Effect of N-Aryl Para-Benzhydryl Substituent on the Thermal Stability of α-Diimine Nickel Catalyst.

The thermal stability of α-diimine nickel catalysts has always been the focus of research. The introduction of large groups in the backbone or N-aryl ortho-position is a relatively mature solution. However, the question of whether the N-aryl bond rotation is a factor affecting the thermal stability of nickel catalysts is still open. In this work, the effects of N-aryl para-benzhydryl substitutes on catalyst thermal stability are investigated, and the results of ethylene polymerization and the factors affecting thermal stability (steric effect, electronic effect, five-membered coordination ring stability, N-aryl bond rotation, etc.) are systematically analyzed. It is believed that the introduction of large steric hindrance groups at the N-aryl para-position hinders the rotation of the N-aryl bond. This obstacle effect is beneficial to improving catalyst thermal stability, and the obstacle capacity is weakened with the increase of ortho-substituent size.

Clip-assisted endoloop ligation of the mucosal defect after resection of colorectal polyps decreased postprocedural delayed bleeding.

Background: Postprocedural delayed bleeding (PDB) remains the most common major complication of colorectal polypectomy. Incomplete clip closure of mucosal defect and unclosed injured blood vessels in gaps between clips may be the risk factors for PDB. Objectives: To observe whether completely no-gap closure of mucosal defect after polypectomy can reduce PDB occurrence. Design: Single-center, retrospective case-control study. Methods: In this study based on historical comparisons of patients in 2 time periods, only the patients with polyps sized between 6 and 15 mm were included. A new clip-assisted endoloop ligation (CAEL, treatment group) method was used between January 2019 and December 2020, and a traditional simple clip closure (SCC, control) was used Between January 2017 and December 2018 to prevent PDB after polypectomy. The rate of PDB of two groups and risk factors for PDB were evaluated. Results: Totally 4560 patients were included in the study; 2418 patients belong to CAEL group, and 2142 patients belong to SCC group. The overall rate of PDB was significantly lower in CAEL group compared to SCC group (0.6% versus 1.5%, p < 0.00). On multivariate logistic analysis, CAEL was a significant independent preventive factor for PDB (odds ratio (OR), 0.092; 95% confidence interval (CI), 0.029-0.3335; p = 0.000). Polyps located at rectum (colon versus rectum) represented a significant independent risk factor for PDB (OR, 11.888; 95% CI, 3.343-42.269; p = 0.001). Conclusion: Completely no-gap closure of mucosal defect after polypectomy further reduced the rate of PDB for polyps sized between 6 and 15 mm. CAEL may be a significant independent preventive factor for PDB. Polyps located at the rectum may be a significant independent risk factor for PDB.

Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes.

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes.

"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.

Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a "fix and click" method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The "fix and click" strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using "fix and click" paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes.

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

Clear colonoscopy as a surveillance tool in the prediction and reduction of advanced neoplasms: a randomized controlled trial.

BACKGROUND: Missed and incompletely resected lesions remain the main cause of the recurrence of advanced neoplasms (ANs) in post-polypectomy patients. This study aims to determine whether the recurrence of ANs can be predicted and reduced by the newly developed clear colonoscopy procedure. METHODS: Between 2006 and 2010, a total of 1350 participants with colorectal neoplasm were equally randomized to clear colonoscopy surveillance (CCS) and routine colonoscopy surveillance (RCS) in our center. Clear colonoscopy was achieved through repeat colonoscopy. On surveillance colonoscopy at 3 (for high-risk patients) and 5 (for low-risk patients) years, the recurrence of ANs and the relationship between the frequency of repeat examinations required for a clear colonoscopy and the recurrence of ANs were analyzed. RESULTS: Surveillance colonoscopy at 3 and 5 years showed that the incidence of ANs in patients belonging to the CCS group was 1.7%, which was lower than 4.7% in patients belonging to the RCS group (P = 0.012) for both high- and low-risk patients. Out of the 1126 patients who achieved clear colonoscopy on first repeat examination, only 5 ( 0.4%) were found to have ANs on surveillance examination, whereas 12 out of the 217 (5.4%) patients on second repeat examination and 5 out of the 29 (17.2%) patients on third repeat examination were found to have ANs (P < 0.001). CONCLUSION: Surveillance based on clear colonoscopy decreased the incidence of ANs in post-polypectomy patients. The number of repeat examinations required for a clear colonoscopy is an important factor in the prediction of the recurrence of ANs.

A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC-γ Isozymes Operating at Membranes.

The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.

Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations.

Direct activation of the human phospholipase C-γ isozymes (PLC-γ1, -γ2) by tyrosine phosphorylation is fundamental to the control of diverse biological processes, including chemotaxis, platelet aggregation, and adaptive immunity. In turn, aberrant activation of PLC-γ1 and PLC-γ2 is implicated in inflammation, autoimmunity, and cancer. Although structures of isolated domains from PLC-γ isozymes are available, these structures are insufficient to define how release of basal autoinhibition is coupled to phosphorylation-dependent enzyme activation. Here, we describe the first high-resolution structure of a full-length PLC-γ isozyme and use it to underpin a detailed model of their membrane-dependent regulation. Notably, an interlinked set of regulatory domains integrates basal autoinhibition, tyrosine kinase engagement, and additional scaffolding functions with the phosphorylation-dependent, allosteric control of phospholipase activation. The model also explains why mutant forms of the PLC-γ isozymes found in several cancers have a wide spectrum of activities, and highlights how these activities are tuned during disease.

Many enzymes are poised to receive signals from the surrounding environment and translate them into responses inside the cell. One such enzyme is phospholipase C-γ1 (PLC-γ1), which controls how cells grow, divide and migrate.When activating signals are absent, PLC-γ1 usually inhibits its own activity, a mechanism called autoinhibition. This prevents the enzyme from binding to its targets, which are fat molecules known as lipids. When activating signals are present, a phosphate group serves as a 'chemical tag' and is added onto PLC-γ1, allowing the enzyme to bind to lipids.Failure in the regulation of PLC-γ1 or other closely related enzymes may lead to conditions such as cancer, arthritis and Alzheimer's disease. However, it remains unclear how autoinhibition suppresses the activity of the enzyme, and how it is stopped by the addition of the phosphate group.Here, Hajicek et al. determine in great detail the three-dimensional structure of the autoinhibited form of the enzyme using a method known as X-ray crystallography. This reveals that PLC-γ1 has two major lobes: one contains the active site that modifies lipids, and the other sits on top of the active site to prevent lipids from reaching it. The findings suggest that when the phosphate group attaches to PLC-γ1, it triggers a large shape change that shifts the second lobe away from the active site to allow lipids to bind.The three-dimensional structure also helps to understand how mutations identified in certain cancers may activate PLC-γ1. In particular, these mutations disrupt the interactions between elements that usually hold the two lobes together, causing the enzyme to activate more easily.The work by Hajicek et al. provides a framework to understand how cells control PLC-γ1. It is a first step toward designing new drugs that alter the activity of this enzyme, which may ultimately be useful to treat cancer and other diseases.

A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.

A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-ß3 by Gαq Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution.

Polypectomy for complete endoscopic resection of small colorectal polyps.

BACKGROUND AND AIMS: Small colorectal polyps are encountered frequently and may be incompletely removed during colonoscopy. The optimal technique for removal of small colorectal polyps is uncertain. The aim of this study was to compare the incomplete resection rate (IRR) by using EMR or cold snare polypectomy (CSP) for the removal of small adenomatous polyps. METHODS: This was a prospective randomized controlled study from a tertiary-care referral center. A total of 358 patients who satisfied the inclusion criteria (polyp sized 6-9 mm) were randomized to the EMR (n =179) and CSP (n =179) groups, and their polyps were treated with conventional EMR or CSP, respectively. After polypectomy, an additional 5 forceps biopsies were performed at the base and margins of polypectomy sites to assess the presence of residual polyp tissue. The EMR and CSP samples were compared to assess the IRR. RESULTS: Among a total of 525 polyps, 415 (79.0%) were adenomatous polyps, and 41 (16.4%) were advanced adenomas. The overall IRR for adenomatous polyps was significantly higher in the CSP group compared with the EMR group (18/212, 8.5% vs 3/203, 1.5%; P = .001). Logistic regression analysis revealed that the CSP procedure was a stronger risk factor for the IRR (odds ratio [OR] 6.924; 95% confidence interval [CI], 2.098-24.393; P = .003). In addition, piecemeal resection was the most important risk factor for the IRR (OR 28.696; 95% CI, 3.620-227.497; P = .001). The mean procedure time for polypectomy was not significantly different between the EMR and CSP groups (5.5 ± 2.7 vs 4.7 ± 3.4 minutes; P = .410). None of these patients presented with delayed bleeding. There were no severe adverse events related to the biopsies. CONCLUSIONS: EMR was significantly superior to CSP for achieving complete endoscopic resection of small colorectal polyps. Patients with piecemeal resection of polyps had a higher risk for incomplete resection. (Clinical trial registration number: Hongwei-1102-12.).

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes.

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

A Method to Generate and Analyze Modified Myristoylated Proteins.

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.

Clip closure of defect after endoscopic resection in patients with larger colorectal tumors decreased the adverse events.

BACKGROUND: Clip closure of large colorectal mucosal defects may reduce the rate of adverse events in a cost-effective manner. OBJECTIVE: To assess the adverse events and outcomes of clip closure of defects after endoscopic resection in patients with large colorectal tumors. DESIGN: Prospective, randomized, controlled study. SETTING: Single tertiary referral center. PATIENTS AND INTERVENTIONS: Patients with lesions measuring 1 to 4 cm who were scheduled for endoscopic resection between March 2012 and December 2014 were randomly assigned to a clip-closure group and a no-closure group. In the clip-closure group, the defect of the resection site was completely closed with an endoclip. In the no-closure group, the defect was left open. The following primary outcome measures were assessed: delayed postoperative bleeding, postpolypectomy coagulation syndrome, perforation, and abdominal pain. Secondary outcome measures of length of hospital stay, time required for procedure, and patient's satisfaction were also assessed. RESULTS: Patients and lesions had similar characteristics across both groups. For patients who underwent clip closure (n = 174), the rates of delayed postoperative bleeding (1.1% [2/174]) and postpolypectomy coagulation syndrome (0.6% [1/174]) were lower than those in the no-closure group (6.9% [12/174], P = .01 and 4.6% [8/174], P = .03). Two patients experienced perforation, 1 in each group. In the clip-closure group, 4 patients reported abdominal pain as opposed to 26 in the no-closure group (2.8% vs 16.7%, P < .01). The procedure took longer in the closure group (38.1 minutes vs 30.9 minutes, P = .04). The length of hospitalization was shorter in the closure group (3.1 days vs 4.7 days, P = .03). Total medical expense was similar between the 2 groups. Patients who underwent closure reported greater satisfaction. LIMITATION: This was a single-center analysis. CONCLUSIONS: Clip closure of endoscopic resection defects in patients with large colorectal tumors decreased the rate of procedure-related adverse events and did not increase the cost of hospitalization.

Antimicrobial prophylaxis in patients with colorectal lesions undergoing endoscopic resection.

AIM: To investigate the effect of prophylaxis with antibiotics on clinical adverse events in patients who underwent endoscopic submucosal dissection (ESD) or endoscopic mucosal resection (EMR) for colorectal lesions. METHODS: From June 2011 to December 2013, a total of 428 patients were enrolled into the study, of which 214 patients admitted to hospital underwent EMR or ESD procedures. These patients were randomized to an antibiotic group, in which patients were given cefuroxime 1.5 g iv half an hour before and 6 h after surgery respectively, and a control group, in which patients were not given any antibiotic. A further 214 outpatients with small polyps treated by polypectomy were compared with controls that were matched by age and gender, and operations were performed as outpatient surgery. Recorded patient parameters were demographics, characteristics of lesions and treatment modality, and the size of the wound area. The primary outcome measures were clinical adverse events, including abdominal pain, diarrhea, hemotachezia, and fever. Secondary outcome measures were white blood cell count, C-reactive protein and blood culture. Additionlly, the relationship between the size of the wound area and clinical adverse events was analyzed. RESULTS: A total of 409 patients were enrolled in this study, with 107 patients in the control group, 107 patients in the antibiotic group, and another 195 cases in the follow-up outpatient group. The patients' demographic characteristics, including age, gender, characteristics of lesions, treatment modality, and the size of the wound area were similar between the 2 groups. The rates of adverse events in the antibiotic group were significantly lower than in the control group: abdominal pain (2.8% vs 14.9%, P < 0.01), diarrhea (2.0% vs 9.3%, P < 0.05), and fever (0.9% vs 8.4%, P < 0.05) respectively. The levels of inflammatory markers also decreased significantly in the antibiotic group compared with the control group: leukocytosis (2.0% vs 11.2%, P < 0.01), and C-reactive protein (2.0% vs 10.7%, P < 0.05). Additionally, clinical adverse events were related to the size of the surgical wound area. When the surgical wound area was larger than 10 mm × 10 mm, there were more clinical adverse events. CONCLUSION: Clinical adverse events are not uncommon after EMR or ESD procedures. Prophylactic antibiotics can reduce the incidence of clinical adverse events. This should be further explored.

Small molecule inhibitors of phospholipase C from a novel high-throughput screen.

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.

Isotope-coded, fluorous photoaffinity labeling reagents.

A pair of isotope-coded, fluorous photoaffinity labeling reagents has been developed and coupled with a peptide. The modified peptides form adducts with methanol upon light illumination, which show characteristic isotope labeling patterns in mass spectra and can be separated from other peptides through fluorous silica.

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives.

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.

High-throughput fluorescence polarization assay for the enzymatic activity of GTPase-activating protein of ADP-ribosylation factor (ARFGAP).

GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs) play key cellular roles in vesicle production and trafficking, adhesion, migration, and development. Dysfunctional regulation of ARFGAPs has been implicated in various diseases, including cancer, Alzheimer disease, and autism. Unfortunately, there are few mechanistic details describing how ARFGAPs contribute to disease states. In this regard, it would be extremely helpful to have a set of small molecules that selectively and directly modulate specific ARFGAPs as probes to dissect ARFGAP-regulated cell signaling under various conditions. Currently, such probes are lacking, and their identification is hampered by the lack of a suitable high-throughput assay to monitor ARFGAP activity. Here, the authors describe and validate a robust high-throughput assay using fluorescence polarization to monitor the ability of diverse ARFGAPs to enhance the capacity of ARF1 to hydrolyze guanosine triphosphate.

A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes.

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer's disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable K(m) and V(max) with the endogenous PIP(2). This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high-throughput screening of PLC inhibitors.