Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression.

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Rapid Visual Authentication Based on DNA Strand Displacement.

Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment. The system is driven by toehold-mediated strand-displacement reactions where matching oligonucleotide sequences drive the generation of a fluorescent signal through the potential energy of base pairing. By pooling different "input" oligonucleotide sequences in a taggant and spatially separating "reporter" oligonucleotide sequences on a paper ticket, unique, sequence-driven patterns emerge for different taggant formulations. Algorithmically generated oligonucleotide sequences show no crosstalk and ink-embedded taggants maintain activity for at least 99 days at 60 °C (equivalent to nearly 2 years at room temperature). The resulting fluorescent signals can be analyzed by the eye or a smartphone when paired with a UV flashlight and filtered glasses.

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability.

Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.

The structural and biochemical impacts of monomerizing human acetylcholinesterase.

Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit-subunit interactions on the structure of hAChE, a 2.1 Å X-ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S ) - VX (ethyl({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site.

Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors.

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) ß-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/ß (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKß-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKß knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKß-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.

Interactions between bortezomib and romidepsin and belinostat in chronic lymphocytic leukemia cells.

PURPOSE: The goal of this study was to characterize interactions between the proteasome inhibitor bortezomib and the histone deacetylase (HDAC) inhibitors (HDACI) romidepsin or belinostat in chronic lymphocytic leukemia (CLL) cells. EXPERIMENTAL DESIGN: Primary and cultured (JVM-3 and MEC-2) CLL cells were exposed to agents alone or in combination, after which cell death was determined by 7-aminoactinomycin D staining/flow cytometry. Acetylation of target proteins, activation of caspase cascades, and expression of apoptosis-regulatory proteins were monitored by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) activity was determined by luciferase reporter assay. Cells were transiently transfected with wild-type and acetylation site-mutated (inactive) RelA(p65) (e.g., K221R, K310R, or K281/221/310R) and assessed for HDACI sensitivity. RESULTS: Combined exposure to very low concentrations of romidepsin or belinostat (i.e., low nanomolar and submicromolar, respectively) in combination with low nanomolar concentrations of bortezomib synergistically induced cell death in primary and cultured CLL cells. These events were likely associated with prevention of HDACI-mediated RelA acetylation and NF-kappaB activation by bortezomib, down-regulation of antiapoptotic proteins (i.e., Bcl-xL, Mcl-1, and XIAP), as well as up-regulation of the proapoptotic protein Bim, resulting in activation of caspase cascade. Finally, CLL cells transfected with inactive RelA displayed a significant increase in HDACI lethality. CONCLUSIONS: Coadministration of the clinically relevant HDACIs romidepsin or belinostat with bortezomib synergistically induces cell death in CLL cells, likely through mechanisms involving, among other factors, NF-kappaB inactivation and perturbation in the expression of proapoptotic and antiapoptotic proteins. A strategy combining HDAC with proteasome inhibition warrants further attention in CLL.