Characterizing and Improving pET Vectors for Cell-free Expression.

Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow.

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

The quality of life index: a pilot study integrating treatment efficacy and quality of life in oncology.

The majority of women diagnosed with breast cancer will experience some form of drug-related toxicity and subsequent impairments in Health-related Quality of Life (HRQoL). Despite this, HRQoL is assessed inconsistently and there is no validated method to integrate HRQoL data into the assessment of therapeutic agents. This proof of concept study utilizes data from the neoadjuvant I-SPY 2 clinical trial to describe the development of the Quality of Life Index (QoLI) measure. The QoLI represents a single composite score that incorporates validated longitudinal measures of clinical efficacy and QoL and one that permits a more comprehensive, direct comparison of individual therapeutic agents. Preliminary data suggest the QoLI is able to distinguish between agents based on their efficacy and toxicity; with further validation, the QoLI has the potential to provide more patient-centered evaluations in clinical trials and help guide treatment decision making in breast cancer and other oncologic diseases.

Chiral terahertz wave emission from the Weyl semimetal TaAs.

Weyl semimetals host chiral fermions with distinct chiralities and spin textures. Optical excitations involving those chiral fermions can induce exotic carrier responses, and in turn lead to novel optical phenomena. Here, we discover strong coherent terahertz emission from Weyl semimetal TaAs, which is demonstrated as a unique broadband source of the chiral terahertz wave. The polarization control of the THz emission is achieved by tuning photoexcitation of ultrafast photocurrents via the photogalvanic effect. In the near-infrared regime, the photon-energy dependent nonthermal current due to the predominant circular photogalvanic effect can be attributed to the radical change of the band velocities when the chiral Weyl fermions are excited during selective optical transitions between the tilted anisotropic Weyl cones and the massive bulk bands. Our findings provide a design concept for creating chiral photon sources using quantum materials and open up new opportunities for developing ultrafast opto-electronics using Weyl physics.

Distribution models for koalas in South Australia using citizen science-collected data.

The koala (Phascolarctos cinereus) occurs in the eucalypt forests of eastern and southern Australia and is currently threatened by habitat fragmentation, climate change, sexually transmitted diseases, and low genetic variability throughout most of its range. Using data collected during the Great Koala Count (a 1-day citizen science project in the state of South Australia), we developed generalized linear mixed-effects models to predict habitat suitability across South Australia accounting for potential errors associated with the dataset. We derived spatial environmental predictors for vegetation (based on dominant species of Eucalyptus or other vegetation), topographic water features, rain, elevation, and temperature range. We also included predictors accounting for human disturbance based on transport infrastructure (sealed and unsealed roads). We generated random pseudo-absences to account for the high prevalence bias typical of citizen-collected data. We accounted for biased sampling effort along sealed and unsealed roads by including an offset for distance to transport infrastructures. The model with the highest statistical support (wAIC c ∼ 1) included all variables except rain, which was highly correlated with elevation. The same model also explained the highest deviance (61.6%), resulted in high R (2)(m) (76.4) and R (2)(c) (81.0), and had a good performance according to Cohen's κ (0.46). Cross-validation error was low (∼ 0.1). Temperature range, elevation, and rain were the best predictors of koala occurrence. Our models predict high habitat suitability in Kangaroo Island, along the Mount Lofty Ranges, and at the tips of the Eyre, Yorke and Fleurieu Peninsulas. In the highest-density region (5576 km(2)) of the Adelaide-Mount Lofty Ranges, a density-suitability relationship predicts a population of 113,704 (95% confidence interval: 27,685-199,723; average density = 5.0-35.8 km(-2)). We demonstrate the power of citizen science data for predicting species' distributions provided that the statistical approaches applied account for some uncertainties and potential biases. A future improvement to citizen science surveys to provide better data on search effort is that smartphone apps could be activated at the start of the search. The results of our models provide preliminary ranges of habitat suitability and population size for a species for which previous data have been difficult or impossible to gather otherwise.

Novel indole-3-sulfonamides as potent HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs).

This Letter describes the design, synthesis, and biological evaluation of novel 3-indole sulfonamides as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) with balanced profiles against common HIV RT mutants K103N and Y181C.

Discovery of potent, selective 4-fluoroproline-based thrombin inhibitors with improved metabolic stability.

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4R)- and (4S)-4-fluoroproline was rationalized by analyzing inhibitor-enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2xAPTT=190 nM), excellent selectivity versus the digestive enzyme trypsin (K(i)=3300 nM), and excellent oral bioavailability in dogs with moderate clearance (F=100%, CL=12 mL/min/kg).

3-amino-4-sulfonylpyridinone acetamide and related pyridothiadiazine thrombin inhibitors.

We describe a series of highly potent and efficacious thrombin inhibitors based on a 3-amino-4-sulfonylpyridinone acetamide template. The functionally dense sulfonyl group stabilizes the aminopyridinone, conformationally constrains the 4-substituent, and forms a hydrogen bond to the insertion loop tyrosine OH. We also describe a related series of fused bicyclic dihydrothiadiazinedioxide derivatives, of which one had improved pharmacokinetics in dogs after oral dosing.

Azaindoles: moderately basic P1 groups for enhancing the selectivity of thrombin inhibitors.

Starting from a 2-amino-6-methylpyridine P1 group and following a strategy of enlarging it whilst reducing its polarity, we have developed a series of potent, moderately basic azaindoles which are intrinsically much more selective for thrombin versus trypsin. Certain pyrazinone acetamide azaindole derivatives have pharmacokinetic parameters after oral administration to dogs, or efficacy in vitro, comparable to an optimized pyrazinone acetamide 2-amino-6-methylpyridine derivative.

Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Small, low nanomolar, noncovalent thrombin inhibitors lacking a group to fill the 'distal binding pocket'.

Use of a chlorophenoxyacetamide P1 group with a pyridinone acetamide P2/P3 gave an exceptionally potent thrombin inhibitor (K(i)=40 pM). Truncation of the molecule at the N-terminus gave unique, low nanomolar, non-covalent thrombin inhibitors which do not have a group to fill thrombin's 'distal binding pocket'. A co-crystal structure indicates the importance of an intramolecular hydrogen bond between the P1 side chain and P1/P2 amide link in this series.