Divergent directed evolution of a TetR-type repressor towards aromatic molecules.

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.

A "biphasic glycosyltransferase high-throughput screen" identifies novel anthraquinone glycosides in the diversification of phenolic natural products.

The sugar moieties of many glycosylated small molecule natural products are essential for their biological activity. Glycosyltransferases (GTs) are enzymes responsible for installing these sugar moieties on a variety of biomolecules. Many GTs active on natural products are inherently substrate promiscuous and thus serve as useful tools in manipulating natural product glycosylation to generate new combinations of sugar units (glycones) and scaffold molecules (aglycones) in a process called glycodiversification. It is important to have an effective screening tool to detect the activity of promiscuous enzymes and their resulting glycoside products. Toward this aim, we developed a strategy for screening natural product GTs in a high-throughput fashion enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This involves a solvent extraction step to isolate the resulting polar glycoside product from the unreacted aglycone acceptor substrate and the detection of the formed glycoside by the innate absorbance or fluorescence of the aglycone moiety. Using our approach, we screened a collection of natural product GTs against a panel of precursors to therapeutically important molecules. Three GTs showed previously unreported promiscuity toward anthraquinones resulting in novel ε-rhodomycinone glycosides. Considering the pharmaceutical value of clinically used anthraquinone glycosides that are biosynthesized from an ε-rhodomycinone precursor, and the significance that the sugar moiety has on the biological activity of these drugs, our results are of particular importance toward the glycodiversification of therapeutics in this class. The GTs identified and the novel compounds they produce show promise toward new biocatalytic tools and therapeutics.

Ontologies in the New Computational Age of Radiology: RadLex for Semantics and Interoperability in Imaging Workflows.

From basic research to the bedside, precise terminology is key to advancing medicine and ensuring optimal and appropriate patient care. However, the wide spectrum of diseases and their manifestations superimposed on medical team-specific and discipline-specific communication patterns often impairs shared understanding and the shared use of common medical terminology. Common terms are currently used in medicine to ensure interoperability and facilitate integration of biomedical information for clinical practice and emerging scientific and educational applications alike, from database integration to supporting basic clinical operations such as billing. Such common terminologies can be provided in ontologies, which are formalized representations of knowledge in a particular domain. Ontologies unambiguously specify common concepts and describe the relationships between those concepts by using a form that is mathematically precise and accessible to humans and machines alike. RadLex® is a key RSNA initiative that provides a shared domain model, or ontology, of radiology to facilitate integration of information in radiology education, clinical care, and research. As the contributions of the computational components of common radiologic workflows continue to increase with the ongoing development of big data, artificial intelligence, and novel image analysis and visualization tools, the use of common terminologies is becoming increasingly important for supporting seamless computational resource integration across medicine. This article introduces ontologies, outlines the fundamental semantic web technologies used to create and apply RadLex, and presents examples of RadLex applications in everyday radiology and research. It concludes with a discussion of emerging applications of RadLex, including artificial intelligence applications. © RSNA, 2023 Quiz questions for this article are available in the supplemental material.

Interactive Multimedia Reporting Technical Considerations: HIMSS-SIIM Collaborative White Paper.

Despite technological advances in the analysis of digital images for medical consultations, many health information systems lack the ability to correlate textual descriptions of image findings linked to the actual images. Images and reports often reside in separate silos in the medical record throughout the process of image viewing, report authoring, and report consumption. Forward-thinking centers and early adopters have created interactive reports with multimedia elements and embedded hyperlinks in reports that connect the narrative text with the related source images and measurements. Most of these solutions rely on proprietary single-vendor systems for viewing and reporting in the absence of any encompassing industry standards to facilitate interoperability with the electronic health record (EHR) and other systems. International standards have enabled the digitization of image acquisition, storage, viewing, and structured reporting. These provide the foundation to discuss enhanced reporting. Lessons learned in the digital transformation of radiology and pathology can serve as a basis for interactive multimedia reporting (IMR) across image-centric medical specialties. This paper describes the standard-based infrastructure and communications to fulfill recently defined clinical requirements through a consensus from an international workgroup of multidisciplinary medical specialists, informaticists, and industry participants. These efforts have led toward the development of an Integrating the Healthcare Enterprise (IHE) profile that will serve as a foundation for interoperable interactive multimedia reporting.

Multispecialty Enterprise Imaging Workgroup Consensus on Interactive Multimedia Reporting Current State and Road to the Future: HIMSS-SIIM Collaborative White Paper.

Diagnostic and evidential static image, video clip, and sound multimedia are captured during routine clinical care in cardiology, dermatology, ophthalmology, pathology, physiatry, radiation oncology, radiology, endoscopic procedural specialties, and other medical disciplines. Providers typically describe the multimedia findings in contemporaneous electronic health record clinical notes or associate a textual interpretative report. Visual communication aids commonly used to connect, synthesize, and supplement multimedia and descriptive text outside medicine remain technically challenging to integrate into patient care. Such beneficial interactive elements may include hyperlinks between text, multimedia elements, alphanumeric and geometric annotations, tables, graphs, timelines, diagrams, anatomic maps, and hyperlinks to external educational references that patients or provider consumers may find valuable. This HIMSS-SIIM Enterprise Imaging Community workgroup white paper outlines the current and desired clinical future state of interactive multimedia reporting (IMR). The workgroup adopted a consensus definition of IMR as "interactive medical documentation that combines clinical images, videos, sound, imaging metadata, and/or image annotations with text, typographic emphases, tables, graphs, event timelines, anatomic maps, hyperlinks, and/or educational resources to optimize communication between medical professionals, and between medical professionals and their patients." This white paper also serves as a precursor for future efforts toward solving technical issues impeding routine interactive multimedia report creation and ingestion into electronic health records.

Resources and Methods for Engineering "Designer" Glycan-Binding Proteins.

This review provides information on available methods for engineering glycan-binding proteins (GBP). Glycans are involved in a variety of physiological functions and are found in all domains of life and viruses. Due to their wide range of functions, GBPs have been developed with diagnostic, therapeutic, and biotechnological applications. The development of GBPs has traditionally been hindered by a lack of available glycan targets and sensitive and selective protein scaffolds; however, recent advances in glycobiology have largely overcome these challenges. Here we provide information on how to approach the design of novel "designer" GBPs, starting from the protein scaffold to the mutagenesis methods, selection, and characterization of the GBPs.

Enzymatic Synthesis of a Fluorogenic Reporter Substrate and the Development of a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation.

We report a straightforward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of N-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequentially coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

Unlocking Radiology Reporting Data: an Implementation of Synoptic Radiology Reporting in Low-Dose CT Cancer Screening.

Cancer Care Ontario (CCO) is the clinical advisor to the Ontario Ministry of Health and Long-Term Care for the funding and delivery of cancer services. Data contained in radiology reports are inaccessible for analysis without significant manual cost and effort. Synoptic reporting includes highly structured reporting and discrete data capture, which could unlock these data for clinical and evaluative purposes. To assess the feasibility of implementing synoptic radiology reporting, a trial implementation was conducted at one hospital within CCO's Lung Cancer Screening Pilot for People at High Risk. This project determined that it is feasible to capture synoptic data with some barriers. Radiologists require increased awareness when reporting cases with a large number of nodules due to lack of automation within the system. These challenges may be mitigated by implementation of some report automation. Domains such as pathology and public health reporting have addressed some of these challenges with standardized reports based on interoperable standards, and radiology could borrow techniques from these domains to assist in implementing synoptic reporting. Data extraction from the reports could also be significantly automated to improve the process and reduce the workload in collecting the data. RadLex codes aided the difficult data extraction process, by helping label potential ambiguity with common terms and machine-readable identifiers.

Facile Formation of ß-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase.

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

Chemoenzymatic Synthesis of a Type 2 Blood Group A Tetrasaccharide and Development of High-throughput Assays Enables a Platform for Screening Blood Group Antigen-cleaving Enzymes.

A facile enzymatic synthesis of the methylumbelliferyl ß-glycoside of the type 2 A blood group tetrasaccharide in good yields is reported. Using this compound, we developed highly sensitive fluorescence-based high-throughput assays for both endo-ß-galactosidase and α-N-acetylgalactosaminidase activity specific for the oligosaccharide structure of the blood group A antigen. We further demonstrate the potential to use this assay to screen the expressed gene products of metagenomic libraries in the search for efficient blood group antigen-cleaving enzymes.

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

A plate-based high-throughput activity assay for polysialyltransferase from Neisseria meningitidis.

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiter-plate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z' score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.

Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Engineering the enzyme toolbox to tailor glycosylation in small molecule natural products and protein biologics.

Many glycosylated small molecule natural products and glycoprotein biologics are important in a broad range of therapeutic and industrial applications. The sugar moieties that decorate these compounds often show a profound impact on their biological functions, thus biocatalytic methods for controlling their glycosylation are valuable. Enzymes from nature are useful tools to tailor bioproduct glycosylation but these sometimes have limitations in their catalytic efficiency, substrate specificity, regiospecificity, stereospecificity, or stability. Enzyme engineering strategies such as directed evolution or semi-rational and rational design have addressed some of the challenges presented by these limitations. In this review, we highlight some of the recent research on engineering enzymes to tailor the glycosylation of small molecule natural products (including alkaloids, terpenoids, polyketides, and peptides), as well as the glycosylation of protein biologics (including hormones, enzyme-replacement therapies, enzyme inhibitors, vaccines, and antibodies).

Bariatric Surgery Before Spine Surgery is Associated With Fewer Postsurgical Complications: A Systematic Review and Meta-Analysis.

STUDY DESIGN: Systematic review and meta-analysis. OBJECTIVE: To perform a systematic review and meta-analysis investigating the rate of adverse events after spine surgery in patients who underwent bariatric surgery (BS). SUMMARY OF BACKGROUND DATA: Obesity is an established risk factor for postoperative complications after spine surgery. BS has been associated with improvements in health in patients with severe obesity. However, it is not known whether undergoing BS before spine surgery is associated with reduced adverse outcomes. MATERIALS AND METHODS: PubMed, EMBASE, Scopus, and Web-of-Science were systematically searched according to "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" guidelines. The search included indexed terms and text words from database inception to the date of the search (May 27, 2022). Data and estimates were pooled using the Mantel-Haenszel method for random-effects meta-analysis. Risk of bias was assessed using the Joanna Briggs Institute risk of bias tool. The primary outcome was an all-cause complication rate after surgery. Relative risks for surgical and medical complications were assessed. RESULTS: A total of 4 studies comprising 177,273 patients were included. The pooled analysis demonstrated that the all-cause medical complication rate after spine surgery was lower in patients undergoing BS (relative risk: 0.54, 95% CI: 0.39, 0.74, P < 0.01). There was no difference in rates of surgical complications and 30-day hospital readmission rates between the cohort undergoing BS before spine surgery and the cohort that did not. CONCLUSION: These analyses suggest that obese patients undergoing BS before spine surgery have significantly lower adverse event rates. Future prospective studies are needed to corroborate these findings. LEVEL OF EVIDENCE: 4.

A Versatile Transcription Factor Biosensor System Responsive to Multiple Aromatic and Indole Inducers.

Allosteric transcription factor (aTF) biosensors are valuable tools for engineering microbes toward a multitude of applications in metabolic engineering, biotechnology, and synthetic biology. One of the challenges toward constructing functional and diverse biosensors in engineered microbes is the limited toolbox of identified and characterized aTFs. To overcome this, extensive bioprospecting of aTFs from sequencing databases, as well as aTF ligand-specificity engineering are essential in order to realize their full potential as biosensors for novel applications. In this work, using the TetR-family repressor CmeR from Campylobacter jejuni, we construct aTF genetic circuits that function as salicylate biosensors in the model organisms Escherichia coli and Saccharomyces cerevisiae. In addition to salicylate, we demonstrate the responsiveness of CmeR-regulated promoters to multiple aromatic and indole inducers. This relaxed ligand specificity of CmeR makes it a useful tool for detecting molecules in many metabolic engineering applications, as well as a good target for directed evolution to engineer proteins that are able to detect new and diverse chemistries.

In Vitro Reconstitution of the dTDP-l-Daunosamine Biosynthetic Pathway Provides Insights into Anthracycline Glycosylation.

Many small molecule natural products are decorated with sugar moieties that are essential for their biological activity. A considerable number of natural product glycosides and their derivatives are clinically important therapeutics. Anthracyclines like daunorubicin and doxorubicin are examples of valuable glycosylated natural products used in medicine as potent anticancer agents. The sugar moiety, l-daunosamine (a highly modified deoxyhexose), plays a key role in the bioactivity of these molecules as evidenced by semisynthetic anthracycline derivatives such as epirubicin, wherein alteration in the configuration of a single stereocenter of the sugar unit generates a chemotherapeutic drug with lower cardiotoxicity. The nucleotide activated sugar donor that provides the l-daunosamine group for attachment to the natural product scaffold in the biosynthesis of these anthracyclines is dTDP-l-daunosamine. In an in vitro system, we have reconstituted the enzymes in the daunorubicin/doxorubicin pathway involved in the biosynthesis of dTDP-l-daunosamine. Through the study of the enzymatic steps in this reconstituted pathway, we have gained several insights into the assembly of this precursor including the identification of a major bottleneck and competing reactions. We carried out kinetic analysis of the aminotransferase that catalyzes a limiting step of the pathway. Our in vitro reconstituted pathway also provided a platform to test the combinatorial enzymatic synthesis of other dTDP-activated deoxyhexoses as potential tools for "glycodiversification" of natural products. To this end, we replaced the stereospecific ketoreductase that acts in the last step of dTDP-l-daunosamine biosynthesis with an enzyme from a heterologous pathway with opposite stereospecificity and found that it is active in the in vitro pathway, demonstrating the potential for the enzymatic synthesis of nucleotide-activated sugars with regio- and stereospecific tailoring.

Insights into the stereospecificity of ketoreduction in a modular polyketide synthase.

Ketoreductase enzymes are responsible for the generation of hydroxyl stereocentres during the biosynthesis of complex polyketide natural products. Previous studies of isolated polyketide ketoreductases have shown that the stereospecificity of ketoreduction can be switched by mutagenesis of selected active site amino acids. We show here that in the context of the intact polyketide synthase multienzyme the same changes do not alter the stereochemical outcome in the same way. These findings point towards additional factors that govern ketoreductase stereospecificity on intact multienzymes in vivo.