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Cell‐free protein synthesis (CFPS) is a versatile biotechnology platform enabling a broad range of applications including clinical diagnostics, large‐scale production of officinal therapeutics, small‐scale on‐demand production of personal magistral therapeutics, and exploratory research. The shelf stability and scalability of CFPS systems also have the potential to overcome cost and infrastructure challenges for distributing and using essential medical tests at home in both high‐ and low‐income countries. However, CFPS systems are often more time‐consuming and expensive to prepare than traditional in vivo systems, limiting their broader use. Much work has been done to lower CFPS costs by optimizing cell extract preparation, small molecule reagent recipes, and DNA template preparation. In order to further reduce reagent cost and preparation time, this work presents a CFPS system that does not require separately purified DNA template. Instead, a DNA plasmid encoding the recombinant protein is transformed into the cells used to make the extract, and the extract preparation process is modified to allow enough DNA to withstand homogenization‐induced shearing. The finished extract contains sufficient levels of intact DNA plasmid for the CFPS system to operate. For a 10 mL scale CFPS system expressing recombinant sfGFP protein for a biosensor, this new system reduces reagent cost by more than half. This system is applied to a proof‐of‐concept glutamine sensor compatible with smartphone quantification to demonstrate its viability for further cost reduction and use in low‐resource settings.

 

Abstract As demonstrated at Anak Krakatau on December 22 nd , 2018, tsunamis generated by volcanic flank collapse are incompletely understood and can be devastating. Here, we present the first high-resolution characterisation of both subaerial and submarine components of the collapse. Combined Synthetic Aperture Radar data and aerial photographs reveal an extensive subaerial failure that bounds pre-event deformation and volcanic products. To the southwest of the volcano, bathymetric and seismic reflection data reveal a blocky landslide deposit (0.214 ± 0.036 km 3 ) emplaced over 1.5 km into the adjacent basin. Our findings are consistent with en-masse lateral collapse with a volume ≥0.175 km 3 , resolving several ambiguities in previous reconstructions. Post-collapse eruptions produced an additional ~0.3 km 3 of tephra, burying the scar and landslide deposit. The event provides a model for lateral collapse scenarios at other arc-volcanic islands showing that rapid island growth can lead to large-scale failure and that even faster rebuilding can obscure pre-existing collapse. 

Life history theory is based on the assumption that resources are finite so that traits competing for this common pool of resources will experience a trade-off. The shared resource most commonly studied is food and studies typically manipulate resource acquisition by varying diet quantity or quality without considering the specific nutrients involved. Recent studies using the Geometric Framework (GF), however, suggest that life-history trade-offs are often regulated by the intake of specific nutrients. Despite this, a robust framework documenting the existence and quantifying the strength of nutritionally based trade-offs currently does not exist for studies using the GF. Here, we provide a conceptual framework showing that such trade-offs occur when life-history traits are maximised in different regions of nutrient space and that this divergence can be quantified by the overlap in the 95% confidence region (CR) of the global maxima, the angle (θ) between the linear nutritional vectors and the Euclidean distance (d) between the global maxima for each trait. We then empirically tested this framework by examining the effects of protein (P) and carbohydrate (C) intake on the trade-off between reproduction and immune function in male and female decorated crickets (Gryllodes sigillatus). Encapsulation ability and egg production in females increased with the intake of both nutrients, being maximised at a P:C ratio of 1.04:1 and 1:1.17, respectively. In contrast, encapsulation ability in males only increased with the intake of P being maximised at a P:C ratio of 5.14:1, whereas calling effort increased with the intake of C but decreased with the intake of P and was maximized at a P:C ratio of 1:7.08. Consequently, the trade-off between reproduction and encapsulation ability is much larger in males than females, a view supported by the non-overlapping 95% CRs on the global maxima for these traits in males and the larger estimates of θ and d. The sexes regulated their intake of nutrients in a similar way under dietary choice, at a P:C ratio of 1:2 and 1:1.84 in males and females, respectively. Although this ratio was more closely aligned with the optima for immune function and reproduction in females than males, neither sex optimally regulated their nutrient intake. Collectively, our study highlights that greater consideration should be given to the intake of specific nutrients when examining nutritionally based life-history trade-offs and how this varies across the sexes. 

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life‐saving protein therapeutics more available to the world, the possibility of magistral or point‐of‐care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized “cell‐free” protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA‐approved cancer therapeutics have increased its potential for low‐cost, single‐batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small‐batch, cell‐free protein synthesis (CFPS) reactions through air‐water foams. These “hydrofoam” reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell‐lysis and production losses, hydrofoam CFPS reactions are “cell‐free” and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.

 

Acute lymphocytic leukemia (ALL) is a common childhood cancer in the United States, with over 6000 new cases diagnosed each year. Administration of bacterial asparaginase (ASNase) has improved survival rates to nearly 80%, however these therapeutics have high incidence of immunological neutralization and serum activity must be monitored for most effective treatment regimens. Here, a 72% improvement in cell‐free protein synthesis (CFPS) of FDA approvedl‐asparaginase (crisantaspase) is demonstrated by employing an aspartate‐fed‐batch reactor format. A CFPS‐based ASNase activity assay as a tool for therapeutic regimentation and production quality control is also presented. This work suggests that shelf‐stable and low‐costEscherichia coli‐based CFPS reactions may be employed on‐demand to 1) synthesize biologics on‐site for patient administration, 2) verify biologic activity for dosage calculations, and 3) monitor therapeutic activity in human serum during the treatment regimen. The combination of both therapeutic production and activity assessment introduces a concept of synergistic utility for bacterial cell lysates in modern medical treatment. Indeed, recent work with CFPS biosensors supports a not‐too‐distant future when shelf‐stableE. coliCFPS systems are used to diagnose, treat, and monitor treatment of diseases in the clinical setting.