids

Traffic congestion is a globally widespread problem that causes significant economic losses, delays, and environmental impacts. Monitoring traffic conditions and analyzing congestion factors are the first, challenging steps in optimizing traffic congestion, one of the main causes of which is regional spatiotemporal imbalance. In this article, we propose an improved spatiotemporal hierarchical analysis method whose steps include calculating road carrying capacity based on geospatial data, extracting vehicle information from remote sensing images to reflect instantaneous traffic demand, and analyzing the spatiotemporal matching degree between roads and vehicles in theory and in practice. First, we defined and calculated the ratio of carrying capacity in a regional road network using a nine-cell-grid model composed of nested grids of different sizes. By the conservation law of flow, we determined unbalanced areas in the road network configuration using the ratio of the carrying capacity of the central cell to that of the nine grid cells. Then, we designed a spatiotemporal analysis method for traffic congestion using real-time traffic data as the dependent variables and five selected spatial indicators relative to the spatial grids as the independent variables. The proposed spatiotemporal analysis method was applied to Chengdu, a typical provincial capital city in China. The relationships among regional traffic, impact factors, and spatial heterogeneity were analyzed. The proposed method effectively integrates GIS, remote sensing, and deep learning technologies. It was further demonstrated that our method is reliable and effective and enhances the coordination of congested areas by virtue of a fast calculation speed and an efficient local balance adjustment

Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex

Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context

In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-μm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart