Quantitative Kinetic Analysis Research Articles

Kinetic models of conjugated metabolic cycles

A general method is developed for the quantitative kinetic analysis of conjugated metabolic cycles in the human organism. This method is used as a basis for constructing a kinetic graph and model of the conjugated citric acid and ureapoiesis cycles. The results from a kinetic analysis of the model for these cycles are given.

Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes.

SummaryThe quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

Single-molecule visualization of a formin-capping protein ‘decision complex’ at the actin filament barbed end

Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

Computer-aided breast MR image feature analysis for prediction of tumor response to chemotherapy.

To identify a new clinical marker based on quantitative kinetic image features analysis and assess its feasibility to predict tumor response to neoadjuvant chemotherapy. The authors assembled a dataset involving breast MR images acquired from 68 cancer patients before undergoing neoadjuvant chemotherapy. Among them, 25 patients had complete response (CR) and 43 had partial and nonresponse (NR) to chemotherapy based on the response evaluation criteria in solid tumors. The authors developed a computer-aided detection scheme to segment breast areas and tumors depicted on the breast MR images and computed a total of 39 kinetic image features from both tumor and background parenchymal enhancement regions. The authors then applied and tested two approaches to classify between CR and NR cases. The first one analyzed each individual feature and applied a simple feature fusion method that combines classification results from multiple features. The second approach tested an attribute selected classifier that integrates an artificial neural network (ANN) with a wrapper subset evaluator, which was optimized using a leave-one-case-out validation method. In the pool of 39 features, 10 yielded relatively higher classification performance with the areas under receiver operating characteristic curves (AUCs) ranging from 0.61 to 0.78 to classify between CR and NR cases. Using a feature fusion method, the maximum AUC=0.85±0.05. Using the ANN-based classifier, AUC value significantly increased to 0.96±0.03 (p<0.01). This study demonstrated that quantitative analysis of kinetic image features computed from breast MR images acquired prechemotherapy has potential to generate a useful clinical marker in predicting tumor response to chemotherapy.

From nuclei to micro-structure in colloidal crystallization: Investigating intermediate length scales by small angle laser light scattering.

Hard sphere suspensions are well recognized model systems of statistical physics and soft condensed matter. We here investigate the temporal evolution of the immediate environment of nucleating and growing crystals and/or their global scale distribution using time resolved Small Angle Light Scattering (SALS). Simultaneously performed Bragg scattering measurements provide an accurate temporal gauging of the sequence of events. We apply this approach to studies of re-crystallization in several different shear molten hard sphere and attractive hard sphere samples with the focus being on the diversity of observable signal shapes and their change in time. We demonstrate that depending on the preparation conditions different processes occur on length scales larger than the structural scale, which significantly influence both the crystallization kinetics and the final micro-structure. By careful analysis of the SALS signal evolution and by comparing different suggestions for small angle signal shapes to our data, we can for most cases identify the processes leading to the observed signals. These include form factor scattering from crystals surrounded by depletion zones and structure factor scattering from late stage inter-crystallite ordering. The large variety of different small angle signals thus in principle contains valuable information complementary to that gained from Bragg scattering or microscopy. Our comparison, however, also shows that further refinement and adaptation of the theoretical expressions to the sample specific boundary conditions is desired for a quantitative kinetic analysis of micro-structural evolution.

Regulatory Rebound in IL-12-Treated Tumors Is Driven by Uncommitted Peripheral Regulatory T Cells.

IL-12 promotes a rapid reversal of immune suppression in the tumor microenvironment. However, the adjuvant activity of IL-12 is short-lived due to regulatory T cell (Treg) reinfiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the posttreatment rebound was driven by the CD4(+)CD25(+)Foxp3(+) neuropilin-1(low) peripheral Treg (pTreg), resulting in a 3-5-fold increase in the pTreg to CD4(+)CD25(+)Foxp3(+) neuropilin-1(high) thymic Treg ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region, CTLA-4 exon 2, and glucocorticoid-induced TNFR exon 5, were phenotypically unstable, and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg-like phenotype. In vitro culture of posttherapy Treg populations under Th1-promoting conditions resulted in higher levels of IFN-γ production by pTreg compared with thymic Treg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified IDO-positive dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.

Highly Enhanced Crystallization Kinetics of Poly(l-lactic acid) by Poly(ethylene glycol) Grafted Graphene Oxide Simultaneously as Heterogeneous Nucleation Agent and Chain Mobility Promoter

Nanofillers can act as effective heterogeneous nucleation agents for semicrystalline polymers; however, it never facilitates the crystal growth. In the current work, we proposed a facile strategy to enhance the crystallization kinetics of poly(l-lactic acid) (PLLA) by simultaneously accelerating the crystal nucleation and growth. Herein, we synthesized poly(ethylene glycol) (PEG) grafted graphene oxide (GO) (PEGgGO). Pronounced effects of PEGgGO on the crystalline morphology and crystallization rate of PLLA were explicitly clarified by direct morphological observation and quantitative crystallization kinetics analysis. The results manifested that, in contrast to the unmodified GO, PEGgGO desirably dispersed in PLLA and also preserved the high nucleation ability. More importantly, the flexible PEG chains on GO served as a chain mobility promoter and boosted the crystal growth rate of PLLA. Compared to the PLLA/GO nanocomposite containing 0.5 wt % GO, the nucleation density and crystal growth rate of the PLLA/PEGgGO one were increased by 110% and 14.3% at the crystallization temperature of 130 °C, respectively, leading to 52.2% relative augment in the final crystallinity. Our proposed methodology offers the flexibility of fabricating the polymer nanocomposits with well-dispersed nanofillers and also high crystallinity, by which the step toward the high-performance nanocomposites will be further.

Biochemical characterization of smoothened receptor antagonists by binding kinetics against drug-resistant mutant

Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (koff) rates were determined by kinetic binding studies using [3H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [3H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.

Quantitative kinetic analysis of PET amyloid imaging agents [11C]BF227 and [18F]FACT in human brain

IntroductionThe purpose of this study was to compare two amyloid imaging agents, [11C]BF227 and [18F]FACT (derivative from [11C]BF227) through quantitative pharmacokinetics analysis in human brain. MethodsPositron emission tomography studies were performed on six elderly healthy control (HC) subjects and seven probable Alzheimer’s disease (AD) patients with [11C]BF227 and 10 HC subjects and 10 probable AD patients with [18F]FACT. Data from nine regions of interest were analyzed by several approaches, namely non-linear least-squared fitting methods with arterial input functions (one-tissue compartment model(1TCM), two-tissue compartment model (2TCM)), Logan plot, and linearized methods with reference region (Reference Logan plot (RefLogan), MRTM0, MRTM2). We also evaluated SUV and SUVR for both tracers. The parameters estimated by several approaches were compared between two tracers for detectability of differences between HC and AD patients. ResultsFor [11C]BF227, there were no significant difference of VT (2TCM, 1TCM) and SUV in all regions (Student t-test; p<0.05) and significant differences in the DVRs (Logan, RefLogan, and MRTM2) and SUVRs in six neocortical regions (p<0.05) between the HC and AD groups. For [18F]FACT, significant differences in DVRs (RefLogan, MRTM0, and MRTM2) were observed in more than four neocortical regions between the HC and AD groups (p<0.05), and the significant differences were found in SUVRs for two neocortical regions (inferior frontal coretex and lateral temporal coretex). Our results showed that both tracers can clearly distinguish between HC and AD groups although the pharmacokinetics and distribution patterns in brain for two tracers were substantially different. ConclusionThis study revealed that although the PET amyloid imaging agents [11C]BF227 and [18F]FACT have similar chemical and biological properties, they have different pharmacokinetics, and caution must be paid for usage of the tracers.

Regulatory rebound in Interleukin-12-treated tumors is driven by uncommitted peripheral Treg (TUM9P.1012)

Abstract Interleukin-12 (IL-12) promotes a rapid reversal of immune suppression in the tumor microenvironment. However the adjuvant activity of IL-12 is short-lived due to T-regulatory cell (Treg) re-infiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes (TDLN) revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the post-treatment rebound was driven by the CD4+ CD25+ Foxp3+ Neuropilin-1(Nrp-1)low peripheral Treg (pTreg) resulting in a 5-fold increase in the pTreg to CD4+ CD25+ Foxp3+ Nrp-1high thymic Treg (tTreg) ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region (TSDR), CTLA-4 exon 2 and GITR exon 5; were phenotypically unstable; and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg (iTreg)-like phenotype. In vitro culture of post-therapy Treg populations under TH1-promoting conditions resulted in the production of IFNg by pTreg but not by tTreg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified Indoleamine 2,3 dioxygenase (IDO)-positive Dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.

Alpers disease mutations in human DNA polymerase gamma cause catalytic defects in mitochondrial DNA replication by distinct mechanisms.

The human mitochondrial DNA polymerase gamma (Pol-γ) is nuclearly encoded and is responsible for the replication and repair of the mitochondrial genome. Mutations S305R and P1073L in the POLG gene have been reported to be associated with early childhood Alpers syndrome. One patient harboring both mutations as compound heterozygous died at 2 years of age after disease onset at 9 months. Quantitative kinetic analysis on purified enzyme showed that the S305R mutation reduces the DNA binding affinity by 10-fold, and reduces the specificity constant (kcat/Km) for correct nucleotide incorporation by fourfold. It also causes a ∼threefold reduction in the excision rate to remove mismatched nucleotides. Compared to the wild-type Pol-γ, the S305R mutant showed no product formation in a reconstituted rolling circle replisome assay. Interestingly, the P1073L mutant exhibited wild-type activity in single turnover kinetics to quantify changes in kcat/Km, kcat, kexo, or processivity, and showed a twofold decrease in the net polymerization rate in the reconstituted replisome assay, while in yeast, P1073L caused a 60–70% mtDNA reduction in haploid cells. The heterozygous diploid yeast cells carrying S305R and P1073L mutations in trans showed ∼75% reduction of mtDNA content, relative to homozygous diploid cells with two wild-type alleles. Taken together, we show clearly in both the rolling circle and the humanized yeast system that the P1073L mutation caused significant defects in mtDNA replication, and our results suggest a role for P1073 in the functioning of the Pol-γ with the mitochondrial DNA helicase, and provide a rationale for understanding the physiological consequences of the S305R/P1073L compound heterozygote in humans.

Growth of Au on Pt icosahedral nanoparticles revealed by low-dose in situ TEM.

A growth mode was revealed by an in situ TEM study of nucleation and growth of Au on Pt icosahedral nanoparticles. Quantitative analysis of growth kinetics was carried out based on real-time TEM data, which shows the process involves: (1) deposition of Au on corner sites of Pt icosahedral nanoparticles, (2) diffusion of Au from corners to terraces and edges, and (3) subsequent layer-by-layer growth of Au on Au surfaces to form Pt@Au core-shell nanoparticles. The in situ TEM results indicate diffusion of Au from corner islands to terraces and edges is a kinetically controlled growth, as evidenced by a measurement of diffusion coefficients for these growth processes. We demonstrated that in situ electron microscopy is a valuable tool for quantitative study of nucleation and growth kinetics and can provide new insight into the design and precise control of heterogeneous nanostructures.

A simple routine for quantitative analysis of light and dark kinetics of photochemical and non-photochemical quenching of chlorophyll fluorescence in intact leaves.

Paper describes principles and application of a novel routine that enables the quantitative analysis of the photochemical O–J phase of the variable fluorescence F v associated with the reversible photo-reduction of the secondary electron acceptor QA of photosystem II (PSII) in algae and intact leaves. The kinetic parameters that determine the variable fluorescence F PP(t) associated with the release of photochemical quenching are estimated from 10 µs time-resolved light-on and light-off responses of F v induced by two subsequent light pulses of 0.25 (default) and 1000 ms duration, respectively. Application of these pulses allows estimations of (i) the actual value of the rate constants k L and k AB of the light excitation (photoreduction of QA) and of the dark re-oxidation of photoreduced QA ({text{Q}}_{text{A}}^{ - }), respectively, (ii) the actual maximal normalized variable fluorescence [nF v] associated with 100 % photoreduction of QA of open RCs, and (iii) the actual size β of RCs in which the re-oxidation of {text{Q}}_{text{A}}^{ - } is largely suppressed (QB-nonreducing RC with k AB ~ 0). The rate constants of the dark reversion of Fv associated with the release of photo-electrochemical quenching F PE and photo-electric stimulation F CET in the successive J–I and I–P parts of the thermal phase are in the range of (100 ms)−1 and (1 s)−1, respectively. The kinetics of fluorescence changes during and after the I–P phase are given special attention in relation to the hypothesis on the involvement of a Δµ H+-dependent effect during this phase and thereafter. Paper closes with author’s personal view on the demands that should be fulfilled for chlorophyll fluorescence methods being a correct and unchallenged signature of photosynthesis in algae and plants.

Quantification of 18F-fluorocholine kinetics in patients with prostate cancer.

Choline kinase is upregulated in prostate cancer, resulting in increased (18)F-fluoromethylcholine uptake. This study used pharmacokinetic modeling to validate the use of simplified methods for quantification of (18)F-fluoromethylcholine uptake in a routine clinical setting. Forty-minute dynamic PET/CT scans were acquired after injection of 204 ± 9 MBq of (18)F-fluoromethylcholine, from 8 patients with histologically proven metastasized prostate cancer. Plasma input functions were obtained using continuous arterial blood-sampling as well as using image-derived methods. Manual arterial blood samples were used for calibration and correction for plasma-to-blood ratio and metabolites. Time-activity curves were derived from volumes of interest in all visually detectable lymph node metastases. (18)F-fluoromethylcholine kinetics were studied by nonlinear regression fitting of several single- and 2-tissue plasma input models to the time-activity curves. Model selection was based on the Akaike information criterion and measures of robustness. In addition, the performance of several simplified methods, such as standardized uptake value (SUV), was assessed. Best fits were obtained using an irreversible compartment model with blood volume parameter. Parent fractions were 0.12 ± 0.4 after 20 min, necessitating individual metabolite corrections. Correspondence between venous and arterial parent fractions was low as determined by the intraclass correlation coefficient (0.61). Results for image-derived input functions that were obtained from volumes of interest in blood-pool structures distant from tissues of high (18)F-fluoromethylcholine uptake yielded good correlation to those for the blood-sampling input functions (R(2) = 0.83). SUV showed poor correlation to parameters derived from full quantitative kinetic analysis (R(2) < 0.34). In contrast, lesion activity concentration normalized to the integral of the blood activity concentration over time (SUVAUC) showed good correlation (R(2) = 0.92 for metabolite-corrected plasma; 0.65 for whole-blood activity concentrations). SUV cannot be used to quantify (18)F-fluoromethylcholine uptake. A clinical compromise could be SUVAUC derived from 2 consecutive static PET scans, one centered on a large blood-pool structure during 0-30 min after injection to obtain the blood activity concentrations and the other a whole-body scan at 30 min after injection to obtain lymph node activity concentrations.

Micelle stabilization via entropic repulsion: balance of force directionality and geometric packing of subunit.

Nanoparticles, 10-30 nm in size, have shown great prospects as nanocarriers for drug delivery. We designed amphiphiles based on 3-helix peptide-PEG conjugate forming 15 nm micelles (defined as "3-helix micelles") with good in vivo stability. Here, we investigated the effect of the site of PEG conjugation on the kinetic stability and showed that the conjugation site affects the PEG chain conformation and the overall molecular architecture of the subunit. Compared to the original design where the PEG chain is located in the middle of the 3-helix bundle, micelle kinetic stability was reduced when the PEG chain was attached near the N-terminus (t(1/2) = 35 h) but was enhanced when the PEG chain was attached near the C-terminus (t(1/2) = 80 h). Quantitative structural and kinetic analysis suggest that the kinetic stability was largely dictated by the combined effects of entropic repulsion associated with PEG chain conformation and the geometric packing of the trimeric subunits. The modular design approach coupled with a variety of well-defined protein stucture and functional polymers will significantly expand the utility of these materials as nanocarriers to meet current demands in nanomedine.

Interaction of Metal–Dipeptide Complex with Ninhydrin in the Absence and Presence of Conventional CTAB Surfactant

The present work is aimed at studying the interaction between copper-glycyltyrosine [(Cu(II)-Gly-Tyr)]+ and ninhydrin in water and in micelles formed by cetyltrimethylammonium bromide (CTAB) using spectrophometric measurements at 80°C and pH 5.0. The order of reaction remains the same in the two systems, that is, first- and fractional-order kinetics with respect to [Cu(II)-Gly-Tyr]+ and [ninhydrin], respectively, in the excess of ninhydrin over [Cu(II)-Gly-Tyr]+. It was observed that the product formed is same in both the media. The reaction is catalyzed by CTAB, and the maximum rate enhancement is about three fold. Quantitative kinetic analysis of kψ–[CTAB] data was explained in terms of pseudo-phase of the micelles (assuming the association/incorporation of both the reactants at the micellar surface).

Dynamic Contrast-Enhanced MRI of Mouse Liver: A Feasibility Study Using a Dual-Input Two-Compartment Tracer Kinetic Model

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been widely applied to evaluate microcirculatory parameters in clinical settings. However, pre-clinical studies involving DCE-MRI of small animals remain challenging with the requirement for high spatial and temporal resolution for quantitative tracer kinetic analysis. This study illustrates the feasibility of applying a high temporal resolution (2 s) protocol for liver imaging in mice by analyzing the DCE-MRI datasets of mice liver with a dual-input two-compartment tracer kinetic model. Phantom studies were performed to validate the T1 estimates derived by the proposed protocol before applying it in mice studies. The DCE-MRI datasets of mice liver were amendable to tracer kinetic analysis using a dual-input two-compartment model. Estimated micro-circulatory parameters were consistent with liver physiology, indicating viability of applying the technique for pre-clinical drug developments.

Interaction of Nickel-Dipeptide Complex with Ninhydrin in Aqueous and Micellar Media

Ninhydrin reactions using manual and automated techniques as well as ninhydrin spray reagents are wide ly used to analyze and characterize amino acids, peptides, and proteins as well as numerous other ninhydrin po sitive compounds in biomedical, clinical, food, forensic, histochemical , microbiological, and nutritional and plant studie s. Ninhydrin in aqueous solution reacts with α-amino acids to give a compound known as Ruhemann's purple (diketohydrindylidenediketohydrindamine, DYDA). In order to elucidate the mechanism of the reactions o f ninhydrin with (Ni(II)-Gly-Leu) + complexes, systematic kinetic studies were perform ed by following appearance of the respective colore d condensation products at different (surfactant), (r eactant), (salt) and temperature. The rates of the reaction were determined in both water and surfactant micelles in the absence and presence of various org anic and inorganic salts at 70 °C and pH 5.0. The result shows that the reaction follows the same fir st-order and fractional-order kinetics with respect to (Ni(II)-Gly-Leu) + and (ninhydrin), respectively, in both aqueous and mice llar medium. Quantitative kinetic analysis of k ψ(CTAB) data was performed on the basis of the pseudo-phase model of the micelles. + , CTAB

Interaction of the Molecular Chaperone DNAJB6 with Growing Amyloid-beta 42 (Aβ42) Aggregates Leads to Sub-stoichiometric Inhibition of Amyloid Formation

The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in Alzheimer disease) in a highly efficient manner. By monitoring fibril formation using Thioflavin T fluorescence and far-UV CD spectroscopy, we have found that the aggregation of Aβ42 is retarded by DNAJB6 in a concentration-dependent manner, extending to very low sub-stoichiometric molar ratios of chaperone to peptide. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of Aβ42 rather than the monomeric form of the peptide. This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. A low dissociation rate of DNAJB6 from Aβ42 aggregates leads to its incorporation into growing fibrils and hence to its gradual depletion from solution with time. When DNAJB6 is eventually depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction. These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form.

In situ drug-receptor binding kinetics in single cells: a quantitative label-free study of anti-tumor drug resistance.

Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.