An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria.

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non-human primates, demonstrating the strain's ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.

Development of a mechanistic model to predict synthetic biotic activity in healthy volunteers and patients with phenylketonuria.

The development of therapeutics depends on predictions of clinical activity from pre-clinical data. We have previously described SYNB1618, an engineered bacterial therapeutic (synthetic biotic) for the treatment of Phenylketonuria (PKU), a rare genetic disease that leads to accumulation of plasma phenylalanine (Phe) and severe neurological complications. SYNB1618 consumes Phe in preclinical models, healthy human volunteers, and PKU patients. However, it remains unclear to what extent Phe consumption by SYNB1618 in the gastrointestinal tract lowers plasma Phe levels in PKU patients. Here, we construct a mechanistic model that predicts SYNB1618 function in non-human primates and healthy subjects by combining in vitro simulations and prior knowledge of human physiology. In addition, we extend a model of plasma Phe kinetics in PKU patients, in order to estimate plasma Phe lowering by SYNB1618. This approach provides a framework that can be used more broadly to define the therapeutic potential of synthetic biotics.

Advancing lightweight mirror design: a paradigm shift in mirror preforms by utilizing design for additive manufacturing.

Additive manufacturing is a disruptive technology that can be leveraged by the redesign of components in most engineering fields. Fundamental engineering resources for lightweight mirrors were developed more than 30 years ago with a main design limitation, state of the art manufacturing. Here, we present two design methodologies for the design of lightweight mirrors. The first method utilizes analytical expressions to design a traditional isogrid mirror, which provided the foundation for most lightweight mirrors to date. The second method employs a combination of topology optimization, lattice infill, and analytical estimation to develop an advanced lightweight mirror designed for additive manufacturing. The advanced mirror design outperforms the traditional design for each functional requirement, including a 94% reduction in predicted surface quilting and a higher specific stiffness. The manufacturing of the advanced mirror is only possible with an additive manufacturing process.

Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli.

In this study, we developed a dynamic mathematical model of E. coli cell-free protein synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and protein product, chloramphenicol acetyltransferase (CAT) measurements. The model was successfully trained to simulate these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the performance, e.g., the yield and rates of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity was most sensitive to the oxidative phosphorylation and glycolysis/gluconeogenesis pathways. Amino acid biosynthesis was also important for productivity, while overflow metabolism and TCA cycle affected the overall system state. In addition, translation was more important to productivity than transcription. Finally, CAT production was robust to allosteric control, as were most of the predicted metabolite concentrations; the exceptions to this were the concentrations of succinate and malate, and to a lesser extent pyruvate and acetate, which varied from the measured values when allosteric control was removed. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and could provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.

Physiologically Based Pharmacokinetic Modeling to Supplement Nilotinib Pharmacokinetics and Confirm Dose Selection in Pediatric Patients.

In adult patients, nilotinib is indicated for chronic myeloid leukemia at an approved oral dose of 300 or 400 mg BID. Physiologically based pharmacokinetic (PBPK) model was developed to describe and supplement limited PK data in the pediatric population ranging from 2 to less than 6 years of age and ultimately inform dosing regimen. An adult Simcyp PBPK model was established and verified with clinical pharmacokinetic data after a single or multiple oral doses of 400 mg nilotinib (230 mg/m2). The model was then applied to a pediatric PBPK model, taking account of ontogeny profiles of metabolizing enzymes and pediatric physiological parameters. The model was further verified using observed pediatric PK data in 12- to <18-year-old and from 6- to <12-year-old patients. The PBPK models were able to recover, describe, and supplement the limited nilotinib concentration-time data profile in 2- to <6-year-old patients after a single dose and Cmin,ss after BID dosing. The exposure (Cmax,ss, Cmin,ss, and AUCtau,ss) was predicted to be similar across age groups. PBPK model simulations confirmed that body surface area-normalized dosing regimen of 230 mg/m2 is considered appropriate for pediatric patients >2 to <18 years of age.

Sequence Specific Modeling of E. coli Cell-Free Protein Synthesis.

Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance of E. coli cell-free protein synthesis. A core E. coli metabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis, and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function. Model parameters were largely taken from literature; thus the constraint based approach coupled the transcription and translation of the protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have data sets; we then expanded the simulations to a range of additional proteins. Protein expression simulations were consistent with measurements for a variety of cases. The constraint based simulations confirmed that oxidative phosphorylation was active in the CAT cell-free extract, as without it there was no feasible solution within the experimental constraints of the system. We then compared the metabolism of theoretically optimal and experimentally constrained CFPS reactions, and developed parameter free correlations which could be used to estimate productivity as a function of carbon number and promoter type. Lastly, global sensitivity analysis identified the key metabolic processes that controlled CFPS productivity and energy efficiency. In summary, sequence specific constraint based modeling of CFPS offered a novel means to a priori estimate the performance of a cell-free system, using only a limited number of adjustable parameters. While we modeled the production of a single protein in this study, the approach could easily be extended to multiprotein synthetic circuits, RNA circuits, or the cell-free production of small molecule products.

Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin.

Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences. It is unknown to what extent the plant villin headpiece structure and function resemble those of the well-studied vertebrate counterparts. Here we present the first solution NMR structure and backbone dynamics characterization of a headpiece from plants, villin isoform 4 from Arabidopsis thaliana. The villin 4 headpiece is a 63-residue domain (V4HP63) that adopts a typical headpiece fold with an aromatics core and a tryptophan-centered hydrophobic cap within its C-terminal subdomain. However, V4HP63 has a distinct N-terminal subdomain fold as well as a novel, high mobility loop due to the insertion of serine residue in the canonical sequence that follows the variable length loop in headpiece sequences. The domain binds actin filaments with micromolar affinity, like the vertebrate analogues. However, the V4HP63 surface charge pattern is novel and lacks certain features previously thought necessary for high-affinity F-actin binding. Utilizing the updated criteria for strong F-actin binding, we predict that the headpiece domains of all other villin isoforms in A. thaliana have high affinity for F-actin.

Expanding the Scope of Sortase-Mediated Ligations by Using Sortase Homologues.

Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.

Interlocked positive and negative feedback network motifs regulate ß-catenin activity in the adherens junction pathway.

The integrity of epithelial tissue architecture is maintained through adherens junctions that are created through extracellular homotypic protein-protein interactions between cadherin molecules. Cadherins also provide an intracellular scaffold for the formation of a multiprotein complex that contains signaling proteins, including ß-catenin. Environmental factors and controlled tissue reorganization disrupt adherens junctions by cleaving the extracellular binding domain and initiating a series of transcriptional events that aim to restore tissue homeostasis. However, it remains unclear how alterations in cell adhesion coordinate transcriptional events, including those mediated by ß-catenin in this pathway. Here were used quantitative single-cell and population-level in vitro assays to quantify the endogenous pathway dynamics after the proteolytic disruption of the adherens junctions. Using prior knowledge of isolated elements of the overall network, we interpreted these data using in silico model-based inference to identify the topology of the regulatory network. Collectively the data suggest that the regulatory network contains interlocked network motifs consisting of a positive feedback loop, which is used to restore the integrity of adherens junctions, and a negative feedback loop, which is used to limit ß-catenin-induced gene expression.