Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

National Institutes of Health Funding for Tuberculosis Comorbidities Is Disproportionate to Their Epidemiologic Impact.

Tuberculosis (TB) is a leading infectious killer worldwide. We systematically searched the National Institutes of Health Research, Portfolio Online Reporting Tools Expenditures and Results (RePORTER) website to compare research funding for key TB comorbidities-undernutrition, alcohol use, human immunodeficiency virus, tobacco use, and diabetes-and found a large mismatch between the population attributable fraction of these risk factors and the funding allocated to them.

Resistive electrode array (REA) for radiotherapy beam monitoring and quality assurance.

We have developed a new type of detector array for monitoring of radiation beams in radiotherapy. The detector has parallel-plane architecture with multiple large-area uniform thin-film electrodes. At least one of the electrodes is resistive and has multiple signal readouts spread out along its perimeter. The integral dose deposited in the detector gives rise to multiple signals that depend on the distribution of radiation with respect to resistive electrode array (REA) geometry. The purpose of the present study was to experimentally determine basic detector response to MLC collimated x-ray fields. Two detector arrays have been characterized: circular and rectangular. The current and electrostatic potential distribution within the resistive electrode are governed by the Laplace and continuity equations with boundary conditions at the border with the readouts. Measurements for pencil beams showed that signal strength depends primarily on the distances between the location of the pencil beam and the readouts. Measurements for larger irregular MLC showed that signals as a function of time are quasi-linear with respect to MLC position and are proportional to the MLC area. Derivation of clinically relevant radiation beam parameters from REA signals, such as MLC position, MLC gap size and monitor unit per MLC segment relies on the detector response model with empirical model parameters. An approximate analytical detector response model was proposed and used to fit experiment data.

Stable recombinant production of codon-scrambled lubricin and mucin in human cells.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or "synDNAs," for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.