Safety and feasibility pilot study of continuous low-dose maternal supplemental oxygen in fetal single ventricle heart disease.

OBJECTIVES: Fetuses with single ventricle physiology (SVP) exhibit reductions in fetal cerebral oxygenation with associated delays in fetal brain growth and neurodevelopmental outcomes. Maternal supplemental oxygen (MSO) has been proposed to improve fetal brain growth but current evidence on dosing, candidacy, and outcomes are limited. In this pilot study, we evaluated the safety and feasibility of continuous low-dose MSO in the setting of SVP. METHODS: This single-centre, open-label, pilot phase 1 safety and feasibility clinical trial included 25 pregnant individuals with a fetal diagnosis of SVP. Participants self-administered continuous supplemental oxygen using medical-grade oxygen concentrators for up to 24 hours per day from the second half of gestation until delivery. The primary aim was the evaluation of the safety profile and feasibility of MSO. A secondary preliminary analysis was performed to assess the impact of MSO on the fetal circulation by echocardiography and late-gestational cardiovascular magnetic resonance, early outcomes including brain growth and pre-operative brain injury, and 18-month neurodevelopmental outcomes by the Bayley Scales of Infant and Toddler Development 3rd Edition compared to a contemporary fetal SVP cohort that received standard of care (SOC). RESULTS: Among 25 participants, the average maternal age at conception was 35 years, and fetal SVP diagnoses included 16 right ventricle dominant, 8 left ventricle dominant, and 1 indeterminant ventricular morphology. Participants started the trial at approximately 29.3 gestational weeks and took MSO for a median 16.1 hours per day for 63 days, cumulating a median 1029 hours of oxygen intake from enrollment until delivery. The only treatment-associated adverse events were nasal complications that were typically resolved by attaching a humidifier unit to the oxygen concentrator. No premature closure of the ductus arteriosus or unexpected fetal demise was observed. In the secondary analysis, MSO was not associated with any changes in fetal growth, middle cerebral artery pulsatility index, cerebroplacental ratio, nor head circumference to abdominal circumference ratio Z-scores over gestation compared to SOC. Although MSO was associated with changes in umbilical artery pulsatility index Z-score over gestation compared to SOC (p=0.02), this was likely due to initial baseline differences in placental resistance. At late-gestational cardiovascular magnetic resonance, MSO was not associated with any significant increase in umbilical vein oxygen saturation, fetal oxygen delivery, or fetal cerebral oxygen delivery. Similarly, we observed no differences in newborn outcomes including brain volume and pre-operative brain injury, nor mortality by 18 months of age, nor neurodevelopmental outcomes at 18 months of age. CONCLUSIONS: This pilot phase 1 clinical trial indicates low-dose maternal supplemental oxygen therapy is safe and well tolerated in pregnancies diagnosed with fetal SVP. However, our protocol was not associated with any significant changes in fetal circulatory physiology or improvements in early neurologic or neurodevelopmental outcomes. This article is protected by copyright. All rights reserved.

Evaluation of high dose N- Acetylcysteine on airway inflammation and quality of life outcomes in adults with bronchiectasis: A randomised placebo-controlled pilot study.

BACKGROUND: High dose N acetylcysteine (NAC), a mucolytic, anti-inflammatory and antioxidant agent has been shown to significantly reduce exacerbations, and improve quality of life in placebo controlled, double blind randomised (RCT) studies in patients with COPD, and in an open, randomised study in bronchiectasis. In this pilot, randomised, double-blind, placebo-controlled study, we wished to investigate the feasibility of a larger clinical trial, and the anti-inflammatory and clinical benefits of high dose NAC in bronchiectasis. AIMS: Primary outcome: to assess the efficacy of NAC 2400 mg/day at 6 weeks on sputum neutrophil elastase (NE), a surrogate marker for exacerbations. Secondary aims included assessing the efficacy of NAC on sputum MUC5B, IL-8, lung function, quality of life, and adverse effects. METHODS: Participants were randomised to receive 2400 mg or placebo for 6 weeks. They underwent 3 visits: at baseline, week 3 and week 6 where clinical and sputum measurements were assessed. RESULTS: The study was stopped early due to the COVID pandemic. In total 24/30 patients were recruited, of which 17 completed all aspects of the study. Given this, a per protocol analysis was undertaken: NAC (n = 9) vs placebo (n = 8): mean age 72 vs 62 years; male gender: 44% vs 50%; baseline median FEV11.56 L (mean 71.5 % predicted) vs 2.29L (mean 82.2% predicted). At 6 weeks, sputum NE fell by 47% in the NAC group relative to placebo (mean fold difference (95%CI: 0.53 (0.12,2.42); MUC5B increased by 48% with NAC compared with placebo. Lung function, FVC improved significantly with NAC compared with placebo at 6 weeks (mean fold difference (95%CI): 1.10 (1.00, 1.20), p = 0.045. Bronchiectasis Quality of life measures within the respiratory and social functioning domains demonstrated clinically meaningful improvements, with social functioning reaching statistical significance. Adverse effects were similar in both groups. CONCLUSION: High dose NAC exhibits anti-inflammatory benefits, and improvements in aspects of quality of life and lung function measures. It is safe and well tolerated. Further larger placebo controlled RCT's are now warranted examining its role in reducing exacerbations.

Maximum likelihood estimation of renal transporter ontogeny profiles for pediatric PBPK modeling.

Optimal treatment of infants with many renally cleared drugs must account for maturational differences in renal transporter (RT) activity. Pediatric physiologically-based pharmacokinetic (PBPK) models may incorporate RT activity, but this requires ontogeny profiles for RT activity in children, especially neonates, to predict drug disposition. Therefore, RT expression measurements from human kidney postmortem cortical tissue samples were normalized to represent a fraction of mature RT activity. Using these data, maximum likelihood estimated the distributions of RT activity across the pediatric age spectrum, including preterm and term neonates. PBPK models of four RT substrates (acyclovir, ciprofloxacin, furosemide, and meropenem) were evaluated with and without ontogeny profiles using average fold error (AFE), absolute average fold error (AAFE), and proportion of observations within the 5-95% prediction interval. Novel maximum likelihood profiles estimated ontogeny distributions for the following RT: OAT1, OAT3, OCT2, P-gp, URAT1, BCRP, MATE1, MRP2, MRP4, and MATE-2 K. Profiles for OAT3, P-gp, and MATE1 improved infant furosemide and neonate meropenem PBPK model AFE from 0.08 to 0.70 and 0.53 to 1.34 and model AAFE from 12.08 to 1.44 and 2.09 to 1.36, respectively, and improved the percent of data within the 5-95% prediction interval from 48% to 98% for neonatal ciprofloxacin simulations, respectively. Even after accounting for other critical population-specific maturational differences, novel RT ontogeny profiles substantially improved neonatal PBPK model performance, providing validated estimates of maturational differences in RT activity for optimal dosing in children.

Anakinra Removal by Continuous Renal Replacement Therapy: An Ex Vivo Analysis.

OBJECTIVES: Patients with sepsis are at significant risk for multiple organ dysfunction, including the lungs and kidneys. To manage the morbidity associated with kidney impairment, continuous renal replacement therapy (CRRT) may be required. The extent of anakinra pharmacokinetics in CRRT remains unknown. The objectives of this study were to investigate the anakinra-circuit interaction and quantify the rate of removal from plasma. DESIGN: The anakinra-circuit interaction was evaluated using a closed-loop ex vivo CRRT circuit. CRRT was performed in three phases based on the method of solute removal: 1) hemofiltration, 2) hemodialysis, and 3) hemodiafiltration. Standard control samples of anakinra were included to assess drug degradation. SETTING: University research laboratory. PATIENTS: None. INTERVENTIONS: Anakinra was administered to the CRRT circuit and serial prefilter blood samples were collected along with time-matched control and hemofiltrate samples. Each circuit was run in triplicate to assess inter-run variability. Concentrations of anakinra in each reference fluid were measured by enzyme-linked immunosorbent assay. Transmembrane filter clearance was estimated by the product of the sieving coefficient/dialysate saturation constant and circuit flow rates. MEASUREMENTS AND MAIN RESULTS: Removal of anakinra from plasma occurred within minutes for each CRRT modality. Average drug remaining (%) in plasma following anakinra administration was lowest with hemodiafiltration (34.9%). The average sieving coefficient was 0.34, 0.37, and 0.41 for hemodiafiltration, hemofiltration, and hemodialysis, respectively. Transmembrane clearance was fairly consistent across each modality with the highest during hemodialysis (5.53 mL/min), followed by hemodiafiltration (4.99 mL/min), and hemofiltration (3.94 mL/min). Percent drug remaining within the control samples (93.1%) remained consistent across each experiment, indicating negligible degradation within the blood. CONCLUSIONS: The results of this analysis are the first to demonstrate that large molecule therapeutic proteins such as anakinra, are removed from plasma with modern CRRT technology. Current dosing recommendations for patients with severe renal impairment may result in subtherapeutic anakinra concentrations in those receiving CRRT.

Meropenem extraction by ex vivo extracorporeal life support circuits.

BACKGROUND: Meropenem is a broad-spectrum carbapenem-type antibiotic commonly used to treat critically ill patients infected with extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. As many of these patients require extracorporeal membrane oxygenation (ECMO) and/or continuous renal replacement therapy (CRRT), it is important to understand how these extracorporeal life support circuits impact meropenem pharmacokinetics. Based on the physicochemical properties of meropenem, it is expected that ECMO circuits will minimally extract meropenem, while CRRT circuits will rapidly clear meropenem. The present study seeks to determine the extraction of meropenem from ex vivo ECMO and CRRT circuits and elucidate the contribution of different ECMO circuit components to extraction. METHODS: Standard doses of meropenem were administered to three different configurations (n = 3 per configuration) of blood-primed ex vivo ECMO circuits and serial sampling was conducted over 24 h. Similarly, standard doses of meropenem were administered to CRRT circuits (n = 4) and serial sampling was conducted over 4 h. Meropenem was administered to separate tubes primed with circuit blood to serve as controls to account for drug degradation. Meropenem concentrations were quantified, and percent recovery was calculated for each sample. RESULTS: Meropenem was cleared at a similar rate in ECMO circuits of different configurations (n = 3) and controls (n = 6), with mean (standard deviation) recovery at 24 h of 15.6% (12.9) in Complete circuits, 37.9% (8.3) in Oxygenator circuits, 47.1% (8.2) in Pump circuits, and 20.6% (20.6) in controls. In CRRT circuits (n = 4) meropenem was cleared rapidly compared with controls (n = 6) with a mean recovery at 2 h of 2.36% (1.44) in circuits and 93.0% (7.1) in controls. CONCLUSION: Meropenem is rapidly cleared by hemodiafiltration during CRRT. There is minimal adsorption of meropenem to ECMO circuit components; however, meropenem undergoes significant degradation and/or plasma metabolism at physiological conditions. These ex vivo findings will advise pharmacists and physicians on the appropriate dosing of meropenem.

Streamlining the Detection of Human Thyroid Receptor Ligand Interactions with XL1-Blue Cell-Free Protein Synthesis and Beta-Galactosidase Fusion Protein Biosensors.

Thyroid receptor signaling controls major physiological processes and disrupted signaling can cause severe disorders that negatively impact human life. Consequently, methods to detect thyroid receptor ligands are of great toxicologic and pharmacologic importance. Previously, we reported thyroid receptor ligand detection with cell-free protein synthesis of a chimeric fusion protein composed of the human thyroid receptor beta (hTRß) receptor activator and a ß-lactamase reporter. Here, we report a 60% reduction in sensing cost by reengineering the chimeric fusion protein biosensor to include a reporter system composed of either the full-length beta galactosidase (ß-gal), the alpha fragment of ß-gal (ß-gal-α), or a split alpha fragment of the ß-gal (split ß-gal-α). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the ß-gal background activity abundant in BL21 cell extract and (2) facilitate ß-gal complementation reporter activity to detect human thyroid receptor ligands. These results constitute a promising platform for high throughput screening and potentially the portable detection of human thyroid receptor ligands.

Interaction of ceftazidime and clindamycin with extracorporeal life support.

BACKGROUND: Ceftazidime and clindamycin are commonly prescribed to critically ill patients who require extracorporeal life support such as ECMO and CRRT. The effect of ECMO and CRRT on the disposition of ceftazidime and clindamycin is currently unknown. METHODS: Ceftazidime and clindamycin extraction were studied with ex vivo ECMO and CRRT circuits primed with human blood. The percent recovery of these drugs over time was calculated to determine the degree of interaction between these drugs and circuit components. RESULTS: Neither ceftazidime nor clindamycin exhibited measurable interactions with the ECMO circuit. In contrast, CRRT cleared 100% of ceftazidime from the experimental circuit within the first 2 h. Clearance of clindamycin from the CRRT circuit was slower, with about 20% removed after 6 h. CONCLUSION: Clindamycin and ceftazidime dosing adjustments are likely required in patients who are supported with CRRT, and future studies to quantify these adjustments should consider the pathophysiology of the patient in combination with the clearance due to CRRT. Dosing adjustments to account for adsorption to ECMO circuit components are likely unnecessary and should focus instead on the pathophysiology of the patient and changes in volume of distribution. These results will help improve the safety and efficacy of ceftazidime and clindamycin in patients requiring ECMO and CRRT.

"Just add small molecules" cell-free protein synthesis: Combining DNA template and cell extract preparation into a single fermentation.

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.

Challenging the highstand-dormant paradigm for land-detached submarine canyons.

Sediment, nutrients, organic carbon and pollutants are funnelled down submarine canyons from continental shelves by sediment-laden flows called turbidity currents, which dominate particulate transfer to the deep sea. Post-glacial sea-level rise disconnected more than three quarters of the >9000 submarine canyons worldwide from their former river or long-shore drift sediment inputs. Existing models therefore assume that land-detached submarine canyons are dormant in the present-day; however, monitoring has focused on land-attached canyons and this paradigm remains untested. Here we present the most detailed field measurements yet of turbidity currents within a land-detached submarine canyon, documenting a remarkably similar frequency (6 yr-1) and speed (up to 5-8 ms-1) to those in large land-attached submarine canyons. Major triggers such as storms or earthquakes are not required; instead, seasonal variations in cross-shelf sediment transport explain temporal-clustering of flows, and why the storm season is surprisingly absent of turbidity currents. As >1000 other canyons have a similar configuration, we propose that contemporary deep-sea particulate transport via such land-detached canyons may have been dramatically under-estimated.

Assessing site-specific PEGylation of TEM-1 ß-lactamase with cell-free protein synthesis and coarse-grained simulation.

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites. Emerging site-specific PEGylation technology provides control of PEG placement by conjugating PEG polymers via click chemistry reaction to genetically encoded non-canonical amino acids. Unfortunately, a method to rapidly determine the optimal PEGylation location has yet to be established. Here we seek to address this challenge. In this work, coarse-grained molecular dynamic simulations are paired with high-throughput experimental screening utilizing cell-free protein synthesis to investigate the effect of site-specific PEGylation on the two-state folder protein TEM-1 ß-lactamase. Specifically, the conjugation efficiency, thermal stability, and enzymatic activity are studied for the enzyme PEGylated at several different locations. The results of this analysis confirm that the physical properties of the PEGylated protein vary considerably with PEGylation site and that traditional design recommendations are insufficient to predict favorable PEGylation sites. In this study, the best predictor of the most favorable conjugation site is coarse-grained simulation. Thus, we propose a dual combinatorial screening approach in which coarse-grained molecular simulation informs site selection for high-throughput experimental verification.

A True Positive and a False Negative? The Dilemma of Negative Colonoscopy After a Positive Fecal Occult Blood Test.

INTRODUCTION: Many colonoscopies following a positive fecal immunochemical test (FIT) will not identify a probable cause for fecal blood, and missed neoplasia is a concern. The study determined whether the absence of neoplasia at a FIT positive diagnostic colonoscopy was due to a missed lesion and whether the initial FIT hemoglobin (f-Hb) concentration could predict missed lesions. METHODS: This was a retrospective audit of patients who had undergone diagnostic colonoscopy after FIT screening (2 sample ≥ 20 µg Hb/g feces). Probable bleeding lesions including cancer, advanced adenoma, colitis, and angiodysplasia were considered a "positive colonoscopy outcome." For those with a negative outcome, findings at the subsequent colonoscopy were assessed. RESULTS: There were 1087 good quality colonoscopies within 12 months of a positive FIT. In total, 171 (15.7%) patients had a positive outcome at the diagnostic colonoscopy. Subsequent colonoscopies of negative outcome cases (n = 418, median of 3.1y later) were reviewed; of these, there were 57 (13.6%) cases with a positive outcome. This included CRC in 0.5% (n = 2) and advanced adenoma in 11.7% (n = 49). High f-Hb and having both FIT samples ≥ 20 µg/g feces were associated with a positive outcome at the original diagnostic colonoscopy (p < 0.05). However, f-Hb was not predictive for a positive outcome at the subsequent colonoscopy by either maximum f-Hb (p = 0.768), total f-Hb (p = 0.459), or both FIT samples ≥ 20 µg/g (p = 0.091). CONCLUSION: A small proportion of "false" positive FIT results had cancer or advanced adenoma found at the subsequent colonoscopy. A missed lesion could not be predicted by the initial FIT f-Hb.

Towards detection of SARS-CoV-2 RNA in human saliva: A paper-based cell-free toehold switch biosensor with a visual bioluminescent output.

The COVID-19 pandemic has illustrated the global demand for rapid, low-cost, widely distributable and point-of-care nucleic acid diagnostic technologies. Such technologies could help disrupt transmission, sustain economies and preserve health and lives during widespread infection. In contrast, conventional nucleic acid diagnostic procedures require trained personnel, complex laboratories, expensive equipment, and protracted processing times. In this work, lyophilized cell-free protein synthesis (CFPS) and toehold switch riboregulators are employed to develop a promising paper-based nucleic acid diagnostic platform activated simply by the addition of saliva. First, to facilitate distribution and deployment, an economical paper support matrix is identified and a mass-producible test cassette designed with integral saliva sample receptacles. Next, CFPS is optimized in the presence of saliva using murine RNase inhibitor. Finally, original toehold switch riboregulators are engineered to express the bioluminescent reporter NanoLuc in response to SARS-CoV-2 RNA sequences present in saliva samples. The biosensor generates a visible signal in as few as seven minutes following administration of 15 µL saliva enriched with high concentrations of SARS-CoV-2 RNA sequences. The estimated cost of this test is less than 0.50 USD, which could make this platform readily accessible to both the developed and developing world. While additional research is needed to decrease the limit of detection, this work represents important progress toward developing a diagnostic technology that is rapid, low-cost, distributable and deployable at the point-of-care by a layperson.

Rapid RNase inhibitor production to enable low-cost, on-demand cell-free protein synthesis biosensor use in human body fluids.

Human body fluids contain biomarkers which are used extensively for prognostication, diagnosis, monitoring, and evaluation of different treatments for a variety of diseases and disorders. The application of biosensors based on cell-free protein synthesis (CFPS) offers numerous advantages including on-demand and at-home use for fast, accurate detection of a variety of biomarkers in human fluids at an affordable price. However, current CFPS-based biosensors use commercial RNase inhibitors to inhibit different RNases present in human fluids and this reagent is approximately 90% of the expense of these biosensors. Here the flexible nature of Escherichia coli-lysate-based CFPS was used for the first time to produce murine RNase Inhibitor (m-RI) and to optimize its soluble and active production by tuning reaction temperature, reaction time, reduced potential, and addition of GroEL/ES folding chaperons. Furthermore, RNase inhibition activity of m-RI with the highest activity and stability was determined against increasing amounts of three human fluids of serum, saliva, and urine (0%-100% v/v) in lyophilized CFPS reactions. To further demonstrate the utility of the CFPS-produced m-RI, a lyophilized saliva-based glutamine biosensor was demonstrated to effectively work with saliva samples. Overall, the use of CFPS-produced m-RI reduces the total reagent costs of CFPS-based biosensors used in human body fluids approximately 90%.

Submarine landslide megablocks show half of Anak Krakatau island failed on December 22nd, 2018.

As demonstrated at Anak Krakatau on December 22nd, 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 km3) emplaced over 1.5 km into the adjacent basin. Our findings are consistent with en-masse lateral collapse with a volume ≥0.175 km3, resolving several ambiguities in previous reconstructions. Post-collapse eruptions produced an additional ~0.3 km3 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.

The impact of postoperative radiological surveillance intensity on disease free and overall survival from primary retroperitoneal, abdominal and pelvic soft-tissue sarcoma.

AIM: This observational study aimed to evaluate the impact of intensity of radiological surveillance on survival following resection of retroperitoneal sarcoma. METHOD: Retrospective cohort study of patients undergoing primary resection of soft tissue sarcoma arising in the retroperitoneum, abdomen or pelvis at a single, high-volume sarcoma centre. Intensity of follow-up regimes up to 5 postoperative years were categorized as 'European Society for Medical Oncology (ESMO) compliant' (intense), or 'non-ESMO compliant' (less-intense). The primary outcome measure was overall survival (OS). The secondary outcome measures were disease-free survival (DFS) and reoperation rate. Analyses were stratified by high (grade 2 or 3) or low (grade 1) tumour grade. RESULTS: Of 168 patients, 67.1% had high-grade and 32.9% had low-grade disease. Overall, 40.0% of patients had ESMO-compliant radiological follow-up (high-grade:25.7%, low-grade:66.7%). 41.7% of patients died and 48.2% suffered local or distant recurrence by cessation of follow up. Upon univariable analysis for high-grade tumours, ESMO compliance reduced DFS (p = 0.066) but had no impact on OS. There was no significant difference in the reoperation rate in patients with ESMO-compliant and non-compliant follow-up (p = 0.097). In low-grade tumours, ESMO compliance significantly reduced DFS (p < 0.001), but without effecting OS. In risk-adjusted models for high-grade tumours, ESMO compliant follow-up was associated with reduced OS (HR:3.47, 1.40-8.61, p = 0.007) and no difference in DFS. In low-grade tumours, there was no association between overall ESMO compliance and OS or DFS. CONCLUSION: This study did not find a benefit for high-intensity radiological surveillance and overall survival in patients undergoing primary resection for high or low-grade retroperitoneal sarcoma.

Rapid sensing of clinically relevant glutamine concentrations in human serum with metabolically engineered E. coli-based cell-free protein synthesis.

Bioavailable glutamine (Gln) is critical for metabolism, intestinal health, immune function, and cell signaling. Routine measurement of serum Gln concentrations could facilitate improved diagnosis and treatment of severe infections, anorexia nervosa, chronic kidney disease, diabetes, and cancer. Current methods for quantifying tissue Gln concentrations rely mainly on HPLC, which requires extensive sample preparation and expensive equipment. Consequently, patient Gln levels may be clinically underutilized. Cell-free protein synthesis (CFPS) is an emerging sensing platform with promising clinical applications, including detection of hormones, amino acids, nucleic acids, and other biomarkers. In this work, in vitro E. coli amino acid metabolism is engineered with methionine sulfoximine to inhibit glutamine synthetase and create a CFPS Gln sensor. The sensor features a strong signal-to-noise ratio and a detection range ideally suited to physiological Gln concentrations. Furthermore, it quantifies Gln concentration in the presence of human serum. This work demonstrates that CFPS reactions which harness the metabolic power of E. coli lysate may be engineered to detect clinically relevant analytes in human samples. This approach could lead to transformative point-of-care diagnostics and improved treatment regimens for a variety of diseases including cancer, diabetes, anorexia nervosa, chronic kidney disease, and severe infections.

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport.

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.