A comprehensive assessment of a prototype high ratio antiscatter grid in interventional cardiology using experimental measurements and Monte Carlo simulations.

Objective. To assess the performance of a new antiscatter grid design in interventional cardiology for image quality improvement and dose reduction using experimental measurements and Monte Carlo (MC) simulation.Approach.Experimental measurements were performed on an angiography system, using a multi-layered tissue simulating composite phantom made from of poly(methyl methacrylate), aluminium and expanded polystyrene (2/0.2/0.7 cm). The total phantom thickness ranged from 20.3 cm to 40.6 cm. Four conditions were compared; (A) 105 cm source-image receptor distance (SID) without grid, (Bi) 105 cm SID with grid ratio (r) and strip density (N) (r15N80), (Bii) 120 cm SID without grid, and (Biii) 120 cm SID with high ratio grid (r29N80). The system efficiency (η), defined by the signal-to-noise ratio, was compared from theBconditions against caseA. These conditions were also simulated with MC techniques, allowing additional phantom compositions to be explored. Weighted image quality improvement factor (ηw(u)) was studied experimentally at a specific spatial frequency due to the SID change. Images were simulated with an anthropomorphic chest phantom for the different conditions, and the system efficiency was compared for the different anatomical regions.Main results.Good agreement was found between theηandηw(u) methods using both measured and simulated data, with average relative differences between 2%-11%. CaseBiiiprovided higherηvalues compared toA, andBifor thicknesses larger than 20.3 cm. In addition, caseBiiialso provided higherηvalues for high attenuating areas in the anthropomorphic phantom, such as behind the spine.Significance.The new antiscatter grid design provided higher system efficiency compared to the standard grid for the parameters explored in this work.

Limited impact of adding digital breast tomosynthesis to full field digital mammography in an elevated breast cancer risk population.

PURPOSE: To investigate the impact of adding digital breast tomosynthesis (DBT) to full field digital mammography (FFDM) in screening asymptomatic women with an elevated breast cancer life time risk (BCLTR) but without known genetic mutation. METHODS: This IRB-approved single-institution multi-reader study on prospectively acquired FFDM + DBT images included 429 asymptomatic women (39-69y) with an elevated BC risk on their request form. The BCLTR was calculated for each patient using the IBISrisk calculator v8.0b. The screening protocol and reader study consisted of 4-view FFDM + DBT, which were read by four independent radiologists using the BI-RADS lexicon. Standard of care (SOC) included ultrasound (US) and magnetic resonance imaging (MRI) for women with > 30 % BCLTR. Breast cancer detection rate (BCDR), sensitivity and positive predictive value were assessed for FFDM and FFDM + DBT and detection outcomes were compared with McNemar-test. RESULTS: In total 7/429 women in this clinically elevated breast cancer risk group were diagnosed with BC using SOC (BCDR 16.3/1000) of which 4 were detected with FFDM. Supplemental DBT did not detect additional cancers and BCDR was the same for FFDM vs FFDM + DBT (9.3/1000, McNemar p = 1). Moderate inter-reader agreement for diagnostic BI-RADS score was found for both study arms (ICC for FFDM and FFDM + DBT was 0.43, resp. 0.46). CONCLUSION: In this single institution study, supplemental screening with DBT in addition to standard FFDM did not increase BCDR in this higher-than-average BC risk group, objectively documented using the IBISrisk calculator.

Moment-based metrics for molecules computable from cryogenic electron microscopy images.

Single-particle cryogenic electron microscopy (cryo-EM) is an imaging technique capable of recovering the high-resolution three-dimensional (3D) structure of biological macromolecules from many noisy and randomly oriented projection images. One notable approach to 3D reconstruction, known as Kam's method, relies on the moments of the two-dimensional (2D) images. Inspired by Kam's method, we introduce a rotationally invariant metric between two molecular structures, which does not require 3D alignment. Further, we introduce a metric between a stack of projection images and a molecular structure, which is invariant to rotations and reflections and does not require performing 3D reconstruction. Additionally, the latter metric does not assume a uniform distribution of viewing angles. We demonstrate the uses of the new metrics on synthetic and experimental datasets, highlighting their ability to measure structural similarity.

Optimized signal of calcifications in wide-angle digital breast tomosynthesis: a virtual imaging trial.

OBJECTIVES: Evaluate microcalcification detectability in digital breast tomosynthesis (DBT) and synthetic 2D mammography (SM) for different acquisition setups using a virtual imaging trial (VIT) approach. MATERIALS AND METHODS: Medio-lateral oblique (MLO) DBT acquisitions on eight patients were performed at twice the automatic exposure controlled (AEC) dose. The noise was added to the projections to simulate a given dose trajectory. Virtual microcalcification models were added to a given projection set using an in-house VIT framework. Three setups were evaluated: (1) standard acquisition with 25 projections at AEC dose, (2) 25 projections with a convex dose distribution, and (3) sparse setup with 13 projections, every second one over the angular range. The total scan dose and angular range remained constant. DBT volume reconstruction and synthetic mammography image generation were performed using a Siemens prototype algorithm. Lesion detectability was assessed through a Jackknife-alternative free-response receiver operating characteristic (JAFROC) study with six observers. RESULTS: For DBT, the area under the curve (AUC) was 0.97 ± 0.01 for the standard, 0.95 ± 0.02 for the convex, and 0.89 ± 0.03 for the sparse setup. There was no significant difference between standard and convex dose distributions (p = 0.309). Sparse projections significantly reduced detectability (p = 0.001). Synthetic images had a higher AUC with the convex setup, though not significantly (p = 0.435). DBT required four times more reading time than synthetic mammography. DISCUSSION: A convex setup did not significantly improve detectability in DBT compared to the standard setup. Synthetic images exhibited a non-significant increase in detectability with the convex setup. Sparse setup significantly reduced detectability in both DBT and synthetic mammography. CLINICAL RELEVANCE STATEMENT: This virtual imaging trial study allowed the design and efficient testing of different dose distribution trajectories with real mammography images, using a dose-neutral protocol. KEY POINTS: • In DBT, a convex dose distribution did not increase the detectability of microcalcifications compared to the current standard setup but increased detectability for the SM images. • A sparse setup decreased microcalcification detectability in both DBT and SM images compared to the convex and current clinical setups. • Optimal microcalcification cluster detection in the system studied was achieved using either the standard or convex dose setting, with the default number of projections.

A combined analytical and Monte Carlo method for detailed simulations of antiscatter grids in x-ray medical imaging: implementing scatter within the grid.

Objective. To implement a hybrid method, which combines analytical tracking and interaction simulation using Monte Carlo (MC) techniques, in order to model photon transport inside antiscatter grids (ASG) for x-ray imaging.Approach. A new tally was developed for PENELOPE (v.2018) and penEasy (v. 2020) MC code to simulate photon transmission through ASGs. Two established analytical algorithms from the literature were implemented in this tally. In addition, a new hybrid method was introduced by extending one of the analytical algorithms to include photon-interactions inside the grid, while preserving the imaged grid structure. Calculations of primary(TP),scatter(TS),and total(TT)grid transmissions in addition to theQfactor (Q=TP2/TT) were performed. The new tally was validated for a quadric geometry ASG, and experimental measurements with a PMMA phantom of several thicknesses. In addition, the contribution of the scatter inside the grid was studied for three interspace materials, and a high resolution image of the grid was simulated.Main results. An excellent agreement was found between the two analytical models compared with the quadric grid without scatter, and the hybrid method with the geometrical grid with scatter. Average deviations of 0.2% and 1.4% were found betweenTPandTSfor the hybrid method and quadric grid, while for the hybrid method and experimental measurements these values were 1% and 20%. Antiscatter grids with aluminium as interspace material had the highest amount of scatter from inside the grid to the final image, followed up by paper fibre and air. The high resolution image of the grid was equivalent using the quadric geometry or the hybrid mode.Significance. The hybrid method provides a means of studying scattered radiation from the antiscatter grid with the advantage of higher performance, with results that are consistent with a full quadric geometry simulation of the ASG.

Safety-Net Hospital Status Is Associated With Coronary Artery Bypass Grafting Outcomes at an Urban Academic Medical Center.

INTRODUCTION: Socioeconomic disparities impact outcomes after cardiac surgery. At our institution, cardiac surgery cases from the safety-net, county funded hospital (CH), which primarily provides care for underserved patients, are performed at the affiliated university hospital. We aimed to investigate the association of socioeconomic factors and CH referral status with outcomes after coronary artery bypass grafting (CABG). METHODS: The institutional Adult Cardiac Surgery database was queried for perioperative and demographic data from patients who underwent isolated CABG between January 2014 and June 2020. The primary outcome was major adverse cardiovascular event (MACE), a composite of postoperative myocardial infarction, stroke, or death. Secondary outcomes included individual complications. Chi-square, Wilcoxon rank-sum, and logistic regression analyses were used to compare differences between CH and non-CH cohorts. RESULTS: We included 836 patients with 472 (56.5%) from CH. Compared to the non-CH cohort, CH patients were younger, more likely to be Hispanic, non-English speaking, and be completely uninsured or require state-specific financial assistance. CH patients were more likely to have a history of tobacco and drug use, liver disease, diabetes, prior myocardial infarction, and greater degrees of left main coronary and left anterior descending artery stenosis. CH cases were less likely to be elective. The incidence of MACE was significantly higher in the CH cohort (16.3% versus 8.2%, P = 0.001). There were no significant differences in 30-d mortality, home discharge, prolonged mechanical ventilation, bleeding, sepsis, pneumonia, new dialysis requirement, cardiac arrest, or multiorgan system failure between cohorts. CH patients were more likely to develop renal failure and less likely to develop atrial fibrillation. On multivariable analysis, CH status (odds ratio 2.39, 95% confidence interval 1.25-4.55, P = 0.008) was independently associated with MACE. CONCLUSIONS: CH patients undergoing CABG presented with greater comorbidity burden, more frequently required nonelective surgery, and are at significantly higher risk of postoperative MACE.

Special Section Guest Editorial: Introduction to the JMI Special Issue on Advances in Breast Imaging.

The editorial introduces the JMI Special Issue on Advances in Breast Imaging.

Development and validation of a 3D anthropomorphic phantom for dental CBCT imaging research.

BACKGROUND: Optimization of dental cone beam computed tomography (CBCT) imaging is still in a preliminary stage and should be addressed using task-based methods. Dedicated models containing relevant clinical tasks for image quality studies have yet to be developed. PURPOSE: To present a methodology to develop and validate a virtual adult anthropomorphic voxel phantom for use in task-based image quality optimization studies in dental CBCT imaging research, focusing on root fracture (RF) detection tasks in the presence of metal artefacts. METHODS: The phantom was developed from a CBCT scan with an isotropic voxel size of 0.2 mm, from which the main dental structures, mandible and maxilla were segmented. The missing large anatomical structures, including the spine, skull and remaining soft tissues, were segmented from a lower resolution full skull scan. Anatomical abnormalities were absent in the areas of interest. Fine detailed dental structures, that could not be segmented due to the limited resolution and noise in the clinical data, were modelled using a-priori anatomical knowledge. Model resolution of the teeth was therefore increased to 0.05 mm. Models of RFs as well as dental restorations to create the artefacts, were developed, and could be inserted in the phantom in any desired configuration. Simulated CBCT images of the models were generated using a newly developed multi-resolution simulation framework that incorporated the geometry, beam quality, noise and spatial resolution characteristics of a real dental CBCT scanner. Ray-tracing and Monte Carlo techniques were used to create the projection images, which were reconstructed using the classical FDK algorithm. Validation of the models was assessed by measurements of different tooth lengths, the pulp volume and the mandible, and comparison with reference values. Additionally, the simulated images were used in a reader study in which two oral radiologists had to score the realism level of the model's normal anatomy, as well as the modelled RFs and restorations. RESULTS: A model of an adult head, as well as models of RFs and different types of dental restorations were created. Anatomical measurements were consistent with ranges reported in literature. For the tooth length measurements, the deviations from the mean reference values were less than 20%. In 77% of all the measurements, the deviations were within 10.1%. The pulp volumes, and mandible measurements were within one standard deviation of the reference values. Regarding the normal anatomy, both readers considered the realism level of the dental structures to be good. Background structures received a lower realism score due to the lack of detailed enough trabecular bone structure, which was expected but not the focus of this study. All modelled RFs were scored at least adequate by at least one of the readers, both in appearance and position. The realism level of the modelled restorations was considered to be good. CONCLUSIONS: A methodology was proposed to develop and validate an anthropomorphic voxel phantom for image quality optimization studies in dental CBCT imaging, with a main focus on RF detection tasks. The methodology can be extended further to create more models representative of the clinical population.

Fast principal component analysis for cryo-electron microscopy images.

Principal component analysis (PCA) plays an important role in the analysis of cryo-electron microscopy (cryo-EM) images for various tasks such as classification, denoising, compression, and ab initio modeling. We introduce a fast method for estimating a compressed representation of the 2-D covariance matrix of noisy cryo-EM projection images affected by radial point spread functions that enables fast PCA computation. Our method is based on a new algorithm for expanding images in the Fourier-Bessel basis (the harmonics on the disk), which provides a convenient way to handle the effect of the contrast transfer functions. For N images of size L × L, our method has time complexity O(NL3 + L4) and space complexity O(NL2 + L3). In contrast to previous work, these complexities are independent of the number of different contrast transfer functions of the images. We demonstrate our approach on synthetic and experimental data and show acceleration by factors of up to two orders of magnitude.

Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin.

In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°') of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from Pseudomonas aeruginosa. These variants were found to differentially influence the E°' of T1Cu, with M13F Az decreasing E°', M44F Az increasing E°', and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases E°' by 26 mV relative to WT-Az, which is very close to the combined effect of E°' by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.

Burnout in Surgical Residents of Underrepresented in Medicine Backgrounds: Key Influencing Factors and Possible Solutions.

INTRODUCTION: Alarming rates of burnout in surgical training pose a concern due to its deleterious effects on both patients and providers. Datum remains lacking on rates of burnout in surgical residents based on race and ethnicity. This study aims to document the frequency of burnout in surgical residents of racially underrepresented backgrounds and elucidate contributing factors. METHODS: A 35-question anonymized survey was distributed to general surgery residents from 23 programs between August 2018 and May 2019. This survey was designed from the validated Maslach Burnout Inventory, and included additional questions assessing participant demographics, educational, and social backgrounds. Responses were analyzed utilizing chi-square tests and Wilcoxon rank sum tests. There was also a free response portion of the survey which was evaluated using thematic analysis. RESULTS: We received 243 responses from 23 general surgery programs yielding a 9% (23/246) program response rate and 26% (243/935) response rate by surgical residents. One hundred and eighty-five participants (76%) identified as nonunderrepresented in medicine and 58 (24%) of participants identified as underrepresented in medicine. Fifty-three percent were male and 47% female. Overall, sixty-six percent of all surgical residents (n = 161) endorsed burnout with racially underrepresented residents reporting higher rates of burnout at 76% compared to 63% in their nonunderrepresented counterparts (P = 0.07). CONCLUSIONS: Although the generalizability of these results is limited, higher rates of reported burnout in racially underrepresented trainees noted in our study illuminates the need for continual dialogue on potential influencing factors and mitigation strategies.

Uncertainty estimation for deep learning-based pectoral muscle segmentation via Monte Carlo dropout.

Objective. Deep Learning models are often susceptible to failures after deployment. Knowing when your model is producing inadequate predictions is crucial. In this work, we investigate the utility of Monte Carlo (MC) dropout and the efficacy of the proposed uncertainty metric (UM) for flagging of unacceptable pectoral muscle segmentations in mammograms.Approach. Segmentation of pectoral muscle was performed with modified ResNet18 convolutional neural network. MC dropout layers were kept unlocked at inference time. For each mammogram, 50 pectoral muscle segmentations were generated. The mean was used to produce the final segmentation and the standard deviation was applied for the estimation of uncertainty. From each pectoral muscle uncertainty map, the overall UM was calculated. To validate the UM, a correlation between the dice similarity coefficient (DSC) and UM was used. The UM was first validated in a training set (200 mammograms) and finally tested in an independent dataset (300 mammograms). ROC-AUC analysis was performed to test the discriminatory power of the proposed UM for flagging unacceptable segmentations.Main results. The introduction of dropout layers in the model improved segmentation performance (DSC = 0.95 ± 0.07 versus DSC = 0.93 ± 0.10). Strong anti-correlation (r= -0.76,p< 0.001) between the proposed UM and DSC was observed. A high AUC of 0.98 (97% specificity at 100% sensitivity) was obtained for the discrimination of unacceptable segmentations. Qualitative inspection by the radiologist revealed that images with high UM are difficult to segment.Significance. The use of MC dropout at inference time in combination with the proposed UM enables flagging of unacceptable pectoral muscle segmentations from mammograms with excellent discriminatory power.

FAST EXPANSION INTO HARMONICS ON THE DISK: A STEERABLE BASIS WITH FAST RADIAL CONVOLUTIONS.

We present a fast and numerically accurate method for expanding digitized L × L images representing functions on [-1, 1]2 supported on the disk x ∈ R 2 : | x | < 1 in the harmonics (Dirichlet Laplacian eigenfunctions) on the disk. Our method, which we refer to as the Fast Disk Harmonics Transform (FDHT), runs in 𝒪 L 2  log  L operations. This basis is also known as the Fourier-Bessel basis, and it has several computational advantages: it is orthogonal, ordered by frequency, and steerable in the sense that images expanded in the basis can be rotated by applying a diagonal transform to the coefficients. Moreover, we show that convolution with radial functions can also be efficiently computed by applying a diagonal transform to the coefficients.

MULTI-TARGET DETECTION WITH ROTATIONS.

We consider the multi-target detection problem of estimating a two-dimensional target image from a large noisy measurement image that contains many randomly rotated and translated copies of the target image. Motivated by single-particle cryo-electron microscopy, we focus on the low signal-to-noise regime, where it is difficult to estimate the locations and orientations of the target images in the measurement. Our approach uses autocorrelation analysis to estimate rotationally and translationally invariant features of the target image. We demonstrate that, regardless of the level of noise, our technique can be used to recover the target image when the measurement is sufficiently large.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

Tuning reduction potentials of type 1 copper center in azurin by replacing a histidine ligand with its isostructural analogues.

Type 1 copper proteins have a conserved ligand set of one cysteine and two histidines, with many proteins, such as azurin, also containing an axial methionine. While the cysteine and methionine in azurin have been replaced with their respective isostructural analogues of unnatural amino acids to reveal their roles in tuning electronic structures and functional properties, such as reduction potentials (E°'), the histidine ligands have not been probed in this way. We herein report the substitution of His117 in azurin with three unnatural isostructural analogues, 5-nitrohistidine(Ntr), thiazolylalanine(SHis) and 1-methylhistidine(MeH) by expressed protein ligation. While UV-vis absorption and electron paramagnetic resonance spectroscopies confirm that isostructural replacement results in minimal structural change in the Cu(II) state, the E°' of these variants increases with increasing pKa of the δ nitrogens of the imidazole. This counter-intuitive relationship between E°' of the protein and pKa of the sidechain group suggests additional factors may play a role in tuning E°'.

Pharyngeal tongue base augmentation for dysphagia therapy: A prospective case series in patients post head and neck cancer treatment.

BACKGROUND: Dysphagia post head and neck cancer (HNC) multimodality treatment is attributed to reduced pharyngeal strength. We hypothesized that pharyngeal tongue base augmentation for dysphagia (PAD therapy) would increase pharyngeal pressures during swallowing thereby improving swallow symptoms. METHODS: Adults with moderate-severe dysphagia post-HNC treatment had PAD therapy using a temporary filler (hyaluronic acid [HA]), with follow-up long-lasting lipofilling. Swallowing preprocedure and postprocedure was assessed with the Sydney Swallow Questionnaire (SSQ), High-Resolution Pharyngeal Manometry (HRPM), and Videofluoroscopic Swallowing Study (VFSS). Statistical comparison utilized paired tests. RESULTS: Six participants (all male; median age 64 years [IQR 56, 71]) underwent PAD therapy at a median of 47 [IQR 8, 95] months post-treatment. SSQ scores reduced from baseline (mean 1069 [95%CI 703, 1434]) to post-HA (mean 579 [76, 1081], p > 0.05), and post-lipofilling (491 [95%CI 913, 789], p = 0.003, n = 4). Individual participants demonstrated reduced Swallow Risk Index, Bolus Presence Time, and increased Upper Esophageal Sphincter opening, but mesopharyngeal contractile pressures were unchanged. VFSS measures of aspiration, residue, and severity were unchanged. CONCLUSIONS: Novel PAD therapy is safe and improves dysphagia symptoms. Biomechanical swallowing changes are suggestive of more efficacious bolus propulsion with conservative filler volume, but this was unable to resolve residue or aspiration measures.

Spectroscopic investigation of the electronic and excited state properties of para-substituted tetraphenyl porphyrins and their electrochemically generated ions.

Porphyrins play pivotal roles in many crucial biological processes including photosynthesis. However, there is still a knowledge gap in understanding electronic and excited state implications associated with functionalization of the porphyrin ring system. These effects can have electrochemical and spectroscopic signatures that reveal the complex nature of these somewhat minor substitutions, beyond simple inductive or electronic effect correlations. To obtain a deeper insight into the influences of porphyrin functionalization, four free-base, meso-substituted porphyrins: tetraphenyl porphyrin (TPP), tetra(4-hydroxyphenyl) porphyrin (THPP), tetra(4-carboxyphenyl) porphyrin (TCPP), and tetra(4-nitrophenyl) porphyrin (TNPP), were synthesized, characterized, and investigated. The influence of various substituents, (-hydroxy,-carboxy, and -nitro) in the para position of the meso-substituted phenyl moieties were evaluated by spectroelectrochemical techniques (absorption and fluorescence), femtosecond transient absorption spectroscopy, cyclic and differential pulse voltammetry, ultraviolet photoelectron spectroscopy (UPS), and time-dependent density functional theory (TD-DFT). Spectral features were evaluated for the neutral porphyrins and differences observed among the various porphyrins were further explained using rendered frontier molecular orbitals pertaining to the relevant transitions. Electrochemically generated anionic and cationic porphyrin species indicate similar absorbance spectroscopic signatures attributed to a red-shift in the Soret band. Emissive behavior reveals the emergence of one new fluorescence decay pathway for the ionic porphyrin, distinct from the neutral macrocycle. Femtosecond transient absorption spectroscopy analysis provided further analysis of the implications on the excited-state as a function of the para substituent of the free-base meso-substituted tetraphenyl porphyrins. Herein, we provide an in-depth and comprehensive analysis of the electronic and excited state effects associated with systematically varying the induced dipole at the methine bridge of the free-base porphyrin macrocycle and the spectroscopic signatures related to the neutral, anionic, and cationic species of these porphyrins.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.