The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes.

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

Structures and activation mechanism of the Gabija anti-phage system.

Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.

Elucidation of the 1-phenethylisoquinoline pathway from an endemic conifer Cephalotaxus hainanensis.

Cephalotaxines harbor great medical potential, but their natural source, the endemic conifer Cephalotaxus is highly endangered, creating a conflict between biotechnological valorization and preservation of biodiversity. Here, we construct the whole biosynthetic pathway to the 1-phenethylisoquinoline scaffold, as first committed compound for phenylethylisoquinoline alkaloids (PIAs), combining metabolic modeling, and transcriptome mining of Cephalotaxus hainanensis to infer the biosynthesis for PIA precursor. We identify a novel protein, ChPSS, driving the Pictet-Spengler condensation and show that this enzyme represents the branching point where PIA biosynthesis diverges from the concurrent benzylisoquinoline-alkaloids pathway. We also pinpoint ChDBR as crucial step to form 4-hydroxydihydrocinnamaldehyde diverging from lignin biosynthesis. The elucidation of the early PIA pathway represents an important step toward microbe-based production of these pharmaceutically important alkaloids resolving the conflict between biotechnology and preservation of biodiversity.

InferLoop: leveraging single-cell chromatin accessibility for the signal of chromatin loop.

Deciphering cell-type-specific 3D structures of chromatin is challenging. Here, we present InferLoop, a novel method for inferring the strength of chromatin interaction using single-cell chromatin accessibility data. The workflow of InferLoop is, first, to conduct signal enhancement by grouping nearby cells into bins, and then, for each bin, leverage accessibility signals for loop signals using a newly constructed metric that is similar to the perturbation of the Pearson correlation coefficient. In this study, we have described three application scenarios of InferLoop, including the inference of cell-type-specific loop signals, the prediction of gene expression levels and the interpretation of intergenic loci. The effectiveness and superiority of InferLoop over other methods in those three scenarios are rigorously validated by using the single-cell 3D genome structure data of human brain cortex and human blood, the single-cell multi-omics data of human blood and mouse brain cortex, and the intergenic loci in the GWAS Catalog database as well as the GTEx database, respectively. In addition, InferLoop can be applied to predict loop signals of individual spots using the spatial chromatin accessibility data of mouse embryo. InferLoop is available at https://github.com/jumphone/inferloop.

Upregulated Tß4 expression in inflammatory bowel disease impairs the intestinal mucus barrier by inhibiting autophagy in mice.

Disrupted intestinal barrier homeostasis is fundamental to inflammatory bowel disease. Thymosin ß4 (Tß4) improves inflammation and has beneficial effects in dry-eye diseases, but its effects on the intestinal mucus barrier remain unknown. Therefore, this study evaluated the underlying regulatory mechanisms and effects of Tß4 by examining Tß4 expression in a mouse model with dextran sodium sulfate (DSS)-induced colitis and colonic barrier damage. Additionally, we intraperitoneally injected C57BL/6 mice with Tß4 to assess barrier function, microtubule-associated protein 1 light chain 3 (LC3II) protein expression, and autophagy. Finally, normal human colon tissue and colon carcinoma cells (Caco2) were cultured to verify Tß4-induced barrier function and autophagy changes. Mucin2 levels decreased, microbial infiltration increased, and Tß4 expression increased in the colitis mouse model versus the control mice, indicating mucus barrier damage. Moreover, Tß4-treated C57BL/6 mice had damaged intestinal mucus barriers and decreased LC3II levels. Tß4 also inhibited colonic mucin2 production, disrupted tight junctions, and downregulated autophagy; these results were confirmed in Caco2 cells and normal human colon tissue. In summary, Tß4 may be implicated in colitis by compromising the integrity of the intestinal mucus barrier and inhibiting autophagy. Thus, Tß4 could be a new diagnostic marker for intestinal barrier defects.

Multiple myeloma with high expression of SLC7A11 is sensitive to erastin-induced ferroptosis.

Ferroptosis, a nonapoptotic form of cell death marked by iron-dependent peroxidation of phospholipids, is associated with the occurrence and progression of tumors. Erastin, a selective inhibitor of the cystine/glutamate transporter system Xc-, can induce the ferroptosis of cancer cells. Multiple myeloma (MM) has been reported to be insensitive to erastin-induced ferroptosis. However, we found the erastin sensitivity of different MM cells varied widely. Specifically, SLC7A11 abundance determined the sensitivity of MM cells to erastin-induced ferroptosis. MM cells expressing a high SLC7A11 level were more sensitive to erastin-induced ferroptosis than cells expressing a low level of SLC7A11. Moreover, the expression of SLC7A11 gradually increased with the progression of plasma cell dyscrasias. Survival analysis indicated that high levels of SLC7A11 predicted a poor prognosis for MM patients. Knocking down SLC7A11 expression significantly inhibited the proliferation of MM cells and induced ferroptotic cell death. Additionally, we revealed that the long noncoding RNA (lncRNA) SLC7A11-AS1 was a critical regulatory factor of SLC7A11 expression. SLC7A11-AS1 overexpression diminished SLC7A11 levels, leading to the ferroptosis of MM cells. In summary, our data show that heterogeneous SLC7A11 expression affects MM cell sensitivity to ferroptosis, providing a theoretical basis for improving the clinical treatment of MM.

Unraveling Intracellular Protein Corona Components of Nanoplastics via Photocatalytic Protein Proximity Labeling.

Bioaccumulation of nanoplastic particles has drawn increasing attention regarding environmental sustainability and biosafety. How nanoplastic particles interact with the cellular milieu still remains elusive. Herein, we exemplify a general approach to profile the composition of a "protein corona" interacting with nanoparticles via the photocatalytic protein proximity labeling method. To enable photocatalytic proximity labeling of the proteome interacting with particles, iodine-substituted BODIPY (I-BODIPY) is selected as the photosensitizer and covalently conjugated onto amino-polystyrene nanoparticles as a model system. Next, selective proximity labeling of interacting proteins is demonstrated using I-BODIPY-labeled nanoplastic particles in both Escherichia coli lysate and live alpha mouse liver 12 cells. Mechanistic studies reveal that the covalent modifications of proteins by an aminoalkyne substrate are conducted via a reactive oxygen species photosensitization pathway. Further proteomic analysis uncovers that mitochondria-related proteins are intensively involved in the protein corona, indicating substantial interactions between nanoplastic particles and mitochondria. In addition, proteostasis network components are also identified, accompanied by consequent cellular proteome aggregation confirmed by fluorescence imaging. Together, this work exemplifies a general strategy to interrogate the composition of the protein corona of nanomaterials by endowing them with photooxidation properties to enable photocatalytic protein proximity labeling function.

FitDevo: accurate inference of single-cell developmental potential using sample-specific gene weight.

The quantification of developmental potential is critical for determining developmental stages and identifying essential molecular signatures in single-cell studies. Here, we present FitDevo, a novel method for inferring developmental potential using scRNA-seq data. The main idea of FitDevo is first to generate sample-specific gene weight (SSGW) and then infer developmental potential by calculating the correlation between SSGW and gene expression. SSGW is generated using a generalized linear model that combines sample-specific information and gene weight learned from a training dataset covering scRNA-seq data of 17 previously published datasets. We have rigorously validated FitDevo's effectiveness using a testing dataset with scRNA-seq data from 28 existing datasets and have also demonstrated its superiority over current methods. Furthermore, FitDevo's broad application scope has been illustrated using three practical scenarios: deconvolution analysis of epidermis, spatial transcriptomic data analysis of hearts and intestines, and developmental potential analysis of breast cancer. The source code and related data are available at https://github.com/jumphone/fitdevo.

Artificial Intelligence in battling infectious diseases: A transformative role.

It is widely acknowledged that infectious diseases have wrought immense havoc on human society, being regarded as adversaries from which humanity cannot elude. In recent years, the advancement of Artificial Intelligence (AI) technology has ushered in a revolutionary era in the realm of infectious disease prevention and control. This evolution encompasses early warning of outbreaks, contact tracing, infection diagnosis, drug discovery, and the facilitation of drug design, alongside other facets of epidemic management. This article presents an overview of the utilization of AI systems in the field of infectious diseases, with a specific focus on their role during the COVID-19 pandemic. The article also highlights the contemporary challenges that AI confronts within this domain and posits strategies for their mitigation. There exists an imperative to further harness the potential applications of AI across multiple domains to augment its capacity in effectively addressing future disease outbreaks.

Multiplexable all-optical nonlinear activator for optical computing.

As an alternative solution to surpass electronic neural networks, optical neural networks (ONNs) offer significant advantages in terms of energy consumption and computing speed. Despite the optical hardware platform could provide an efficient approach to realizing neural network algorithms than traditional hardware, the lack of optical nonlinearity limits the development of ONNs. Here, we proposed and experimentally demonstrated an all-optical nonlinear activator based on the stimulated Brillouin scattering (SBS). Utilizing the exceptional carrier dynamics of SBS, our activator supports two types of nonlinear functions, saturable absorption and rectified linear unit (Relu) models. Moreover, the proposed activator exhibits large dynamic response bandwidth (∼11.24 GHz), low nonlinear threshold (∼2.29 mW), high stability, and wavelength division multiplexing identities. These features have potential advantages for the physical realization of optical nonlinearities. As a proof of concept, we verify the performance of the proposed activator as an ONN nonlinear mapping unit via numerical simulations. Simulation shows that our approach achieves comparable performance to the activation functions commonly used in computers. The proposed approach provides support for the realization of all-optical neural networks.

Observing the Role of Electron Delocalization in Electronic Transport by Incorporating Actinides into Ligated Metal-Chalcogenide Superatoms.

Since delocalization of electronic states is a prerequisite for exerting unique electron transport properties, early actinides (An) with highly delocalized 5f/6d orbitals are natural candidates. However, given the experimental difficulties of such radioactive compounds and the complex relativistic effects in theoretical studies, understanding the electronic structure and bonding of actinides is underdeveloped on the periodic table. A further challenge is the very complicated electronic structures encountered in the confinement of actinides, as vividly illustrated by the weakly radioactive Th(Thorium)-encapsulated metal chalcogenide clusters, Th@Co6Te8L6 (L = PH3, PMe3, PEt3). Here we report the electronic structure and the electron transport properties of the Th@Co6Te8L6 clusters and compare them with those of the hollow Co6Te8L6 clusters using the nonequilibrium Green's function combined with relativistic density functional theory (NEGF-DFT). We found that the equilibrium conductance in Th@Co6Te8(PH3)6 (0.76 G0) has been greatly improved over that in Co6Te8(PH3)6 (0.03 G0), which has also been verified under an applied different bias voltage. The covalent bonding character between 6d (Th) and 3d (Co) atomic orbitals resulting from steric confinement is the source of the performance enhancement and a most important factor governing the accessibility of such 5f/6d orbitals. The results are of significance to the rapidly developing field of molecular nanoelectronics.

The effect of a couple-based posttraumatic growth intervention in supporting couples coping with colorectal cancer: A randomized controlled study.

OBJECTIVES: To promote posttraumatic growth (PTG) in colorectal cancer (CRC) couples, a couple-based PTG intervention was conducted, and the intervention had previously proved be feasible in CRC couples. The current study was conducted to validate the effects of intervention in CRC couples. METHOD: This is a randomized controlled study that included 174 CRC couples. All participants were randomized to either the intervention (usual care plus 5-week PTG intervention, n = 87) or the control group (usual care, n = 87). Data were collected from CRC couple dyads at baseline and immediately post-intervention periods. Primary outcome refers to positive changes, and secondary outcomes include marital satisfaction, quality of life (QOL), and anxiety and depression. Multilevel modeling was applied to analyze the intervention effects. RESULTS: Participants in the program showed increased PTG, marital satisfaction, and QOL both physically and mentally, and decreased levels of anxiety and depression over time. And spousal caregivers showed greater improvement in marital satisfaction and physical QOL compared with patients. In addition, significant intervention effects were shown in the participants' benefit finding, physical health and depressive symptoms. CONCLUSION: The study confirmed the effect of the PTG intervention on CRC couples' benefit finding, physical health and depressive symptoms. However, this study only measured outcome variables at two time-points. Future studies should add follow-up assessments to evaluate long-term effects of the intervention in CRC couples. REGISTRATION NUMBER: ChiCTR2300067809.

Synthesis of Fluorine-Containing Multisubstituted Oxa-Spiro[4,5]cyclohexadienones via a Fluorinated Alcohol-Catalyzed One-Pot Sequential Cascade Strategy.

In recent years, the application of fluorinated alcohols as solvents, cosolvents, or additives has become important in modern organic synthesis. However, their potential as efficient catalysts in organic synthesis has not been well-explored. In this article, we report on the development of a one-pot sequential cascade reaction of p-quinone methides with difluoroenoxysilanes using hexafluoroisopropanol as catalyst. This reaction allows for the preparation of fluorinated multisubstituted oxa-spiro[4,5]cyclohexadienones. By using 50 mol % 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), the reaction proceeds smoothly to yield 1,6-conjugated products, which are then subjected to oxidative dearomatization/hemiacetalization using PhI(OAc)2. The overall process affords moderate to high yields and excellent diastereoselectivities.

Chemical sensors detect and resolve proteome aggregation in peripheral neuropathy cell model induced by chemotherapeutic agents.

As a consequence of somatosensory nervous system injury or disease, neuropathic pain is commonly associated with chemotherapies, known as chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN-induced proteome aggregation in neuronal cells remain elusive due to limited detection tools. Herein, we present series sensors for fluorescence imaging (AggStain) and proteomics analysis (AggLink) to visualize and capture aggregated proteome in CIPN neuronal cell model. The environment-sensitive AggStain imaging sensor selectively binds and detects protein aggregation with 12.3 fold fluorescence enhancement. Further, the covalent AggLink proteomic sensor captures cellular aggregated proteins and profiles their composition via LC-MS/MS analysis. This integrative sensor platform reveals the presence of proteome aggregation in CIPN cell model and highlights its potential for broader applications in assessing proteome stability under various cellular stress conditions.

Clinical outcomes of serial endoscopic balloon dilation for duodenal Crohn's disease-associated strictures.

BACKGROUND: Endoscopic balloon dilation (EBD) is a safe and effective treatment for Crohn's disease (CD)-associated strictures. However, serial EBDs have rarely been reported. This study aimed to evaluate the efficacy and safety of serial EBDs for treating CD-associated duodenal strictures compared with intermittent EBDs. METHODS: Patients with CD-associated duodenal strictures who underwent EBD were recruited. The clinical data, stricture characteristics, number of EBDs, dilation diameter, complications, surgical interventions, and follow-up periods were recorded. Patients were divided into a serial dilation group and an intermittent dilation group to analyze the differences in safety and efficacy. RESULTS: Forty-five patients with duodenal CD-associated strictures underwent a total of 139 dilations. A total of 23 patients in the serial dilation group underwent 72 dilations, for a median of 3 (range 3 ~ 4) dilations per patient, and 22 patients in the intermittent dilation group underwent 67 dilations, for a median of 3 (range 1 ~ 6) dilations per patient. Technical success was achieved in 97.84% (136/139) of the patients. During the follow-up period, three patients in the intermittent dilation group underwent surgery, and the total clinical efficacy was 93.33% (42/45). No difference in safety or short-term efficacy was noted between the two groups, but serial EBDs exhibited significantly greater clinical efficacy between 6 months and 2 years. No significant difference in recurrence-free survival was observed, but the median longest recurrence-free survival and recurrence-free survival after the last EBD in the serial dilation group were 693 days (range 298 ~ 1381) and 815 days (range 502 ~ 1235), respectively, which were significantly longer than the 415 days (range 35 ~ 1493) and 291 days (range 34 ~ 1493) in the intermittent dilation group (p = 0.013 and p = 0.000, respectively). At the last follow-up, the mean diameter of the duodenal lumen was 1.17 ± 0.07 cm in the serial dilation group, which was greater than the 1.11 ± 0.10 cm in the intermittent dilation group (p = 0.018). We also found that the Simple Endoscopic Score for Crohn's Disease was associated with an increased risk of surgical intervention (HR 2.377, 95% CI 1.125-5.020; p = 0.023) and recurrence at 6 months after the last EBD (HR 0.698, 95% CI 0.511-0.953; p = 0.024), as assessed by univariate analysis. CONCLUSIONS: Compared to the intermittent EBDs, serial EBDs for duodenal CD-associated strictures exhibit greater clinical efficacy within two years and could delay stricture recurrence. We suggest that serial EBDs can be a novel option for endoscopic treatment of duodenal CD-associated strictures.

Stratum affects the distribution of soil selenium bioavailability by modulating the soil physicochemical properties: A case study in a Se-enriched area, China.

The soil selenium (Se) content and bioavailability are important for human health. In this regard, knowing the factors driving the concentration of total Se and bioavailable Se in soils is essential to map Se, enhance foodstuffs' Se content, and improve the Se nutritional status of humans. In this study, total Se and Se bioavailability (i.e., phosphate extracted Se) in surface soils (0-20 cm) developed on different strata were analyzed in a Se-enriched region of Southwest China. Furthermore, the interaction between the stratum and soil properties was assessed and how did the stratum effect on the concentration and spatial distribution of Se bioavailability in soils was investigated. Results showed that the median concentration of total Se in soils was 0.308 mg/kg, which is higher than China's soil background. The mean proportion of phosphate extracted Se in total Se was 12.2 %. The values of total Se, phosphate extracted Se, and soil organic matter (SOM) in soils increased with the increasing stratum age. In contrast, the coefficient of weathering and eluviation (BA) values decreased. The analysis of statistics and Geodetector revealed that the SOM, stratum, and BA were the dominant controlling factors for the contents and distributions of soil total Se and phosphate extracted Se. This study provided strong evidence that the soil properties that affected the total Se and Se bioavailability were modulated by the local geological background, and had important practical implications for addressing Se malnutrition and developing the Se-rich resource in the study region and similar geological settings in different parts of the globe.

In situ Production of Hydroxyl Radicals via Three-Electron Oxygen Reduction: Opportunities for Water Treatment.

The electro-Fenton (EF) process is an advanced oxidation technology with significant potential; however, it is limited by two steps: generation and activation of H2O2. In contrast to the production of H2O2 via the electrochemical two-electron oxygen reduction reaction (ORR), the electrochemical three-electron (3e-) ORR can directly activate molecular oxygen to yield the hydroxyl radical (·OH), thus breaking through the conceptual and operational limitations of the traditional EF reaction. Therefore, the 3e- ORR is a vital process for efficiently producing ·OH in situ, thus charting a new path toward the development of green water-treatment technologies. This review summarizes the characteristics and mechanisms of the 3e- ORR, focusing on the basic principles and latest progress in the in situ generation and efficient utilization of ·OH through the modulation of the reaction pathway, shedding light on the rational design of 3e- ORR catalysts, mechanistic exploration, and practical applications for water treatment. Finally, the future developments and challenges of efficient, stable, and large-scale utilization of ·OH are discussed based on achieving optimal 3e- ORR regulation and the potential to combine it with other technologies.

Aggregation Effect on Multiperformance Improvement in Aryl-Armed Phenazine-Based Emitters.

The concept of aggregate science was proposed to explain changes in materials performance that accompany the generation of aggregates, but aggregation-triggered multifunction improvements in a class of materials have rarely been reported. Herein, we present the first report of a new class of multifunctional aggregation-induced emission (AIE) luminogens (AIEgens) based on 5,10-diarylphenazine (DPZ) derivates with full-wavelength emission. Intriguingly, multiple properties, such as fluorescence intensity and free radical and type I reactive oxygen species (ROS) efficiencies, could be simultaneously activated from the unimolecular level to the aggregate state. The mechanisms of this multiple performance improvement are discussed in detail based on sufficient performance characterization, and some of the newly prepared AIEgens exhibited toxicity to cancer cells during photodynamic therapy. This work systematically demonstrates the positive effect of aggregation on improving multiple functions of materials, which is expected to promote the development of aggregate science theory for the design of multifunctional materials.

Dysregulation of alternative splicing contributes to multiple myeloma pathogenesis.

BACKGROUND: Dysregulation of alternative splicing (AS) triggers many tumours, understanding the roles of splicing events during tumorigenesis would open new avenues for therapies and prognosis in multiple myeloma (MM). METHODS: Molecular, genetic, bioinformatic and statistic approaches are used to determine the mechanism of the candidate splicing factor (SF) in myeloma cell lines, myeloma xenograft models and MM patient samples. RESULTS: GSEA reveals a significant difference in the expression pattern of the alternative splicing pathway genes, notably enriched in MM patients. Upregulation of the splicing factor SRSF1 is observed in the progression of plasma cell dyscrasias and predicts MM patients' poor prognosis. The c-indices of the Cox model indicated that SRSF1 improved the prognostic stratification of MM patients. Moreover, SRSF1 knockdown exerts a broad anti-myeloma activity in vitro and in vivo. The upregulation of SRSF1 is caused by the transcription factor YY1, which also functions as an oncogene in myeloma cells. Through RNA-Seq, we systematically verify that SRSF1 promotes the tumorigenesis of myeloma cells by switching AS events. CONCLUSION: Our results emphasise the importance of AS for promoting tumorigenesis of MM. The candidate SF might be considered as a valuable therapeutic target and a potential prognostic biomarker for MM.

Clickable Photoreactive ATP-Affinity Probe for Global Profiling of ATP-Binding Proteins.

Adenosine triphosphate (ATP) is the major energy carrier in organisms, and there are many cellular proteins that can bind to ATP. Among these proteins, kinases are key regulators in several cell signaling processes, and aberrant kinase signaling contributes to the development of many human diseases, including cancer. Hence, small-molecule kinase inhibitors have been successfully used for the treatment of various diseases. Since the ATP-binding pockets are similar for many kinases, it is very important to evaluate the selectivity of different kinase inhibitors. We report here a clickable ATP photoaffinity probe for the global profiling of ATP-binding proteins. After incubating the protein lysate with the ATP probe followed by ultraviolet (UV) irradiation, ATP-binding proteins were labeled with an alkyne handle for subsequent biotin conjugation through click chemistry. Labeled proteins were enriched with streptavidin beads, digested with trypsin, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). More than 400 ATP-binding proteins, including approximately 200 kinases, could be identified in a single LC-MS/MS run in the data-dependent acquisition mode. We then applied this method to the analysis of targets of three selected ATP-competitive kinase inhibitors. We were able to successfully identify some of their reported target proteins from label-free quantification results and validated the results using Western blot analyses. Together, we developed a clickable ATP photoaffinity probe for proteome-wide profiling of ATP-binding proteins and demonstrated that this chemoproteomic method is amenable to high-throughput target identification of kinase inhibitors.