[Study on changes of Styrax varieties].

Styrax is a commonly used imported traditional Chinese medicinal material in China. It was introduced to China in the Han Dynasty and was first described as a traditional Chinese medicine in Miscellaneous Records of Famous Physicians(Ming Yi Bie Lu). In this paper, by combing ancient and modern Chinese and foreign herbal medicine books and modern literature, combined with the results of field investigations on the origin of Styrax, the changes of Styrax involving the name, quality evaluation, origin, place of origin, and harvesting and processing were systematically verified. The results show that since ancient times, the origin and place of origin of Styrax have been unclear. The medical scientists of all dynasties in China have evaluated the quality of Styrax from four aspects: texture, viscosity, odor concentration, and color. The varieties of Styrax changed twice. The first change may have occurred during the Sui and Tang Dynasties, and the base changed from Styrax officinalis to Liquidambar orientalis. The second change was in modern times, and the base changed from L. orientalis to L. styraciflua. At the same time, the place of origin changed for the first time, from Turkey, Syria, and other countries in southern Asia Minor to Honduras, Guatemala, and other countries in Central America and southern North America. This paper studied the historical evolution of Styrax in terms of quality evaluation, origin, place of origin, character, and harvesting and processing. At the same time, it summarized the application of Styrax in the western countries, which can provide a historical basis for the further development and utilization of Styrax.

Mediator subunit MED1 deficiency prevents carbon tetrachloride-induced hepatic fibrosis in mice.

Mediator subunit mediator 1 (MED1) mediates ligand-dependent binding of the mediator coactivator complex to various nuclear receptors and plays a critical role in embryonic development, lipid and glucose metabolism, liver regeneration, and tumorigenesis. However, the precise role of MED1 in the development of liver fibrosis has been unclear. Here, we showed that MED1 expression was increased in livers from nonalcoholic steatohepatitis (NASH) patients and mice and positively correlated with transforming growth factor ß (TGF-ß) signaling and profibrotic factors. Upon treatment with carbon tetrachloride (CCl4), hepatic fibrosis was much less in liver-specific MED1 deletion (MED1ΔLiv) mice than in MED1fl/fl littermates. TGF-ß/Smad2/3 signaling pathway was inhibited, and gene expression of fibrotic markers, including α-smooth muscle actin (α-SMA), collagen type 1 α 1 (Col1a1), matrix metalloproteinase-2 (Mmp2), and metallopeptidase inhibitor 1 (Timp1) were decreased in livers of MED1ΔLiv mice with CCl4 injection. Transcriptomic analysis revealed that the differentially expressed genes in livers of CCl4-administered MED1ΔLiv mice were enriched in the pathway of oxidoreductase activity, followed by robustly reduced oxidoreductase activity-related genes, such as Gm4756, Txnrd3, and Etfbkmt. More importantly, we found that the reduction of reactive oxygen species (ROS) in MED1 knockdown hepatocytes blocked the activation of TGF-ß/Smad2/3 pathway and the expression of fibrotic genes in LX2 cells. These results indicate that MED1 is a positive regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for the regression of liver fibrosis.NEW & NOTEWORTHY In this study, we present the first evidence that liver mediator 1 (MED1) deficiency attenuated carbon tetrachloride-induced hepatic fibrosis in mouse. The underlying mechanism is that MED1 deficiency reduces reactive oxygen species (ROS) production in hepatocytes, thus restricts the activation of TGF-ß/Smad2/3 signaling pathway and fibrogenic genes expression in hepatic stellate cells (HSCs). These data suggest that MED1 is an essential regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for liver fibrosis.

Enhanced Sonodynamic Cancer Therapy through Iron-Doping and Oxygen-Vacancy Engineering of Piezoelectric Bismuth Tungstate Nanosheets.

Sonodynamic therapy (SDT) is regarded as a new-rising strategy for cancer treatment with low invasiveness and high tissue penetration, but the scarcity of high-efficiency sonosensitizers has seriously hindered its application. Herein, the iron-doped and oxygen-deficient bismuth tungstate nanosheets (BWO-Fe NSs) with piezotronic effect are synthesized for enhanced SDT. Due to the existence of oxygen defects introduced through Fe doping, the bandgap of BWO-Fe is significantly narrowed so that BWO-Fe can be more easily activated by exogenous ultrasound (US). The oxygen defects acting as the electron traps inhibit the recombination of US-induced electrons and holes. More importantly, the dynamically renewed piezoelectric potential facilitates the migration of electrons and holes to opposite side and causes energy band bending, which further promotes the production of reactive oxygen species. Furthermore, Fe doping endows BWO-Fe with Fenton reactivity, which converts hydrogen peroxide (H2 O2 ) in tumor microenvironment into hydroxyl radicals (•OH), thereby amplifying the cellular oxidative damage and enhancing SDT. Both in vitro and in vivo experiments illustrate their high cytotoxicity and tumor suppression rate against refractory breast cancer in mice. This work may provide an alternative strategy to develop oxygen-deficient piezoelectric sonosensitizers for enhanced SDT via doping metal ions.

MED1 Deficiency in Macrophages Aggravates Isoproterenol-Induced Cardiac Fibrosis in Mice.

Mediator 1 (MED1), a key subunit of the mediator complex, interacts with various nuclear receptors and functions in lipid metabolism and energy homeostasis. Dilated cardiomyopathy-related ventricular dilatation and heart failure have been reported in mice with cardiomyocyte-specific Med1 deficiency. However, the contribution of macrophage-specific MED1 in cardiac remodeling remains unclear. In this study, macrophage-specific Med1 knockout (Med1ΔMac) mice were generated and exposed to isoproterenol (ISO) to induce cardiac fibrosis; these mice showed aggravated cardiac fibrosis compared with Med1fl/fl mice. The levels of expression of marker genes for myofibroblast transdifferentiation [α-smooth muscle actin (SMA)] and of profibrotic genes, including Col1a1, Col3a1, Postn, Mmp2, Timp1, and Fn1, were significantly increased in the cardiac tissues of Med1ΔMac mice with ISO-induced myocardial fibrosis. In particular, the transforming growth factor (TGF)-ß-Smad2/3 signaling pathway was activated. In bone marrow-derived and peritoneal macrophages, Med1 deficiency was also associated with elevated levels of expression of proinflammatory genes, including Il6, Tnfa, and Il1b. These findings indicate that macrophage-specific MED1 deficiency may aggravate ISO-induced cardiac fibrosis via the regulation of the TGF-ß-SMAD2/3 pathway, and the underlying mechanism may involve MED1 deficiency triggering the activation of inflammatory cytokines in macrophages, which in turn may stimulate phenotypic switch of cardiac fibroblasts and accelerate cardiac fibrosis. Thus, MED1 is a potential therapeutic target for cardiac fibrosis.

A robust t1 noise suppression method in NMR spectroscopy.

Artefacts in high-resolution multidimensional nuclear magnetic resonance (NMR) spectra, known as t1 noise, can significantly downgrade the spectral quality and remain a significant noise source, limiting the sensitivity of most two-dimensional NMR experiments. In addition to highly sensitive hardware and experimental designs, data post-processing is a relatively simple and cost-effective method for suppressing t1 noise. In this study, histograms and quantiles were used to obtain a robust estimation of noise level. We constructed a weighted matrix to suppress the t1 noise. The weighted matrix was calculated from the logistic functions, which were adaptively computed from the spectrum. The proposed method is robust and effective in both simulations and actual experiments. Further, it can maintain the quantitative relationship of the spectrogram and is suitable for various complex peak types.

Comprehensive analysis of the LncRNAs, MiRNAs, and MRNAs acting within the competing endogenous RNA network of LGG.

Messenger RNA (mRNA) and long noncoding RNA (lncRNA) targets interact via competitive microRNA (miRNA) binding. However, the roles of cancer-specific lncRNAs in the competing endogenous RNA (ceRNA) networks of low-grade glioma (LGG) remain unclear. This study obtained RNA sequencing data for normal solid tissue and LGG primary tumour tissue from The Cancer Genome Atlas database. We used a computational method to analyse the relationships among the mRNAs, lncRNAs, and miRNAs in these samples. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to predict the biological processes (BPs) and pathways associated with these genes. Kaplan-Meier survival analysis was used to evaluate the association between the expression levels of specific mRNAs, lncRNAs, and miRNAs and overall survival. Finally, we created a ceRNA network describing the relationships among these mRNAs, lncRNAs, and miRNAs using Cytoscape 3.5.1. A total of 2555 differentially expressed (DE) mRNAs, 218 DElncRNAs, and 192 DEmiRNAs were identified using R. In addition, GO and KEGG pathway analysis of the mRNAs and lncRNAs in the ceRNA network identified 10 BPs, 10 cell components, 10 molecular functions, and 48 KEGG pathways as selectively enriched. A total of 55 lncRNAs, 50 miRNAs, and 10 mRNAs from this network were shown to be closely associated with overall survival in LGG. Finally, 59 miRNAs, 235 mRNAs, and 17 lncRNAs were used to develop a ceRNA network comprising 313 nodes and 1046 edges. This study helps expand our understanding of ceRNA networks and serves to clarify the underlying pathogenesis mechanism of LGG.

A Method for Autonomous Multi-Motion Modes Recognition and Navigation Optimization for Indoor Pedestrian.

The indoor navigation method shows great application prospects that is based on a wearable foot-mounted inertial measurement unit and a zero-velocity update principle. Traditional navigation methods mainly support two-dimensional stable motion modes such as walking; special tasks such as rescue and disaster relief, medical search and rescue, in addition to normal walking, are usually accompanied by running, going upstairs, going downstairs and other motion modes, which will greatly affect the dynamic performance of the traditional zero-velocity update algorithm. Based on a wearable multi-node inertial sensor network, this paper presents a method of multi-motion modes recognition for indoor pedestrians based on gait segmentation and a long short-term memory artificial neural network, which improves the accuracy of multi-motion modes recognition. In view of the short effective interval of zero-velocity updates in motion modes with fast speeds such as running, different zero-velocity update detection algorithms and integrated navigation methods based on change of waist/foot headings are designed. The experimental results show that the overall recognition rate of the proposed method is 96.77%, and the navigation error is 1.26% of the total distance of the proposed method, which has good application prospects.

Severity evaluation of obstructive sleep apnea based on speech features.

PURPOSE: There are upper airway abnormalities in patients with obstructive sleep apnea (OSA), and their speech signal characteristics are different from those of unaffected people. In this study, the severity of OSA was evaluated automatically by machine learning technology based on the speech signals of Chinese people. METHODS: In total, 151 adult male Mandarin native speakers who had suspected OSA completed polysomnography to assess the severity of the disease. Chinese vowels and nasal sounds were recorded in sitting and supine positions, and the accuracy of predicting the apnea-hypopnea index (AHI) of the participants using a machine learning method was analyzed based on features extracted from the speech signals. RESULTS: Among the 151 participants, 75 had AHI > 30 events/h, and 76 had AHI ≤ 30 events/h. Various features including linear prediction cepstral coefficients (LPCC) were extracted from the data collected from participants recorded in the sitting and supine positions and by using a linear support vector machine (SVM); we classified the participants with thresholds of AHI = 30 and AHI = 10 events/h. The accuracies of the classifications were both 78.8%, the sensitivities were 77.3% and 79.1%, and the specificities were 80.3% and 78.0%, respectively. CONCLUSION: This study constructed a severity evaluation model of OSA based on speech signal processing and machine learning, which can be used as an effective method to screen patients with OSA. In addition, it was found that Chinese pronunciation can be used as an effective feature to predict OSA.

Bioinspired Network Analysis Enabled Divergent Syntheses and Structure Revision of Pentacyclic Cytochalasans.

We accomplished the divergent total syntheses of ten pentacyclic cytochalasans (aspergillin PZ, trichodermone, trichoderones, flavipesines, and flavichalasines) from a common precursor aspochalasin D and revised the structures of trichoderone B, spicochalasin A, flavichalasine C, aspergilluchalasin based on structure network analysis of the cytochalasans biosynthetic pathways and DFT calculations. The key steps of the syntheses include transannular alkene/epoxyalkene and carbonyl-ene cyclizations to establish the C/D ring of pentacyclic aspochalasans. Our bioinspired approach to these pentacyclic cytochalasans validate the proposed biosynthetic speculation from a chemical view and provide a platform for the synthesis of more than 400 valuable cytochalasans bearing different macrocycles and amino-acid residues.

Pedestrian Navigation System with Trinal-IMUs for Drastic Motions.

The combination of biomechanics and inertial pedestrian navigation research provides a very promising approach for pedestrian positioning in environments where Global Positioning System (GPS) signal is unavailable. However, in practical applications such as fire rescue and indoor security, the inertial sensor-based pedestrian navigation system is facing various challenges, especially the step length estimation errors and heading drift in running and sprint. In this paper, a trinal-node, including two thigh-worn inertial measurement units (IMU) and one waist-worn IMU, based simultaneous localization and occupation grid mapping method is proposed. Specifically, the gait detection and segmentation are realized by the zero-crossing detection of the difference of thighs pitch angle. A piecewise function between the step length and the probability distribution of waist horizontal acceleration is established to achieve accurate step length estimation both in regular walking and drastic motions. In addition, the simultaneous localization and mapping method based on occupancy grids, which involves the historic trajectory to improve the pedestrian's pose estimation is introduced. The experiments show that the proposed trinal-node pedestrian inertial odometer can identify and segment each gait cycle in the walking, running, and sprint. The average step length estimation error is no more than 3.58% of the total travel distance in the motion speed from 1.23 m/s to 3.92 m/s. In combination with the proposed simultaneous localization and mapping method based on the occupancy grid, the localization error is less than 5 m in a single-story building of 2643.2 m2.

A Deep Learning Model to Predict a Diagnosis of Alzheimer Disease by Using 18F-FDG PET of the Brain.

Purpose To develop and validate a deep learning algorithm that predicts the final diagnosis of Alzheimer disease (AD), mild cognitive impairment, or neither at fluorine 18 (18F) fluorodeoxyglucose (FDG) PET of the brain and compare its performance to that of radiologic readers. Materials and Methods Prospective 18F-FDG PET brain images from the Alzheimer's Disease Neuroimaging Initiative (ADNI) (2109 imaging studies from 2005 to 2017, 1002 patients) and retrospective independent test set (40 imaging studies from 2006 to 2016, 40 patients) were collected. Final clinical diagnosis at follow-up was recorded. Convolutional neural network of InceptionV3 architecture was trained on 90% of ADNI data set and tested on the remaining 10%, as well as the independent test set, with performance compared to radiologic readers. Model was analyzed with sensitivity, specificity, receiver operating characteristic (ROC), saliency map, and t-distributed stochastic neighbor embedding. Results The algorithm achieved area under the ROC curve of 0.98 (95% confidence interval: 0.94, 1.00) when evaluated on predicting the final clinical diagnosis of AD in the independent test set (82% specificity at 100% sensitivity), an average of 75.8 months prior to the final diagnosis, which in ROC space outperformed reader performance (57% [four of seven] sensitivity, 91% [30 of 33] specificity; P < .05). Saliency map demonstrated attention to known areas of interest but with focus on the entire brain. Conclusion By using fluorine 18 fluorodeoxyglucose PET of the brain, a deep learning algorithm developed for early prediction of Alzheimer disease achieved 82% specificity at 100% sensitivity, an average of 75.8 months prior to the final diagnosis. © RSNA, 2018 Online supplemental material is available for this article. See also the editorial by Larvie in this issue.

Total Synthesis of (-)-Perezoperezone through an Intermolecular [5+2] Homodimerization of Hydroxy p-Quinone.

The first copper-catalyzed intermolecular [5+2] homodimerization of hydroxy p-quinone is presented, furnishing bicyclo[3.2.1]octadienone core structures in typically good yields and excellent diastereoselectivities. Applying this synthetic approach enables a concise nine-step total synthesis of (-)-perezoperezone from commercially available 3,5-dimethoxytoluene.

Dynamics of the floating nematic phase formation in platelet suspension with thickness polydispersity by sedimentation.

An inverted phase coexistence, where an ordered phase appears on top of a disordered phase, has been observed in polydisperse colloidal suspensions. Herein, we studied the dynamics of this phenomenon in a suspension of a mixture of two types of platelets, namely, thick and thin. We show that the thick platelets preferentially sediment first excluding the thin platelets, and create a region enriched with thin platelets at some place above the bottom, which eventually gives the inverted configuration. We show that such interplay between the sedimentation and the isotropic-nematic phase transition can cause a rich and complex sedimentation dynamic. Depending on the initial concentration and the gravity strength, the interface between the isotropic phase and the nematic phase can move up or down during the sedimentation, and a variety of final equilibrium structures can appear.

Dynamics of liquid crystalline phase transition in sedimenting platelet-like particles.

When a suspension of platelet-like particles sediment in a closed container, the particles undergo isotropic-nematic phase transition (I-N transition), and there appears a clear interface between the isotropic phase and the nematic phase. Usually the interface moves from bottom to top since the nematic phase appears and grows at the bottom, but it has been observed that in some situations the interface moves from top to bottom. Here, we study the dynamics of the interface by solving the non-equilibrium diffusion equation for the concentration of platelet-like particles, and show that the I-N interface can move upward (rising interface) or downward (falling interface) depending on whether the initial concentration is less than the critical concentration of I-N transition or more than it. We give a simple analysis theory for the motion of the interface in each case, which agrees well with the numerical calculations. We also show that the numerical results are in reasonable agreement with existing experimental measurements.

[Genotype, environment and their interactions of functional components in Rheum tanguticum].

Six kinds of provenance of Rheum tanguticum collected from Qinghai province as the test materials, which were transplanted under 3 different environments by using complete randomized block design with three replicates. The contents of the chemical components was determined by HPLC. This study aimed at analyzing the effect of genotype, environment and their interactions on the 4 kinds of functional components (phenolic acids, bianthrone, free anthraquinones and combined anthraquinones) in 14 kinds of active components of Rh. tanguticum, in order to provide a theoretical basis for the selection of cultivated Rh. tanguticum in high quality producing area and excellent provenance. The provenance trial showed that the genotype and environment influence on the effect of all kinds of functional components in Rh. tanguticum were significant (P<0.05). The content of phenolic acids was mainly influenced by environment, and the other three kinds of functional components were affected by environment and their interactions. The proportion of environment was larger. The cultivation quality of Rh. tanguticum should give priority to environment, then choose a provenance. Sichuan may be beneficial in accumulation of combined anthraquinones in Rh. tanguticum, Gansu may facilitate the binding of free anthraquinone, phenolic acids and bianthrone content. Preliminary inference based on the content and proportion of efficacy components, T4 could be potential special medicinal germplasm that have function of heat-clearing and detoxifying drugs and activate blood circulation to dissipate blood stasis; T3 and T6 could all be potential specialmedicinal germplasms that exist diarrhea attack characters. The results of this study have certain guiding significance for the production of rhubarb precision medicinal materials.

Total Synthesis of Asperchalasines†A, D, E, and H.

The first total syntheses of the cytochalasan dimers asperchalasines A, D, E, and H have been accomplished. The key steps of the synthesis include a highly stereoselective intermolecular Diels-Alder reaction and a Horner-Wadsworth-Emmons macrocyclization to establish the key monomer aspochalasin B, and an intermolecular Diels-Alder reaction followed by a biomimetic oxidative heterodimerization by 5+2 cycloaddition to furnish asperchalasine A. The synthetic efforts provide insight into the biosynthetic pathway of cytochalasan dimers and enables the further study of their biological properties.

CDC20 with malignant progression and poor prognosis of astrocytoma revealed by analysis on gene expression.

The malignant transformation of astrocytoma may result from the accumulation of multiple genetic alterations. Current research shows that diffuse astrocytoma (AIIs, WHO grade II) is inherently predisposed to recur locally, and to spontaneously progress to anaplastic astrocytoma (AAIIIs, WHO grade III) and eventually secondary glioblastoma (sGBMIVs, WHO grade IV). The aim of the study was to identify and validate the important gene(s) associated with malignant progression and poor prognosis of astrocytoma. Average expression levels of 82 samples (35 AIIs, 13 AAIIIs and 34 sGBMIVs) were compared to each other through no-paired student test. Candidate genes were screened by DAVID and Kaplan-Meier survival analysis. Further, the significant candidate genes were validated through real-time PCR(qPCR), western blot and immunohistochemistry (IHC) in different grades of glioma. Finally, the association of target gene and clinical molecular characterization was analyzed by Chi-squared analysis. The cell-division cycle protein 20(CDC20, p = 0.0129) and the polo-like kinase 1(PLK1, p = 0.0046) were screened by statistical and Kaplan-Meier survival analysis. The expression levels of CDC20 and PLK1 rose significantly through real-time PCR(qPCR), western blot and IHC. A chi-squared analysis showed that patients with CDC20 high-expression differ from patients with CDC20 low-expression in terms of WHO classification (p < 0.0001), karnofsky performance score (KPS, p < 0.0001), isocitrate dehydrogenase mutation (IDH1, p < 0.0001), phosphatase and tensin homolog mutation (PTEN, p = 0.027) and epidermal growth factor receptor protein amplification (EGFR, p = 0.048). Moreover, the biological processes analyses indicate CDC20 might have an essential role in astrocyte cell proliferation. We demonstrated that the expression level of CDC20 increases significantly along with malignant progression and poor prognosis of astrocytoma.

Design, synthesis, and biological activity of 4-(imidazo[1,2-b]pyridazin-3-yl)-1H-pyrazol-1-yl-phenylbenzamide derivatives as BCR-ABL kinase inhibitors.

A series of 4-((pyrazolo[1,5-a]pyrimidin-6-yl)-1H-pyrazol-1-yl)phenyl-3-benzamide derivatives and 4-((imidazo[1,2-b]pyridazin-3-yl)-1H-pyrazol-1-yl-)phenyl-3-benzamide derivatives were designed, synthesized as new BCR-ABL tyrosine kinase inhibitors by using combinational strategies of scaffold hopping and conformational constraint. These new compounds were screened for BCR-ABL1 kinase inhibitory activity, and most of them appeared good inhibitory activity against BCR-ABL1 kinase. One of the most potent compounds 16a strongly suppressed BCR-ABL1 kinase with IC50 value of 8.5nM. The tested compounds 16a and 16i showed strong inhibitory activities against K562 with IC50 value of less than 2nM. Molecular docking studies indicated that these compounds fitted well with the active site of BCR-ABL1 protein. The results showed these inhibitors may serve as lead compounds for further developing new drugs targeted BCR-ABL kinase.

Enhancement of tunability of MAPK cascade due to coexistence of processive and distributive phosphorylation mechanisms.

The processive phosphorylation mechanism becomes important when there is macromolecular crowding in the cytoplasm. Integrating the processive phosphorylation mechanism with the traditional distributive one, we propose a mixed dual-site phosphorylation (MDP) mechanism in a single-layer phosphorylation cycle. Further, we build a degree model by applying the MDP mechanism to a three-layer mitogen-activated protein kinase (MAPK) cascade. By bifurcation analysis, our study suggests that the crowded-environment-induced pseudoprocessive mechanism can qualitatively change the response of this biological network. By adjusting the degree of processivity in our model, we find that the MAPK cascade is able to switch between the ultrasensitivity, bistability, and oscillatory dynamical states. Sensitivity analysis shows that the theoretical results remain unchanged within a reasonably chosen variation of parameter perturbation. By scaling the reaction rates and also introducing new connections into the kinetic scheme, we further construct a proportion model of the MAPK cascade to validate our findings. Finally, it is illustrated that the spatial propagation of the activated MAPK signal can be improved (or attenuated) by increasing the degree of processivity of kinase (or phosphatase). Our research implies that the MDP mechanism makes the MAPK cascade become a flexible signal module, and the coexistence of processive and distributive phosphorylation mechanisms enhances the tunability of the MAPK cascade.

Visualizing an ultra-weak protein-protein interaction in phosphorylation signaling.

Proteins interact with each other to fulfill their functions. The importance of weak protein-protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra-weak interactions remain to be characterized. Phosphorylation can take place via a K(D)≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd(III)-based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra-weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro- to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra-weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration.