Jeffrey Varner | Cornell University (original) (raw)

Papers by Jeffrey Varner

PLoS ONE, 2009

Intracellular calcium dynamics are critical to cellular functions like pain transmission. Extrace... more Intracellular calcium dynamics are critical to cellular functions like pain transmission. Extracellular ATP plays an important role in modulating intracellular calcium levels by interacting with the P2 family of surface receptors. In this study, we developed a mechanistic mathematical model of ATP-induced P2 mediated calcium signaling in archetype sensory neurons. The model architecture, which described 90 species connected by 162 interactions, was formulated by aggregating disparate molecular modules from literature. Unlike previous models, only mass action kinetics were used to describe the rate of molecular interactions. Thus, the majority of the 252 unknown model parameters were either association, dissociation or catalytic rate constants. Model parameters were estimated from nine independent data sets taken from multiple laboratories. The training data consisted of both dynamic and steady-state measurements. However, because of the complexity of the calcium network, we were unable to estimate unique model parameters. Instead, we estimated a family or ensemble of probable parameter sets using a multi-objective thermal ensemble method. Each member of the ensemble met an error criterion and was located along or near the optimal trade-off surface between the individual training data sets. The model quantitatively reproduced experimental measurements from dorsal root ganglion neurons as a function of extracellular ATP forcing. Hypothesized architecture linking phosphoinositide regulation with P2X receptor activity explained the inhibition of P2X-mediated current flow by activated metabotropic P2Y receptors. Sensitivity analysis using individual and the whole system outputs suggested which molecular subsystems were most important following P2 activation. Taken together, modeling and analysis of ATP-induced P2 mediated calcium signaling generated qualitative insight into the critical interactions controlling ATP induced calcium dynamics. Understanding these critical interactions may prove useful for the design of the next generation of molecular pain management strategies.

Cell-free protein expression has become a widely used research tool in systems and synthetic biol... more Cell-free protein expression has become a widely used research tool in systems and synthetic biology and a promising technology for protein biomanufacturing. Cell-free protein synthesis relies onin-vitrotranscription and translation processes to produce a protein of interest. However, transcription and translation depend upon the operation of complex metabolic pathways for precursor and energy regeneration. Toward understanding the role of metabolism in a cell-free system, we developed a dynamic constraint-based simulation of protein production in the myTXTLE. colicell-free system with and without electron transport chain inhibitors. Time-resolved absolute metabolite measurements for ℳ = 63 metabolites, along with absolute concentration measurements of the mRNA and protein abundance and measurements of enzyme activity, were integrated with kinetic and enzyme abundance information to simulate the time evolution of metabolic flux and protein production with and without inhibitors. The...

Cell-free synthetic systems are composed of the parts required for transcription and translation ... more Cell-free synthetic systems are composed of the parts required for transcription and translation processes in a buffered solution. Thus, unlike living cells, cell-free systems are amenable to rapid adjustment of the reaction composition and easy sampling. Further, because cellular growth and maintenance requirements are absent, all resources can go toward synthesizing the product of interest. Recent improvement in key performance metrics, such as yield, reaction duration, and portability, has increased the space of possible applications open to cell-free systems and lowered the time required to design-build-test new circuitry. One promising application area is biosensing. This study describes developing and modeling a D-gluconate biosensor circuit operating in a reconstituted cell-free system. Model parameters were estimated using time-resolved measurements of the mRNA and protein concentration with and without the addition of D-gluconate. Sensor performance was predicted using the ...

Journal of Surgical Research, 2022

INTRODUCTION Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting... more INTRODUCTION Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting potential due to factor consumption and proteolytic inactivation of factor V (FV) and activated factor V (FVa) by activated protein C (aPC). The role of FV/FVa depletion or inactivation in burn-induced coagulopathy is not well characterized. This study evaluates FV dynamics following burn and nonburn trauma. METHODS Burn and trauma patients were prospectively enrolled. Western blotting was performed on admission plasma to quantitate levels of FV antigen and to assess for aPC or other proteolytically derived FV/FVa degradation products. Statistical analysis was performed with Spearman's, Chi-square, Mann-Whitney U test, and logistic regression. RESULTS Burn (n = 60) and trauma (n = 136) cohorts showed similar degrees of FV consumption with median FV levels of 76% versus 73% (P = 0.65) of normal, respectively. Percent total body surface area (TBSA) was not correlated with FV, nor were significant differences in median FV levels observed between low and high TBSA groups. The injury severity score (ISS) in trauma patients was inversely correlated with FV (ρ = -0.26; P = 0.01) and ISS ≥ 25 was associated with a lower FV antigen level (64% versus. 93%; P = 0.009). The proportion of samples showing proteolysis-derived FV was greater in trauma than burn patients (42% versus. 16%; P = 0.0006). CONCLUSIONS Increasing traumatic injury severity is associated with decreased FV antigen levels, and a greater proportion of trauma patient samples exhibit proteolytically degraded FV fragments. These associations are not present in burns, suggesting that mechanisms underlying FV depletion in burn and nonburn trauma are not identical.

Journal of Burn Care & Research, 2021

Burn injury induces a systemic hyperinflammatory response with detrimental side effects. Studies ... more Burn injury induces a systemic hyperinflammatory response with detrimental side effects. Studies have described the biochemical changes induced by severe burns, but the transcriptome response is not well characterized. The goal of this work is to characterize the blood transcriptome after burn injury. Burn patients presenting to a regional center between 2012 and 2017 were prospectively enrolled. Blood was collected on admission and at predetermined time points (hours 2, 4, 8, 12, and 24). RNA was isolated and transcript levels were measured with a gene expression microarray. To identify differentially regulated genes (false-discovery rate ≤0.1) by burn injury severity, patients were grouped by TBSA above or below 20% and statistically enriched pathways were identified. Sixty-eight patients were analyzed, most patients were male with a median age of 41 (interquartile range, 30.5–58.5) years, and TBSA of 20% (11%–34%). Thirty-five patients had % TBSA injury ≥20%, and this group exper...

IFAC-PapersOnLine, 2019

Many markup languages can be used to encode biological networks, each with strengths and weakness... more Many markup languages can be used to encode biological networks, each with strengths and weaknesses. Model specifications written in these languages can then used, in conjunction with proprietary software packages e.g., MATLAB, or open community alternatives, to simulate the behavior of biological systems. In this study, we present the Simplified English Modeling Language (SEML) and associated compiler, as an alternative to existing approaches. SEML supports the specification of biological reaction systems in a simple natural language like syntax. Models encoded in SEML are transformed into executable code using a compiler written in the open-source Julia programming language. The compiler performs a sequence of operations, including tokenization, syntactic and semantic error checking, to convert SEML into an intermediate representation (IR). From the intermediate representation, the compiler then generates executable code in one of three programming languages: Julia, Python or MATLAB. Currently, SEML supports both kinetic and constraint based model generation for signal transduction and metabolic modeling. In this study, we demonstrate SEML by modeling two proof-of-concept prototypical networks: a constraint-based model solved using flux balance analysis (FBA) and a kinetic model encoded as Ordinary Differential Equations (ODEs). SEML is a promising tool for encoding and sharing human-readable biological models, however it is still in its infancy. With further development, SEML has the potential to handle more unstructured natural language inputs, generate more complex models types and convert its natural language markup to currently used model interchange formats such systems biology markup language.

Metastasis is the leading cause of breast cancer-related deaths and often driven by invasion and ... more Metastasis is the leading cause of breast cancer-related deaths and often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion have been associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process remains unclear due in part to the lack of convergent approaches that integrate engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis (FBA), and bioinformatic analysis of patient data we investigated the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis. Our results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induced...

ACS Synthetic Biology, 2022

Cell-free systems for gene expression have gained attention as platforms for the facile study of ... more Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low-and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203−424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Journal of Visualized Experiments, 2019

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for... more Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13 C-aniline, while compounds in the sample are derivatized with 12 C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Biotechnology Progress, 1999

An ensemble of model parameters was estimated using multiobjective optimization from dyn... more An ensemble of model parameters was estimated using multiobjective optimization from dynamic C3a and C5a measurements with and without zymosan. The model was trained using C3a and C5a measurements in the absence of zymosan (A–B) or in the presence of 1 mg/ml zymosan (C–D). The solid black lines show the simulated mean value of C3a or C5a for the ensemble, while the dark shaded region denotes the 99% confidence interval of mean. The light shaded region denotes the 99% confidence interval of the simulated C3a and C5a concentration. The experimental training data (points) was taken from Morad et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187373#pone.0187373.ref035" target="_blank">35</a>]. All initial conditions not specified by the experimental condition were assumed to be at zero or their physiological serum levels unless otherwise noted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187373#pone.0187373.s001" target="_blank">S1 Table</a>).</p

Biotechnology and Bioengineering, 1998

A cybernetic model is proposed to examine generic features of storage pathways. This model is cap... more A cybernetic model is proposed to examine generic features of storage pathways. This model is capable of describing synthesis of carbon and non-carbon storage polymers. The effect of environmental conditions is evaluated using storage polymer level as a fraction of total biomass as a gauge of pathway performance. The base wild-type pathway is then analyzed to determine the effect of genetic alterations upon system performance. Proposed modifications are tested using the cybernetic model as a diagnostic tool to ascertain the ramifications of potential genetic alterations. A methodology is developed within the cybernetic framework to describe alterations of enzyme activity and overexpression of pathway enzymes.

☯ These authors contributed equally to this work.

Sensitivity analysis was conducted on the two objectives used for model training. <b&... more Sensitivity analysis was conducted on the two objectives used for model training. A: Sensitivity of the C3a and C5a residual w/o zymosan. B: Sensitivity of the C3a and C5a residual with 1 mg/ml zymosan. The bars denote the mean total sensitivity index for each parameter, while the error bars denote the 95% confidence interval. C: Pathways controlled by the sensitivity parameters. Bold black lines indicate the pathway involves one or more sensitive parameters, while the red lines show current therapeutics targets. Current complement therapeutics were taken from the review of Morgan and Harris [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187373#pone.0187373.ref039" target="_blank">39</a>].</p

Current Opinion in Biotechnology, 1999

Annals of Biomedical Engineering, 2012

Breast cancer initiation, invasion and metastasis span multiple length and time scales. Molecular... more Breast cancer initiation, invasion and metastasis span multiple length and time scales. Molecular events at short length scales lead to an initial tumorigenic population, which left unchecked by immune action, acts at increasingly longer length scales until eventually the cancer cells escape from the primary tumor site. This series of events is highly complex, involving multiple cell types interacting with (and shaping) the microenvironment. Multiscale mathematical models have emerged as a powerful tool to quantitatively integrate the convective-diffusion-reaction processes occurring on the systemic scale, with the molecular signaling processes occurring on the cellular and subcellular scales. In this study, we reviewed the current state of the art in cancer modeling across multiple length scales, with an emphasis on the integration of intracellular signal transduction models with pro-tumorigenic chemical and mechanical microenvironmental cues. First, we reviewed the underlying biomolecular origin of breast cancer, with a special emphasis on angiogenesis. Then, we summarized the development of tissue engineering platforms which could provide highfidelity ex vivo experimental models to identify and validate multiscale simulations. Lastly, we reviewed top-down and bottom-up multiscale strategies that integrate subcellular networks with the microenvironment. We present models of a variety of cancers, in addition to breast cancer specific models. Taken together, we expect as the sophistication of the simulations increase, that multiscale modeling and bottom-up agent-based models in particular will become an increasingly important platform technology for basic scientific discovery, as well as the identification and validation of potentially novel therapeutic targets.

(A-I) The population was generated using JuPOETs and trained using 11 different objectiv... more (A-I) The population was generated using JuPOETs and trained using 11 different objective functions (41 data sets) taken from Medici et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005251#pcbi.1005251.ref007" target="_blank">7</a>]. The model captured the simulated experiments for all cases when compared to randomized controls. (J-L) The model populations were also compared against untrained temporal data to measure the effectiveness as a pure prediction. The western blot data was reproduced from Medici et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005251#pcbi.1005251.ref007" target="_blank">7</a>]. The intensity of each band was estimated using the ImageJ program. These blot intensities were then used as the training data for the EMT parameter estimation studies.</p

Nature Chemical Biology, 2020