Engineering a Human P2X2 Receptor with Altered Ligand Selectivity in Yeast.

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker's yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity towards the ATP analog AMP-PNP. The F303Y A304 variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304 P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Changes in Coding and Efficiency through Modular Modifications to a One Pot PURE System for In Vitro Transcription and Translation.

The incorporation of unnatural amino acids is an attractive method for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE to obtain a robust and modular system for enzymatic single-site-specific incorporation of an unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two different orthogonal aminoacyl tRNA synthetase:tRNA pairs that suppressed two distinctive stop codons in separate reaction mixtures.

Empathy of Medical Students and Compassionate Care for Dying Patients: An Assessment of "No One Dies Alone" Program.

The "No One Dies Alone" (NODA) program was initiated to provide compassionate companions to the bedside of dying patients. This study was designed to test the following hypotheses: (1) Empathy scores would be higher among medical students who volunteered to participate in the NODA program than nonvolunteers; (2) Spending time with dying patients would enhance empathy in medical students. Study sample included 525 first- and second-year medical students, 54 of whom volunteered to participate in the NODA program. Of these volunteers, 26 had the opportunity to visit a dying patient (experimental group), and 28 did not, due to scheduling conflicts (volunteer control group). The rest of the sample (n = 471) comprised the "nonvolunteer control group." Comparisons of the aforementioned groups on scores of the Jefferson Scale of Empathy confirmed the first research hypothesis (P < .05, Cohen d = 0.37); the second hypothesis was not confirmed. This study has implications for the assessment of empathy in physicians-in-training, and timely for recruiting compassionate companion volunteers (armed with personal protective equipment) at the bedside of lonely dying patients infected by COVID-19.

Liquid nitrogen spray cryotherapy for dysphagia palliation in patients with inoperable esophageal cancer.

BACKGROUND AND AIMS: Dysphagia is a debilitating symptom in patients with inoperable esophageal cancer that contributes to poor quality of life and worsening nutritional status. The 2 most commonly used palliative modalities for dysphagia are radiation therapy and esophageal stent placement. However, radiation therapy is limited by adverse events (AEs) and total dose, and stent placement has a high rate of AEs, including reflux, migration, and chest pain. A relatively new modality of liquid nitrogen endoscopic spray cryotherapy has been described as salvage when other options have been exhausted and when patients are no longer receiving systemic therapy. We evaluated the safety and efficacy of cryotherapy as the primary modality for relieving dysphagia in inoperable esophageal cancer including patients receiving systemic cancer therapy. METHODS: This is a retrospective, multicenter, consecutive case series of 49 inoperable esophageal cancer patients undergoing palliative endoscopic cryotherapy at 4 specialized cancer centers from May 2014 to May 2016. The primary outcomes were change in dysphagia scores between pre- and postcryotherapy and AEs. Dysphagia was measured using a 5-point Likert scale: 0, no dysphagia; 1, dysphagia to solids; 2, dysphagia to semisolids; 3, dysphagia to liquids; 4, dysphagia to saliva. RESULTS: Thirty-nine men and 10 women with a mean age of 58 years underwent a total of 120 cryotherapy treatments. The mean dysphagia score improved significantly from 2.4 precryotherapy to 1.7 postcryotherapy (improvement of .7 points; P < .001). Minor AEs were seen in 6 of 120 (5.0%) cryotherapy treatments (1 intraprocedural and 5 postprocedural). In addition, 1 patient developed a severe intraprocedural AE of dilation-related perforation, whereas another patient developed a benign stricture requiring dilation. CONCLUSIONS: This preliminary retrospective study suggests that liquid nitrogen spray cryotherapy may be safe and effective for dysphagia palliation in inoperable esophageal cancer. Large prospective studies are needed to confirm these findings and identify patient and procedure characteristics associated with the greatest benefit.