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Jin Wu,1 Yiting Luo,1 Yan Shen,2 Yuyao Hu,2 Fangyuan Zhu,1 Jiaqian Wu,1 Yingchao Liu3 1The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China; 2Department of Gastroenterology, The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, 310005, People’s Republic of China; 3Academic Affairs Office, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of ChinaCorrespondence: Yingchao Liu, Academic Affairs Office, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China, Email jcjsylyc@163.comBackground: Traditional Chinese medicine (TCM) has the advantage of multi-component and multi-target, which becomes a hot spot in the treatment of numerous diseases. Shaoyao decoction (SYD) is a TCM prescription, which is mainly used to treat damp-heat dysentery clinically, with small side effects and low cost. However, its mechanism remains elusive. The purpose of this study is to explore the mechanism of SYD in the treatment of mice with ulcerative colitis (UC) induced by dextran sulfate sodium (DSS) through metabolomics and network pharmacology, and verify through molecular docking and immunohistochemistry, so as to provide a scientific basis for the role of SYD in the treatment of UC.Materials and Methods: Firstly, DSS-induced UC models were established and then untargeted metabolomics analysis of feces, livers, serum and urine was performed to determine biomarkers and metabolic pathways closely related to the role of SYD. Besides, network pharmacology was applied to screen the active components and UC-related targets, which was verified by molecular docking. Finally, metabonomics and network pharmacology were combined to draw the metabolite-pathway-target network and verified by immunohistochemistry.Results: Metabolomics results showed that a total of 61 differential metabolites were discovered in SYD-treated UC with 3 main metabolic pathways containing glycerophospholipid metabolism, sphingolipid metabolism and biosynthesis of unsaturated fatty acids, as well as 8 core targets involving STAT3, IL1B, IL6, IL2, AKT1, IL4, ICAM1 and CCND1. Molecular docking demonstrated that the first five targets had strong affinity with quercetin, wogonin, kaempferol and baicalein. Combined with metabolomics and network pharmacology, sphingolipid signaling pathway, PI3K/AKT-mTOR signaling pathway and S1P3 pathway were identified as the main pathways.Conclusion: SYD can effectively ameliorate various symptoms and alleviate intestinal mucosal damage and metabolic disorder in DSS induced UC mice. Its effect is mainly related to sphingolipid metabolism, PI3K/AKT-mTOR signaling pathway and S1P3 pathway.Keywords: metabolomics, UPLC-Q-TOF-MS, network pharmacology, Shaoyao decoction, ulcerative colitis

Filopodia, a finger-like structure and actin-rich plasma-membrane protrusion at the leading edge of the cell, has important roles in cell motility. However, the mechanisms of filopodia generation are not well-understood via the actin-related protein 2/3 (ARP2/3) complex in Non-Small Cell Lung Cancer (NSCLC) cells. We previously have demonstrated that PRR11 associates with the ARP2/3 complex to regulate cytoskeleton-nucleoskeleton assembly and chromatin remodeling. In this study, we further demonstrate that PRR11 involves in filopodia formation, focal adhesion turnover and cell motility through ARP2/3 complex. Cell phenotype assays revealed that the silencing of PRR11 increased cellular size and inhibited cell motility in NSCLC cells. Mechanistically, PRR11 recruited and co-localized with Arp2 at the membrane protrusion to promote filopodia formation but not lamellipodia formation. Notably, PRR11 mutant deletion of the proline-rich region 2 (amino acid residues 185–200) abrogated the effect of filopodia formation. In addition, PRR11-depletion inhibited filopodial actin filaments assembly and increased the level of active integrin β1 in the cell surface, whereas reduced the phosphorylation level of focal adhesion kinase (FAKY397) to repress focal adhesion turnover and cell motility in NSCLC cells. Taken together, our findings indicate that PRR11 has critical roles in controlling filopodia formation, focal adhesion turnover and cell motility by recruiting ARP2/3 complex, thus dysregualted expression of PRR11 potentially facilitates tumor metastasis in NSCLC cells.

Yiting Luo,1 Jin Wu,1 Yingchao Liu,2 Yan Shen,3 Fangyuan Zhu,1 Jiaqian Wu,1 Yuyao Hu3 1The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 2Academic Affairs Office, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 3The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of ChinaCorrespondence: Yuyao Hu, The Second Affiliated Hospital of Zhejiang Chinese Medical University, 318 Chaowang Road, Hangzhou, People’s Republic of China, Email 20094017@zcmu.edu.cnPurpose: Shaoyao decoction (SYD) is a traditional Chinese medicine used to treat ulcerative colitis (UC). The exact mechanism of action of SYD in UC treatment is still unclear. Here, we examined the therapeutic effects of SYD in mice with dextran sulfate sodium (DSS)-induced colitis and explored the underlying mechanism.Methods: The experimental group was divided into normal control, UC, and SYD treatment groups. The UC model of C57BL/6 mice was induced using 3% (w/v) DSS for 7 days. SYD was orally administered for 7 days. The proximal and distal colonic metabolic profiles were detected using quadrupole-time-of-flight mass spectrometry-based untargeted metabolomics.Results: SYD significantly increased weight, reduced disease activity index scores, and ameliorated colon length shortening and pathological damage in mice. In the distal colon, SYD increased the abundance of phosphatidic acid and lysophosphatidylethanolamine and decreased the abundance of lactosylceramide, erythrodiol 3-palmitate, and lysophosphatidylcholine. In the proximal colon, SYD increased the abundance of palmitic acid, cyclonormammein, monoacylglyceride, 13S-hydroxyoctadecadienoic acid, and ceanothine C and decreased the abundance of tetracosahexaenoic acid, phosphatidylserine, and diglyceride.Conclusion: Our findings revealed that SYD could alleviate UC by regulating metabolic dysfunction, which provides a reference for further studies on SYD.Keywords: Shaoyao decoction, ulcerative colitis, metabolomics, UPLC-Q-TOF-MS, DSS-induced colitis mouse model

Inherited retinal degenerative diseases such as Retinitis Pigmentosa (RP) result in progressive loss of photoreceptors until an individual is completely blind. A hallmark of these diseases is progressive structural and functional remodeling of the remaining retinal neurons as rod photoreceptors are lost. While many studies focus on regenerative or bionic therapies to restore vision, extensive remodeling of retinal cell types throughout the course of retinal degenerative diseases stands as a barrier for successful implementation of these strategies. As a window onto the molecular basis of remodeling, we have performed a comparative analysis of single-cell transcriptome data from adult Zebrafish retina of wild-type and a P23H mutant rhodopsin model of RP. In addition to providing a benchmark atlas of retinal cell type transcriptomes in the wild-type adult Zebrafish retina, we find transcriptional changes in essentially all retinal cell types in the P23H model. Increased oxidative stress is evident not only in the rods but also in cones, retinal pigmented epithelium (RPE) and to a lesser extent in amacrine and bipolar cells. Metabolic changes increasing oxidative metabolism and glycolysis are found in rods and cones, while evidence of increased activity of the mitochondrial electron transport chain is found in retinal ganglion cells (RGCs). Evidence of synaptic remodeling is found throughout the retina, with changes to increase synaptic transmission in photoreceptors and bipolar cells, increased ionotropic glutamate receptors in amacrine and ganglion cells, and dendritic and axon remodeling throughout. Surprisingly, RPE, cones and bipolar cells in the P23H retinas also have increased expression of genes involved in circadian rhythm regulation. While this model system undergoes continuous regeneration, ongoing remodeling impacts the entire retina. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.

Chronic, often intractable, pain is caused by neuropathic conditions such as traumatic peripheral nerve injury (PNI) and spinal cord injury (SCI). These conditions are associated with alterations in gene and protein expression correlated with functional changes in somatosensory neurons having cell bodies in dorsal root ganglia (DRGs). Most studies of DRG transcriptional alterations have utilized PNI models where axotomy-induced changes important for neural regeneration may overshadow changes that drive neuropathic pain. Both PNI and SCI produce DRG neuron hyperexcitability linked to pain, but contusive SCI produces little peripheral axotomy or peripheral nerve inflammation. Thus, comparison of transcriptional signatures of DRGs across PNI and SCI models may highlight pain-associated transcriptional alterations in sensory ganglia that do not depend on peripheral axotomy or associated effects such as peripheral Wallerian degeneration. Data from our rat thoracic SCI experiments were combined with meta-analysis of published whole-DRG RNA-seq datasets from prominent rat PNI models. Striking differences were found between transcriptional responses to PNI and SCI, especially in regeneration-associated genes (RAGs) and long noncoding RNAs (lncRNAs). Many transcriptomic changes after SCI also were found after corresponding sham surgery, indicating they were caused by injury to surrounding tissue, including bone and muscle, rather than to the spinal cord itself. Another unexpected finding was of few transcriptomic similarities between rat neuropathic pain models and the only reported transcriptional analysis of human DRGs linked to neuropathic pain. These findings show that DRGs exhibit complex transcriptional responses to central and peripheral neural injury and associated tissue damage. Although only a few genes in DRG cells exhibited similar changes in expression across all the painful conditions examined here, these genes may represent a core set whose transcription in various DRG cell types is sensitive to significant bodily injury, and which may play a fundamental role in promoting neuropathic pain.

Objective. Shenling Baizhu San (SLBZS) is reported as an effective drug for ulcerative colitis (UC); however, its effect on intestinal flora remains unknown. In this study, we investigated the effect of SLBZS on intestinal flora in a rat model of UC with spleen deficiency and dampness. Methods. UC was induced in rats using 2,4,6-trinitrobenzene sulfonic acid on the basis of a model of spleen deficiency and dampness. The 16S rDNA sequencing was used to detect structural changes in the intestinal flora; the phylogenetic investigation of communities by reconstruction of unobserved state (PICRUSt) analysis was used to predict the altered pathways. Results. Compared with the model group, rats in the SLBZS group exhibited decreased levels of TNF-α P < 0.05 , and increased abundance and diversity of the intestinal flora. The abundance of Actinobacteria P < 0.001 and Bacteroides P < 0.01 increased and that of Firmicutes decreased P < 0.001 , and the abundance of Bifidobacterium P < 0.05 and Allobaculum increased. PICRUSt analysis showed that the altered pathways between the groups were those of fatty acid and antibiotic biosynthesis, amino acid metabolism, and the pentose phosphate pathway. Conclusions. SLBZS can regulate the structure and function of the intestinal flora, alter expression levels of certain metabolic pathways, and has the potential to treat UC.

Background: Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease with abdominal pain, mucus, pus and blood in the stool as the main clinical manifestations. The pathogenesis of UC is still not completely clear, and multiple factors, such as genetic susceptibility, immune response, intestinal microecological changes and environmental factors, together lead to the onset of UC. In recent years, the role of intestinal microbiota disturbances on the pathogenesis of UC has received widespread attention. Therefore, fecal microbiota transplantation (FMT), which changes the intestinal microecological environment of UC patients by transplantation of normal fecal bacteria, has attracted increasing attention from researchers. However, there are no guidelines to recommend fresh FMT or frozen FMT in the treatment of UC, and there are few studies on this. Therefore, the purpose of this study was to explore the effects of fresh and frozen FMT methods on the treatment of experimental UC models in rats.Results: Compared with the model control group, all FMT groups achieved better efficacy, mainly manifested as weight gain by the rats, improvements in fecal characteristics and blood stools, reduced inflammatory factors and normal bacterial microbiota. The efficacy of the frozen FMT group was better than that of the fresh FMT group in terms of behavior and colon length.Conclusion: FMT method supplements the gut microbiota with beneficial bacteria, such as short-chain fatty acid-producing bacteria. These bacteria can regulate intestinal function, protect the mucosal barrier and reduce harmful bacteria, thus mitigating the damage to the intestinal barrier and the associated inflammatory response, resulting in UC remission. FMT is a feasible method for treating UC, with frozen FMT having a superior therapeutic effect than that of fresh FMT.

Background: Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease with abdominal pain, mucus, pus, and blood in the stool as the main clinical manifestations. The pathogenesis of UC is still not completely clear, and multiple factors such as genetic susceptibility, immune response, intestinal microecological changes, and environmental factors together lead to the onset of UC. In recent years, the role of intestinal flora disturbance on the pathogenesis of UC has received widespread attention. Therefore, fecal microbiota transplantation(FMT), which changes the intestinal microecological environment of UC patients by transplantation of normal fecal bacteria, has attracted increasing attention from researchers. However, there are no guidelines at home and abroad to recommend fresh FMT or frozen FMT in the treatment of UC, and there are a few studies on this. Therefore the purpose of this experiment was to explore the effects of fresh and frozen fecal microbiota transplantation methods on the treatment of experimental ulcerative colitis models in rats.Results: Compared with the model control group, all faecal microbiota transplantation groups achieved better efficacy, mainly manifested as weight gain by the rats, improvements in faecal characteristics and blood stools, reduced inflammatory factors, and normal bacterial flora. The efficacy of the frozen faecal microbiota transplantation group was better than that of the fresh faecal microbiota transplantation group in terms of behaviour and colon length .Conclusions: FMT is a feasible method for treating UC. The mechanism of action may be via competitive inhibition of pathogenic microorganisms, improved immune metabolism, and reduced inflammatory response to mitigate the damage to the intestinal barrier and cause UC remission. Compared with fresh FMT, the therapeutic effect of frozen FMT may be greater.

The purpose of this research article is to explore different factors that can affect the popularity of a song and to predict the potential popularity of one song with the most related factors. Since there are too many factors that can affect a song's popularity and high dimensions caused by large amounts of variables, they may result in significant errors in analysis. To avoid these errors, the methods used in this research are Principal components analysis (PCA) and model blending. By using PCA, we can get lower dimensions; and then model blending to model the relationship between a scalar response and one or more explanatory variables. Different weights to random forest, decision tree and knn are set to get a new model, with mean squared error (MSE) 4.96, which is a comparatively low one. The results provide a relatively precise prediction of the popularity of Spotify top music. And they can be used in music company’s commercial analysis and enrich the field of popularity prediction of the song market.

Hypoxia-acquired radioresitance is considered to be a major factor responsible for the clinical failure of radiotherapy. Carbonic anhydrase IX (CA IX), a hypoxia-induced cell-surface enzyme involved in pH regulation of hypoxic solid tumors, has been acknowledged as a desirable target for cancer therapy. Herein, we developed a dual exogenous/endogenous CA IX inhibition strategy using core/satellite-like metal-organic framework (UiO-66)/gold nanoparticles (Au NPs) nanohybrids as a therapeutic platform for hypoxia-relief enhanced RT of triple negative breast cancer. The UiO-66 matrix supported the Au NPs generation in situ and would decompose inside tumor cells by the high-concentration phosphates to release p-phthalic acid (PTA), a original building skeleton of the UiO-66 matrix, to inhibit CA IX, while the decorated Au NPs served as radiosensitizers to potentiate the sensitivity of tumor cells to X-ray and yet provided active sites for CA IX antisense oligonucleotide (ASO) loading via formation of stable Au-S bond to knockdown CA IX. The dual inhibitory manner, exogenous inhibition from PTA and endogenous inhibition from CA IX ASO, largely alleviated the hypoxia-induced resistance and synergized with Au NPs-mediated radiosensitization to afford super therapeutic outcome in vitro and in vivo, supporting the feasibility of our synergistic RT strategy in hypoxic tumor management.

A composite catalytically active membrane was prepared focusing on the reduction of membrane mass transfer resistance to achieve a better esterification-pervaporation coupling performance. The effect of membrane preparation conditions on the membrane morphology was first evaluated. Under an optimized membrane preparation conditions, a “sandwich-like” composite membrane with a highly inter-connected sponge-like catalytic layer on a polyvinyl alcohol / polyethersulfone bilayer was obtained. The porosity of the membrane was found to be as high as 81.6%. A simple resistance-in-series model was developed to analyze the mass transfer resistance distribution in a traditional inert membrane reactor (IMR), a catalytically active membrane reactor (CAMR) with dense catalytic layer and a catalytically active membrane reactor with porous catalytic layer, respectively. Results showed that the preparation of a highly porous catalytic layer decreased the resistance of catalytic layer from 48.5% to 20.6% of overall resistances, leading to an enhanced water removal ability for the composite membrane. Finally, reaction-separation coupling experiments in IMR, CAMR with porous catalytic layer and CAMR with dense catalytic layer showed that, with a faster reaction kinetics and water removal rate, CAMR with porous catalytic layer exhibited a best coupling performance.

A better conversion enhancement of esterification between acetic acid and n-butanol was achieved in a catalytically active membrane reactor ( p CAMR) when compared to that in a traditional inert membrane reactor (IMR). This enhancement was attributed to a novel composite catalytically active membrane in which a highly porous catalytic layer was introduced. SEM images showed that the membrane consisted of three layers: the top layer was a highly porous catalytic layer with massive macrovoids and “sponge-like” pores, the middle layer was a dense polyvinyl alcohol selective layer, and the bottom layer was a porous polyethersulfone support layer. The preparation of a highly porous catalytic layer instead of a dense one in the composite membrane greatly decreased the overall mass transfer resistance of the reactor from 6.7 × 10 5 to 5.6 × 10 5 s/m, a value which is even comparable to that of IMR (5.1 × 10 5 s/m) where the additional catalytic layer was absent. The effects of operational parameters on the esterification-pervaporation coupling performance in p CAMR were systematically evaluated. Through a reasonable match between reaction rate and water removal rate, a genuine in-situ water removal at a molecular lever was realized. For comparison, coupling performances in an IMR and a catalytically active membrane reactor with a dense composite membrane ( d CAMR) were also investigated. Results showed that the coupling performance in p CAMR outperformed both IMR and d CAMR due to a combination of much lower overall mass transfer resistance and higher mass transfer driving force for water removal in p CAMR. After 45 h at 85 °C, the acid conversion in p CAMR reached almost completion, an approximately 43% of conversion enhancement was achieved when compared to equilibrium conversion.

•Immersion phase inversion is a viable approach for preparing catalytic membrane.•The membrane was extremely catalytically active in the application.•The catalytic membrane enhanced the equilibrium displacement efficiently.•Better synthesis performance obtained with increasing water removal rate.•An approximately 52% of enhancement in glycerol conversion was achieved.

Liquid biofuels from non-grain energy crops on marginal land could become an important substitute of gasoline in the transport sector, and offer the possibility to reduce competition with food crops for land resources. However, the cultivation of energy crops is facing profound challenges due to changing temperature and precipitation in the future. To assess the impact of climate change on the potential of liquid biofuels on marginal land in China, this study used a geographic information system-based approach combined with multiple factor analysis to identify the spatial distribution of marginal land suitable for nine major energy crops in China. Climate scenarios were generated based on bias-corrected results of five different climate models under two representative concentration pathways (RCP2.6 and 8.5). Results show that climate change is projected to have a substantial impact on the land availability for biofuel production in the 2050s under both RCPs. The total amount of marginal land suitable for energy crops was 170.2 million hectares for the period of 2010–2019, and would increase in the 2050s under both RCPs. The changing pattern of area are similar under both RCP 2.6 and 8.5, only the magnitude is different. All the species are projected to have a northward spread in China. The amount of marginal land suitable for all the energy crops is projected to increase in the 2050s, except for Miscanthus floridulus, and Miscanthus lutarioriparius under RCP 8.5. However, the potential productivity of the energy crops is projected to have a substantial decrease in the 2050s. The average yields of the energy crops are only about one fourth of their yields in the 2010s due to climate change. Combined with high costs of producing biofuels and numerous ecological tradeoffs, it is likely that liquid biofuels development using 1.5 and 2-generation energy crops does not have an optimistic perspective in China.

Well-hydrated phospholipid bilayers provide a near-native environment for membrane proteins. They enable the preparation of chemically-defined samples suitable for NMR and other spectroscopic experiments that reveal the structure, dynamics, and functional interactions of the proteins at atomic resolution. The synthetic polymer styrene maleic acid (SMA) can be used to prepare detergent-free samples that form macrodiscs with diameters greater than 30 nm at room temperature, and spontaneously align in the magnetic field of an NMR spectrometer at temperatures above 35 °C. Here we show that magnetically aligned macrodiscs are particularly well suited for solid-state NMR experiments of membrane proteins because the SMA-lipid assembly both immobilizes the embedded protein and provides uniaxial order for oriented sample (OS) solid-state NMR studies. We show that aligned macrodiscs incorporating four different membrane proteins with a wide range of sizes and topological complexity yield high-resolution OS solid-state NMR spectra. The work is dedicated to Michelle Auger who made key contributions to the field of membrane and membrane protein biophysics.

In order to effectively model crude oil spot price with inherently high complexity, a hybrid learning paradigm integrating least squares support vector regression (LSSVR) with a hybrid optimization searching approach for the parameters selection in the LSSVR [consisting of grid method and genetic algorithm (GA)], i.e., a hybrid grid-GA-based LSSVR model, is proposed in this study. In the proposed hybrid learning paradigm, the grid method, a simple but efficient searching method, is first applied to roughly but rapidly determine the proper boundaries of the parameters in the LSSVR; then, the GA, an effective and powerful intelligent searching algorithm, is further implemented to select the most suitable parameters. For illustration and verification, the proposed learning paradigm is used to predict the crude oil spot prices of the West Texas Intermediate and the Brent markets. The empirical results demonstrate that the proposed hybrid grid-GA-based LSSVR learning paradigm can outperform its benchmarking models (including some popular forecasting techniques and similar LSSVRs with other parameter searching algorithms) in terms of both prediction accuracy and time-savings, indicating that it can be utilized as one effective forecasting tool for crude oil price with high volatility and irregularity.

To develop a low-carbon economy, China launched seven pilot programs for carbon emissions trading (CET) in 2011 and plans to establish a nationwide CET mechanism in 2015. This paper formulated a multi-agent-based model to investigate the impacts of different CET designs in order to find the most appropriate one for China. The proposed bottom-up model includes all main economic agents in a general equilibrium framework. The simulation results indicate that (1) CET would effectively reduce carbon emissions, with a certain negative impact on the economy, (2) as for allowance allocation, the grandfathering rule is relatively moderate, while the benchmarking rule is more aggressive, (3) as for the carbon price, when the price level in the secondary CET market is regulated to be around RMB 40 per metric ton, a satisfactory emission mitigation effect can be obtained, (4) the penalty rate is suggested to be carefully designed to balance the economy development and mitigation effect, and (5) subsidy policy for energy technology improvement can effectively reduce carbon emissions without an additional negative impact on the economy. The results also indicate that the proposed novel model is a promising tool for CET policy making and analyses.

Less than 3 % of the human genome encodes protein sequences and the majority of transcribed sequences are noncoding. Long noncoding RNAs (lncRNAs) refer to transcripts lacking in protein-coding potential and longer than 200 nt. lncRNAs are less conserved across species and expressed at a relatively lower level. The expression patterns of lncRNAs are more cell-type-specific than protein-coding genes. Currently, there are 8359 lncRNA genes annotated in Mouse GENCODE version M6 and 15,931 in Human GENCODE version 23. The number of lncRNA genes is still steadily increasing. Many lncRNAs have been shown to play crucial roles in regulating the expression of protein-coding genes during various biological processes. Particularly, lnRNAs can function as regulators during development and cell differentiation. Herein, we discussed the regulated expression and the functions of lncRNAs, as well as the underlying molecular mechanisms. Specifically, we highlighted the importance of lnRNAs in the central nervous system, and their regulatory roles during neural cell-fate determination.

In order to improve the forecasting accuracy for clean energy consumption with inherently high complexity, a hybrid learning paradigm integrating genetic algorithm (GA) and least squares support vector regression (LSSVR), i.e., GA-LSSVR model, is formulated in this study. In this learning paradigm, LSSVR, as a powerful artificial intelligence tool, is employed to forecast clean energy consumption, furthermore, GA is employed to determine the parameters in LSSVR. Taking the Chinese hydropower energy as sample, empirical results indicate that the GA-LSSVR model significantly outperforms other benchmark models, including Artificial neural network (ANN), Autoregressive integrated moving average (ARIMA) and a set of hybrid models based on LSSVR and other searching methods (e.g., particle swarm optimization (PSO) and simulated annealing (SA)), in both level prediction accuracy and directional forecasting. The GA-LSSVR learning paradigm can be extended as an effective prediction technique for other complex data.

Protein kinases play a critical role in the integration of signaling networks in eukaryotic cells. cAMP-dependent protein kinase (PKA) serves as a prototype for this large and highly diverse enzyme family. The catalytic subunit of PKA provides the best example of how a protein kinase recognizes its substrates, as well as inhibitors, and also show how the enzyme moves through the steps of catalysis. Many of the relevant conformational states associated with the catalytic cycle which have been captured in a crystal lattice are summarized here. From these structures, we can begin to appreciate the molecular events of catalysis as well as the intricate orchestration of critical residues in the catalytic subunit that contribute to catalysis. The entire molecule participates. To fully understand signaling by PKA, however, requires an understanding of a large set of related proteins, not just the catalytic subunit. This includes the regulatory subunits that serve as receptors for cAMP and the A kinase anchoring proteins (AKAPs) that serve as scaffolds for PKA. The AKAPs localize PKA to specific sites in the cell by docking to the N-terminus of the regulatory subunits, thus creating microenvironments for PKA signaling. To fully appreciate the diversity and integration of these molecules, one needs not only high-resolution structures but also an appreciation of how these molecules behave in solution. Thus, in addition to obtaining high-resolution structures by X-ray crystallography and NMR, we have used fluorescent tools and also hydrogen/deuterium exchange coupled with mass spectrometry to probe the dynamic properties of these proteins and how they interact with one another. The molecular features of these molecules are described. Finally, we describe a new recombinantly expressed PKA reporter that allows us to monitor PKA activity in living cells.

Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation, modulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is known to be crucial for the development of many human diseases. The discovery of agents that restore this balance has been the subject of many drug research efforts, most of which have focused on tyrosine kinase inhibitors (TKIs), resulting in the development of more than 50 FDA-approved TKIs during the past two decades. More recently, accumulating evidence has suggested that members of the PTP superfamily are also promising drug targets, and efforts to discover tyrosine phosphatase inhibitors (TPIs) have increased dramatically. Here, we provide protocols for determining the potency of TPIs in vitro. We focus on the use of fluorescence-based substrates, which exhibit a dramatic increase in fluorescence emission when dephosphorylated by the PTP, and thus allow setting up highly sensitive and miniaturized phosphatase activity assays using 384-well or 1536-well microplates and a continuous (kinetic) assay format. The protocols cover PTP specific activity assays, Michaelis–Menten kinetics, dose-response inhibition assays, and dose-response data analysis for determining IC ( 50 ) values. Potential pitfalls are also discussed. While advanced instrumentation is utilized for compound spotting and liquid dispensing, all the assays can be adapted to existing equipment in most laboratories. Assays are described for selected PTP drug targets, including SHP2 ( PTPN11 ), PTP1B ( PTPN1 ), STEP ( PTPN5 ), and VHR ( DUSP3 ). However, all protocols are applicable to members of the PTP enzyme family in general. Graphical abstract