Single-Vesicle Electrochemistry Reveals Polysaccharide from Glochidion eriocarpum Champ. Regulates Vesicular Storage and Exocytotic Release of Dopamine.

Polysaccharides, which are well-known natural macromolecules, have been recognized for their protective effects on neurons and their influence on extracellular dopamine levels in the brain. It is crucial to investigate the impact of plant polysaccharides on neurotransmission, particularly regarding the vesicular storage and exocytosis of neurotransmitters. In this study, we demonstrated the possibility of studying how the polysaccharide from Glochidion eriocarpum Champ.(GPS) affects vesicle dopamine content and the dynamics of exocytosis in pheochromocytoma (PC12) cells using single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC). Our results unambiguously demonstrate that GPS effectively enhances vesicular neurotransmitter content and alters the dynamics of exocytosis, favoring a smaller fraction of content released in exocytotic release, thereby inducing the partial release mode. These significant effects are attributed to GPS's efficient elevation of calcium influx, significant alteration in the composition of exocytosis-related membrane lipids, and enhancement of free radical scavenging ability. These findings not only establish GPS as a promising candidate for preventive or therapeutic interventions against neurodegenerative disorders but also reiterate the importance of screening native neurologic drugs with single-vesicle electrochemical approaches, the combination of SCA and IVIEC, from a neurotransmitter-centric perspective.

Single-Vesicle Electrochemistry Reveals Sex Difference in Vesicular Storage and Release of Catecholamine.

Quantitative measurements of sex difference in vesicle chemistry (i.e., chemical storage and release) at the single-vesicle level are essential to understand sex differences in cognitive behaviors; however, such measurements are very challenging to conventional analytical methods. By using single-vesicle electrochemistry, we find the duration of single exocytotic events of chromaffin cells prepared from male rats is statistically longer than that from female rats, leading to more neurotransmitter released in the male group. Further analysis reveals that a higher percentage of vesicles in the female group release part of the neurotransmitter, i.e., partial release, during exocytosis than that in male group. This sex dimorphism in neurotransmitter release in exocytosis might relate to the sex difference in the expression of voltage-dependent calcium channels and membrane lipid composition. Our finding offers the first experimental evidence that sex dimorphism even exists in vesicle chemistry, providing a brand new viewpoint for understanding the sex dimorphism in exocytosis.

Unveiling the Role of DJ-1 Protein in Vesicular Storage and Release of Catecholamine with Nano/Micro-Tip Electrodes.

DJ-1 protein deficiency caused by PARK7 gene mutation has been suggested to closely relate to Parkinson's disease (PD), mainly through the attenuation D2 dopamine receptor activity in mice; however, whether or how it affects the vesicular storage and exocytosis of neurochemicals remains unclear. By using electrochemical methods at a single vesicle/cell level with nano/micro-tip electrodes, we for the first time find that DJ-1 protein deficiency caused by PARK7 gene knockout (KO) in mice has little effect on vesicular catecholamine content but significantly prolongs the exocytotic events, especially the closing time of exocytotic fusion pores. Further studies suggest the inhibition of α-synuclein aggregation by DJ-1 protein might be one way that DJ-1 protein acts on neurotransmission. This finding offers the first direct link between DJ-1 protein deficiency and vesicular chemical storage and release of chemicals, providing a new chemical insight into the pathology of PD caused by PARK7 gene mutation.

Mass Spectrometry Imaging Suggests That Cisplatin Affects Exocytotic Release by Alteration of Cell Membrane Lipids.

We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging to investigate the effect of cisplatin, the first member of the platinum-based anticancer drugs, on the membrane lipid composition of model cells to see if lipid changes might be involved in the changes in exocytosis observed. Platinum-based anticancer drugs have been reported to affect neurotransmitter release resulting in what is called the "chemobrain"; however, the mechanism for the influence is not yet understood. TOF-SIMS imaging was carried out using a high energy 40 keV (CO2)6000+ gas cluster ion beam with improved sensitivity for intact lipids in biological samples. Principal components analysis showed that cisplatin treatment of PC12 cells significantly affects the abundance of different lipids and their derivatives, particularly phosphatidylcholine and cholesterol, which are diminished. Treatment of cells with 2 µM and 100 µM cisplatin showed similar effects on induced lipid changes. Lipid content alterations caused by cisplatin treatment at the cell surface are associated with the molecular and bimolecular signaling pathways of cisplatin-induced apoptosis of cells. We suggest that lipid alterations measured by TOF-SIMS are involved, at least in part, in the regulation of exocytosis by cisplatin.

In Vivo Monitoring of Oxygen Fluctuation Simultaneously at Multiple Sites of Rat Cortex during Spreading Depression.

Spreading depression (SD) is a common pathological process in the brain shown as propagating neuronal depolarization followed by activity depression over the brain, and it is closely related to migraines and epilepsy. Although O2 is known to fluctuate during SD, the difference of O2 responses at different sites in the same brain region remains unknown. In this study, we develop an in vivo electrochemical method with microelectrode arrays (MEAs) to monitor, in real time, O2 fluctuation at multiple sites of rat cortex during SD with high spatial/temporal resolution. Platinum nanoparticles are electrochemically deposited on the multiplexed electrodes of the MEAs to monitor O2 fluctuation simultaneously and selectively via a four-electron reduction process. Configuration of electrode arrays is designed rationally to exclude the probable crosstalk between neighbor recording electrodes during simultaneous measurements. With the MEAs, we find both the basal O2 levels and O2 fluctuations at different sites of the cortex during SD exhibit significant differences, indicating the intensity of energy metabolism and oxidative stress vary at different sites even in the same brain region. Further studies prove that O2 fluctuation is mostly caused by the increase of brain blood flow and the consumption of neuronal O2 during SD. Our study reveals that energy metabolism varies at different sites in brain cortex during SD propagation, which may provide new understanding for SD-related pathological processes.

Electrochemical quantification of transmitter concentration in single nanoscale vesicles isolated from PC12 cells.

We use an electrochemical platform, nanoparticle tracking analysis, and differential centrifugation of single catecholamine vesicles to study the properties of nanometer transmitter vesicles, including the number of molecules, size, and catecholamine concentration inside. Vesicle impact electrochemical cytometry (VIEC) was used to quantify the catecholamine content of single vesicles in different batches isolated from pheochromocytoma (PC12) cells with different ultracentrifugation speeds. We show that, vesicles containing less catecholamine are obtained at subsequent centrifugation steps with higher speed (force). Important to quantification, the cumulative content after subsequent centrifugation steps is equivalent to that of one-step centrifugation at the highest speed, 70 000g. Moreover, as we count molecules in the vesicles, we compared molecular numbers from VIEC, flow VIEC, and intracellular VIEC to corresponding vesicle size measured by nanoparticle tracking analysis to evaluate catecholamine concentration in vesicles. The data suggest that vesicular catecholamine concentration is relatively constant and independent of the vesicular size, indicating vesicular transmitter content as a main factor regulating the vesicle size.

Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry.

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 °C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation.

Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry.

Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday's law and to correlate this to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain-related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.

Zinc Regulates Chemical-Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry.

We applied electrochemical techniques with nano-tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 µm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc-treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.

Using Single-Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis.

The pretreatment of cultured pheochromocytoma (PC12) cells with cis-diamminedichloroplatinum (cisplatin), an anti-cancer drug, influences the exocytotic ability of the cells in a dose-dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single-cell amperometry reflects that 2 µm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 µm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.

Quantitative measurement of transmitters in individual vesicles in the cytoplasm of single cells with nanotip electrodes.

The quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease, and yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nanotip conical carbon-fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only a fraction of the quantal neurotransmitter content is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, thus resulting in incomplete expulsion of the vesicular contents.

Mass spectrometric proteomics reveals that nuclear protein positive cofactor PC4 selectively binds to cross-linked DNA by a trans-platinum anticancer complex.

An MS-based proteomic strategy combined with chemically functionalized gold nanoparticles as affinity probes was developed and validated by successful identification and quantification of HMGB1, which is well characterized to interact selectively with 1,2-cross-linked DNA by cisplatin, from whole cell lysates. The subsequent application of this method to identify proteins responding to 1,3-cross-linked DNA by a trans-platinum anticancer complex, trans-PtTz (Tz = thiazole), revealed that the human nuclear protein positive cofactor PC4 selectively binds to the damaged DNA, implying that PC4 may play a role in cellular response to DNA damage by trans-PtTz.

Single vesicle chemistry reveals partial release happens at the mechanical stress-induced exocytosis.

Neuronal activity can be modulated by mechanical stress in the central nervous system (CNS) in neurodegenerative diseases, for example Alzheimer's disease. However, the impact of mechanical stress on chemical signal transmission, especially the storage and release of neurotransmitter in neuron vesicles, has not been fully clarified. In this study, a nanotip conical carbon fiber microelectrode (CFME) and a disk CFME are placed in and on a cell, respectively. The nanotip conical CFME functions for both the mechanical stress and the quantification of transmitter storage in single vesicles, while the disk CFME is used to monitor the transmitter release during exocytosis induced by mechanical stress at the same cell. By comparing the vesicular transmitter storage with its release during mechanical stress-induced exocytosis at the same cell, we find the release ratio of transmitter in chromaffin cells varies from 27 % to 100 %, while for PC12 cells from 30 % to 100 %. Our results indicate that the exocytosis of cells responding to mechanical stress shows individual difference obviously, with a significant population exhibiting partial release mode. The variation of Ca2+ channels and mechanosensitive ion channels on cell membrane may both contribute to this variation. Our discovery not only shows mechanical stress can change the transmission of cellular chemical signals at the vesicle level, but also provides an important reference perspective for the study of nervous system regulation and nervous system diseases.

Triterpenoid saponins from the roots of Panax notoginseng with protective effects against APAP-induced liver injury.

Five previously undescribed protopanaxatriol-type saponins, notoginsenosides Ta-Te (1-5), together with eighteen known triterpenoid saponins (6-23) were isolated from the roots of Panax notoginseng. The structures of new compounds were determined by HRESIMS and NMR spectroscopic analyses and chemical methods. Compounds 1 and 2 were the first examples of ginsenosides featuring a 6-deoxy-ß-d-glucose moiety from Panax species. Compounds 1-4, 7, 10, 12, 21-22 showed protective effects on L02 cells against the injury of acetaminophen (APAP). Among them, notoginsenoside R1 (12), ginsenoside Rg1 (21), and ginsenoside Re (22) were the most potent ones, with cell viabilities >80%. Moreover, compounds 12 and 22 remarkably alleviated APAP-induced liver injury in mice. These saponins are potential hepatoprotective agents.

Transferrin serves as a mediator to deliver organometallic ruthenium(II) anticancer complexes into cells.

We report herein a systematic study on interactions of organometallic ruthenium(II) anticancer complex [(η(6)-arene)Ru(en)Cl](+) (arene = p-cymene (1) or biphenyl (2), en = ethylenediamine) with human transferrin (hTf) and the effects of the hTf-ligation on the bioavailability of these complexes with cisplatin as a reference. Incubated with a 5-fold excess of complex 1, 2, or cisplatin, 1 mol of diferric hTf (holo-hTf) attached 0.62 mol of 1, 1.01 mol of 2, or 2.14 mol of cisplatin. Mass spectrometry revealed that both ruthenium complexes coordinated to N-donors His242, His273, His578, and His606, whereas cisplatin bound to O donors Tyr136 and Tyr317 and S-donor Met256 in addition to His273 and His578 on the surface of both apo- and holo-hTf. Moreover, cisplatin could bind to Thr457 within the C-lobe iron binding cleft of apo-hTf. Neither ruthenium nor platinum binding interfered with the recognition of holo-hTf by the transferrin receptor (TfR). The ruthenated/platinated holo-hTf complexes could be internalized via TfR-mediated endocytosis at a similar rate to that of holo-hTf itself. Moreover, the binding to holo-hTf well preserved the bioavailability of the ruthenium complexes, and the hTf-bound 1 and 2 showed a similar cytotoxicity toward the human breast cancer cell line MCF-7 to those of the complexes themselves. However, the conjugation with holo-hTf significantly reduced the cellular uptake of cisplatin and the amount of platinated DNA adducts formed intracellularly, leading to dramatic reduction of cisplatin cytotoxicity toward MCF-7. These findings suggest that hTf can serve as a mediator for the targeting delivery of Ru(arene) anticancer complexes while deactivating cisplatin.

Thymines in single-stranded oligonucleotides and G-quadruplex DNA are competitive with guanines for binding to an organoruthenium anticancer complex.

Organometallic ruthenium(II) complexes [(η(6)-arene)Ru(en)Cl](+) (arene = e.g., biphenyl (1), dihydrophenanthrene, tetrahydroanthracene) show promising anticancer activity both in vitro and in vivo and are cytotoxic to cisplatin-resistant cancer cells, implying that these monofunctional complexes have a different mechanism of action from that of bifunctional cisplatin. We demonstrate here that complex 1 binds selectively to the guanine base in the 15-mer single-stranded oligodeoxynucleotides (ODNs) 5'-CTCTCTX7G8Y9CTTCTC-3' [X = Y = T; X = C, Y = A; X = A, Y = T; X = T, Y = A] to form thermodynamically stable adducts, but thymine bases (T7/T11 or T6/T11) compete kinetically with guanine for binding to 1. The T-bound monoruthenated species eventually convert to diruthenated products via a second step of binding at G or/and to G-bound monoruthenated species through dissociation of the diruthenated adducts. Complex 1 was further shown to bind preferentially to the middle T in a sequence rather than to a T near the terminus and favor coordination to a 5'-T compared to a 3'-T. Interestingly, the T bases in the human telomeric G-quadruplex sequence (5'-AGGGTTAGGGTTAGGGTTAGGG-3') were found to be more competitive both kinetically and thermodynamically with G bases for binding to 1. These results suggest that thymine bases play a unique role in the pathways of ruthenation of DNA by organoruthenium anticancer complexes and illustrate that kinetic studies can provide new insight into the mechanism of action of metallodrugs in addition to study of the structures and functions of the thermodynamically stable end products.

Ginsenoside Rg1 modulates vesicular dopamine storage and release during exocytosis revealed with single-vesicle electrochemistry.

Ginsenoside Rg1, a tetracyclic triterpenoid derivative extracted from the roots of Panax ginseng C. A. Meyer, can enhance learning and memory and improve cognitive impairment. However, whether or how it affects vesicular dopamine storage and its release during exocytosis remains unknown. By using single-vesicle electrochemistry, we for the first time find out that Rg1 not only upregulates vesicular dopamine content but also increases exocytosis frequency and modulates dopamine release during exocytosis in PC12 cells, which may relate to the activation of protein kinases, causing a series of biological cascades. This finding offers the possible link between Rg1 and vesicular chemical storage and exocytotic release, which is of significance for understanding the nootropic role of Rg1 from the perspective of neurotransmission.

Continuous electrochemical monitoring of extracellular lactate production from neonatal rat cardiomyocytes following myocardial hypoxia.

Continuous monitoring of lactate production from cardiomyocytes is of great physiological and pathological importance since the level of lactate in extracellular fluid is closely associated with myocardial energy metabolism with implication in the diagnosis and therapeutics of myocardial hypoxia and ischemia. This study demonstrates an electrochemical approach to continuous monitoring of lactate production from neonatal rat cardiomyocytes following myocardial hypoxia with a dehydrogenase-based electrochemical biosensor and a negative pressure driven culture sampling. To eliminate the effect of pH variation occurring following the cardiomyocyte hypoxia on the biosensor response and to supply nicotinamide adenine dinucleotide (NAD(+)) cofactor necessary for the enzymatic reaction of lactate dehydrogenase (LDH), artificial cerebrospinal fluid (aCSF) containing NAD(+) cofactor is externally perfused and mixed online with cell culture before the culture goes to the detector. The method exhibits a high selectivity against the electrochemically active species endogenously existing in the extracellular culture of cardiomyocytes and a high tolerance against the variation of pH following cardiomyocyte hypoxia. The dynamic linear range for lactate detection is from 0.20 to 10 mM (I (nA) = 25.6 C(Lactate) (mM) + 20.1, γ = 0.996) with a detection limit of 0.16 mM (S/N = 3). The physiological level of the extracellular lactate of neonatal rat cardiomyocytes is determined to be 1.1 ± 0.1 mM (n = 3) with the cell density of about 0.5 × 10(3) cells/mm(2). When the cardiomyocytes are subject to hypoxia induced with anoxic reagents, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the extracellular lactate increases to 255 ± 30.3% (n = 3), relative to the physiological level, following 20 min of the hypoxia. This study essentially offers a new and effective electrochemical platform for investigating energy metabolism during cardiac physiological and pathological processes.

An electrochemical method for investigation of conformational flexibility of active sites of Trametes versicolor laccase based on sensitive determination of copper ion with cysteine-modified electrodes.

This study demonstrates a facile yet effective electrochemical method to investigate the conformational flexibility of the active sites of Trametes versicolor (Tv) laccase based on sensitive determination of copper ions (Cu(2+)) dissociated from the enzyme with the cysteine-modified Au electrodes. In the native state, the multicopper active sites are deeply buried in the polypeptide of Tv laccase and are thus not electrochemically detectable even at the cysteine-modified Au electrodes. Upon the unfolding of Tv laccase induced by guanidine hydrochloride (GdnHCl), copper ions dissociate from the peptide chain and, as a consequence, are electrochemically reduced and thus detected at the cysteine-modified Au electrodes. Such a property could be used to investigate the conformational flexibility of multicopper active sites of Tv laccase in a simple way. We find that both the conformation of the multicopper active sites in Tv laccase and the enzyme activity change with the presence of a low concentration of GdnHCl denaturant (midpoint, where 50% of the enzyme is unfolded, at 0.7 M). This concentration is lower than that required to induce the conformational changes of Tv laccase molecule as a whole (midpoint at 3.4 M), as investigated by the intrinsic fluorescence of Tv laccase. This observation suggests that the multicopper active sites are formed by relatively weak interactions and hence may be conformationally more flexible than the intact enzyme. The electrochemical method demonstrated in this study is technically simple yet effective and could be potentially useful for investigation on the thermodynamics and kinetics of the conformational changes of multicopper oxidases induced by different denaturants.

Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by titania coated magnetic mesoporous silica microspheres for screening of protein kinase inhibitors.

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO(2)/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r(2) being 0.99. The IC(50) values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.