Papers by Douglas Lauffenburger
Graphical Abstract Highlights d Glycolysis in T cells/PBMCs from T2D subjects fails to stimulate ... more Graphical Abstract Highlights d Glycolysis in T cells/PBMCs from T2D subjects fails to stimulate T2D inflammation d T cells from T2D subjects have altered mitochondria d Altered import or oxidation of fatty acids activates inflammation in healthy cells d Mitochondrial changes combine with fatty acid metabolites to activate inflammation
Molecular Biology of …, 2008
Epidermal growth factor (EGF) receptor-mediated cell migration plays a vital role in invasion of ... more Epidermal growth factor (EGF) receptor-mediated cell migration plays a vital role in invasion of many tumor types. EGF receptor ligands increase invasiveness in vivo, but it remains unclear how consequent effects on intrinsic cell motility behavior versus effects ...
Journal of biomaterials science. Polymer edition, 2002
In order to elucidate design principles for biocompatible materials that can be created by in sit... more In order to elucidate design principles for biocompatible materials that can be created by in situ transformation from self-assembling oligopeptides, we investigate a class of oligopeptides that can self-assemble in salt solutions to form three-dimensional matrices. This class of peptides possesses a repeated sequence of amino acid residues with the type: hydrophobic/negatively-charged/hydrophobic/positively-charged. We systematically vary three chief aspects of this sequence type: (1) the hydrophobic side chains; (2) the charged side chains; and (3) the number of repeats. Each of these has been previously shown to influence the self-assembly properties of these materials. Employing a rheometric assay we measure the shear modulus of gels created from variants of each of these aspects. First, we observe order-of-magnitude changes in shear moduli when we vary oligopeptide length, with biphasic dependence on length. This result may be due to competition between, in short oligopeptides,...
Immunology and Cell Biology, 2011
Variable sensitivity to T-cell-receptor (TCR)- and IL-7-receptor (IL-7R)-mediated homeostatic sig... more Variable sensitivity to T-cell-receptor (TCR)- and IL-7-receptor (IL-7R)-mediated homeostatic signals among naïve T cells has thus far been largely attributed to differences in TCR specificity. We show here that even when withdrawn from self-peptide-induced TCR stimulation, CD8(+) T cells exhibit heterogeneous responses to interleukin-7 (IL-7) that are mechanistically associated with IL-7R expression differences that correlate with relative CD5 expression. Whereas CD5(hi) and CD5(lo) T cells survive equivalently in the presence of saturating IL-7 levels in vitro, CD5(hi) T cells proliferate more robustly. Conversely, CD5(lo) T cells exhibit prolonged survival when withdrawn from homeostatic stimuli. Through quantitative experimental analysis of signaling downstream of IL-7R, we find that the enhanced IL-7 responsiveness of CD5(hi) T cells is directly related to their greater surface IL-7R expression. Further, we identify a quantitative threshold in IL-7R-mediated signaling capacity required for proliferation that lies well above an analogous threshold requirement for survival. These distinct thresholds allow subtle differences in IL-7R expression between CD5(lo) and CD5(hi) T cells to give rise to significant variations in their respective IL-7-induced proliferation, without altering survival. Heterogeneous IL-7 responsiveness is observed similarly in vivo, with CD5(hi) naïve T cells proliferating preferentially in lymphopenic mice or lymphoreplete mice administered with exogenous IL-7. However, IL-7 in lymphoreplete mice appears to be maintained at an effective level for preserving homeostasis, such that neither CD5(hi) IL-7R(hi) nor CD5(lo) IL-7R(lo) T cells proliferate or survive preferentially. Our findings indicate that IL-7R-mediated signaling not only maintains the size but also impacts the diversity of the naïve T-cell repertoire.
Computational models aid in the quantitative understanding of cell signalling networks. One impor... more Computational models aid in the quantitative understanding of cell signalling networks. One important goal is to ascertain how multiple network components work together to govern cellular responses, that is, to determine cell 'signal-response' relationships. Several methods exist to study steady-state signals in the context of differential equation-based models. However, many biological networks influence cell behaviour through time-varying signals operating during a transient activated state that ultimately returns to a basal steady-state. A computational approach adapted from dynamical systems analysis to discern how diverse transient signals relate to alternative cell fates is described. Direct finite-time Lyapunov exponents (DLEs) are employed to identify phase-space domains of high sensitivity to initial conditions. These domains delineate regions exhibiting qualitatively different transient activities that would be indistinguishable using steady-state analysis but which correspond to different outcomes. These methods are applied to a physicochemical model of molecular interactions among caspase-3, caspase-8 and X-linked inhibitor of apoptosis--proteins whose transient activation determines cell death against survival fates. DLE analysis enabled identification of a separatrix that quantitatively characterises network behaviour by defining initial conditions leading to apoptotic cell death. It is anticipated that DLE analysis will facilitate theoretical investigation of phenotypic outcomes in larger models of signalling networks.
Science signaling, 2015
Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryoti... more Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human α-actinin-4 (ACTN4), Tyr(4) and Tyr(31) are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr(4) site. We found that introducing a phosphomimetic mutation at only Tyr(31) was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr(31) is mostly buried and that phosphorylation of Tyr(4) would increase the solvent exposure and thus kinase accessibility of Tyr(31). In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr(4),...
Nature, 1997
Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that... more Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that link the cell to extracellular-matrix ligands, transmitting forces and signals necessary for locomotion. Whether cells will migrate or not on a given substratum, and also their speed, depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. These and other factors affect the way molecular systems integrate to effect and regulate cell migration. Here we show that changes in cell migration speed resulting from three separate variables-substratum ligand level, cell integrin expression level, and integrin-ligand binding affinity-are all quantitatively predictable through the changes they cause in a single unifying parameter: short-term cell-substratum adhesion strength. This finding is consistent with predictions of a mathematical model for cell migration. The ligand concentration promoting maximum migration speed decreases reciprocally as integrin expression increases. Increases in integrin-ligand affinity similarly result in maximal migration at reciprocally lower ligand concentrations. The maximum speed attainable, however, remains unchanged as ligand concentration, integrin expression, or integrin-ligand affinity vary, suggesting that integrin coupling with intracellular motors remains unaltered.
2008 8th IEEE International Conference on BioInformatics and BioEngineering, 2008
Cell death in hepatocellular carcinoma (HCC) cells as well as in other cell types is driven by a ... more Cell death in hepatocellular carcinoma (HCC) cells as well as in other cell types is driven by a complex signaling transduction network comprised of several different pathways. In this study, we measured cell death of the HCC cell line C3A and we quantify the contribution of several signaling pathways using a reverse approach; namely instead of measuring the activity of
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, Jan 21, 2015
Women in Africa, especially young women, have very high HIV incidence rates that cannot be fully... more Women in Africa, especially young women, have very high HIV incidence rates that cannot be fully explained by behavioural risks. We investigated whether genital inflammation influenced HIV acquisition in this group. Twelve selected cytokines, including 9 inflammatory cytokines and chemokines (IL-1α, IL-1β, IL-6, TNF-α, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β), hematopoeitic IL-7 and GM-CSF, and regulatory IL-10 were measured prior to HIV infection in cervicovaginal lavages from 58 HIV seroconvertors and 58 matched uninfected controls and plasma from a subset of 107 of these women from the CAPRISA 004 tenofovir gel trial. HIV seroconversion was associated with raised genital inflammatory cytokines (including chemokines MIP-1α, MIP-1β and IP-10). The risk of HIV acquisition was significantly higher in women with evidence of genital inflammation, defined by at least 5 of 9 inflammatory cytokines being raised [OR 3.2; 95% confidence interval 1.3-7.9; p=0.014]. Genital cytokine concentrati...
Open forum infectious diseases, 2015
Background. Human immunodeficiency virus (HIV)-1 elite controllers (ECs) represent an ideal popu... more Background. Human immunodeficiency virus (HIV)-1 elite controllers (ECs) represent an ideal population to study the effects of HIV persistence on chronic inflammation in the absence of antiretroviral therapy (ART). Methods. Twenty inflammatory markers measured in cohorts of ECs, HIV suppressed noncontrollers, and HIV-uninfected controls were compared using rank-based tests and partial least squares discriminant analysis (PLSDA). Spearman correlations were determined among the inflammatory markers, residual viremia by the single-copy assay, and CD4(+) T cell slope. Results. Significant differences were seen between cohorts in 15 of the soluble inflammatory markers. Human immunodeficiency virus-1 ECs were found to have the highest levels for all of the markers with the exception of RANTES. In particular, median levels of 7 inflammatory markers (soluble CD14 [sCD14], interferon [IFN]-γ, IFN-γ-inducible protein [IP]-10, interleukin [IL]-4, IL-10, sCD40L, and granulocyte-macrophage co...
We present an integrated microfluidic probe that enables the measurement of multiple kinase activ... more We present an integrated microfluidic probe that enables the measurement of multiple kinase activities in selected phenotypically distinct single cells from large cell populations on standard tissue culture platforms. The contents of a cell are captured without disturbing its extracellular context by creating a small lysis zone at the probe tip by hydrodynamic confinement. Pneumatic micro-valves are then used to separate and mix the captured lysate into different assay mixtures in separate small volume chambers for a fluorimetric assay. We demonstrate here the ability to simultaneously measure the activity of three kinases: Akt, MAPKAPK2, PKA and a loading control enzyme, GAPDH, from a single cell. This single cell assay platform enables the correlation of cellular phenotype to intracellular biochemical state at the single cell level and hence can provide a clearer understanding of cell behavior in heterogeneous cell populations.
Analytical chemistry, Jan 5, 2014
Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high det... more Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.
Computational models aid in quantitative understanding of cell signaling networks. One important ... more Computational models aid in quantitative understanding of cell signaling networks. One important goal is to ascertain how multiple network components work together to govern cellular responses -i.e., to determine cell "signal-response" relationships. Several methods exist to study steady-state signals in the context of differential equation-based models. However, many biological networks influence cell behavior through time-varying signals operating during a transient activated state which ultimately returns to a basal steady-state. We describe a computational approach adapted from dynamical systems analysis to discern how diverse transient signals relate to alternative cell fates. We employ direct finite-time Lyapunov exponents (DLEs) to identify phase-space domains of high sensitivity to initial conditions. These domains delineate regions exhibiting qualitatively different transient activities that would be indistinguishable using steady-state analysis but which correspond to different outcomes. We apply these methods to a physico-chemical model of molecular interactions among caspase-3, caspase-8, and XIAP -proteins whose transient activation determines cell death-vs-survival fates. DLE analysis enabled identification of a separatrix that quantitatively characterizes network behavior by defining initial conditions leading to apoptotic cell death. We anticipate that DLE analysis will facilitate theoretical investigation of phenotypic outcomes in larger models of signaling networks.
Transforming growth factor ~ (TGF~) has been reported to be a more potent agonist when compared t... more Transforming growth factor ~ (TGF~) has been reported to be a more potent agonist when compared to epidermal growth factor (EGF) in several systems while acting via their common receptor, the epidermal growth factor receptor (EGFFt). It has been postulated that this increased potency is mediated by the increased recycling of EGFR upon activation by TGFc~ as against receptor activation by EGF. The authors test this hypothesis by simultaneously measuring mitogenesis and the dynamics of surface receptor number in response to these ligands in NR6 mouse fibroblasts expressing the EGFFt. The data demonstrates that increased receptor recycling due to endosomal dissocation of TGF~ can indeed realise an increased mitogenic potency relative to EGF under appropriate cellular and experimental conditions (i.e. situations in which the increase in the number of occupied receptors clue to receptor sparing by TGF~ represents additional mitogenic signalling capacity). However, this difference in receptor trafficking does not uniquely determine the relative potencies of these ligands since TGFc~ is a less potent mitogen compared to EGF when experimental conditions are dominated by the effects of ligand trafficking on growth factor availability. Thus, the relative potencies of these growth factors are determined in a given context by the relative importance of ligand and receptor trafficking effects which determine the availability of these signalling components. These results are consistent with a suggested model of hormone responsiveness which favours dissociative ligands (such as TGFcO in receptor-limited situations and non-dissociative ligands (such as EGF) in the case of ligand limitation.
PloS one, 2012
K-RAS mutation poses a particularly difficult problem for cancer therapy. Activating mutations in... more K-RAS mutation poses a particularly difficult problem for cancer therapy. Activating mutations in K-RAS are common in cancers of the lung, pancreas, and colon and are associated with poor response to therapy. As such, targeted therapies that abrogate K-RAS-induced oncogenicity would be of tremendous value. We searched for small molecule kinase inhibitors that preferentially affect the growth of colorectal cancer cells expressing mutant K-RAS. The mechanism of action of one inhibitor was explored using chemical and genetic approaches. We identified BAY61-3606 as an inhibitor of proliferation in colorectal cancer cells expressing mutant forms of K-RAS, but not in isogenic cells expressing wild-type K-RAS. In addition to its anti-proliferative effects in mutant cells, BAY61-3606 exhibited a distinct biological property in wild-type cells in that it conferred sensitivity to inhibition of RAF. In this context, BAY61-3606 acted by inhibiting MAP4K2 (GCK), which normally activates NFκβ sig...
Philosophical transactions. Series A, Mathematical, physical, and engineering sciences, Jan 28, 2010
Angiogenesis is crucial during many physiological processes and is influenced by various biochemi... more Angiogenesis is crucial during many physiological processes and is influenced by various biochemical and biomechanical factors. Models have proved useful in understanding the mechanisms of angiogenesis and also the characteristics of the capillaries formed as part of the process. We have developed a three-dimensional hybrid, agent-field model where individual cells are modelled as sprout-forming agents in a matrix field. Cell independence, cell-cell communication and stochastic cell response are integral parts of the model. The model simulations incorporate probabilities of an individual cell to transition into one of four stages--quiescence, proliferation, migration and apoptosis. We demonstrate that several features, such as continuous sprouts, cell clustering and branching, that are observed in microfluidic experiments conducted under controlled conditions using few angiogenic factors can be reproduced by this model. We also identify the transition probabilities that result in sp...
Drug Discovery Today, 2014
Reliable in vitro human disease models that capture the complexity of in vivo tissue behaviors ar... more Reliable in vitro human disease models that capture the complexity of in vivo tissue behaviors are crucial to gain mechanistic insights into human disease and enable the development of treatments that are effective across broad patient populations. The integration of stem cell technologies, tissue engineering, emerging biomaterials strategies and microfabrication processes, as well as computational and systems biology approaches, is enabling new tools to generate reliable in vitro systems to study the molecular basis of human disease and facilitate drug development. In this review, we discuss these recently developed tools and emphasize opportunities and challenges involved in combining these technologies toward regenerative science.
Lab Chip, 2015
Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ... more Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.
Hepatocellular Carcinoma (HCC) is one of the leading causes of death worldwide, with only a handf... more Hepatocellular Carcinoma (HCC) is one of the leading causes of death worldwide, with only a handful of treatments effective in unresectable HCC. Most of the clinical trials for HCC using new generation interventions (drug-targeted therapies) have poor efficacy whereas just a few of them show some promising clinical outcomes [1]. This is amongst the first studies where the mode of action of some of the compounds extensively used in clinical trials is interrogated on the phosphoproteomic level, in an attempt to build predictive models for clinical efficacy. Signaling data are combined with previously published gene expression and clinical data within a consistent framework that identifies drug effects on the phosphoproteomic level and translates them to the gene expression level. The interrogated drugs are then correlated with genes differentially expressed in normal versus tumor tissue, and genes predictive of patient survival. Although the number of clinical trial results considered is small, our approach shows potential for discerning signaling activities that may help predict drug efficacy for HCC.
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Papers by Douglas Lauffenburger