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.

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.

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.

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.

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.

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).

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.

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.

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.

The importance of acquisition learning on nicotine and varenicline drug substitution in a drug-discriminated goal-tracking task.

Nicotine and varenicline (Chantix®; the leading non-nicotine cessation pharmacotherapy) can come to control appetitive behaviors such as goal-tracking. We tested rats (N = 48) in a drug-discriminated goal-tracking (DGT) task where each rat received daily subcutaneous injections of either nicotine (0.4 mg/kg) or saline (0.9% [w/v]) interspersed across the acquisition phase (Phase 1). On saline days, sucrose was intermittently available. On nicotine days, sucrose was withheld. All rats acquired the discrimination with increased goal-tracking rates on saline days relative to nicotine days. Following acquisition, rats were separated into four groups to assess drug-substitution and discrimination reversal in Phase 2. The first group maintained the stimulus-reinforcer relation from acquisition (NIC-). The reversal group was now given access to sucrose on nicotine days (NIC+). The substitution group replaced nicotine with varenicline (1 mg/kg) while maintaining the acquisition stimulus-reinforcer relation (VAR-). The substitution and reversal group had nicotine replaced by varenicline and the stimulus-reinforcer relation reversed (VAR+). Rats in all groups learned or maintained their Phase 1 discriminations. For Phase 2, the reversal groups (+ conditions) acquired their discriminations within 10 sessions. The VAR- group displayed a pattern of disrupted discrimination at the outset of Phase 2 but was reestablished after continued training. In substitution testing, VAR groups received nicotine and NIC groups received varenicline. The NIC- and VAR- groups displayed full substitution of the test stimulus whereas the NIC+ and VAR+ groups displayed partial substitution of the test stimulus. Rats underwent nicotine extinction in Phase 3. Initial responding for each group mimicked Phase 2 training (i.e., higher responding by the reversal groups). All rats maintained similarly low levels of responding after six sessions. In conclusion, initial learning history with nicotine (i.e., + or -) influenced drug-stimulus substitution and the rate at which new learning (e.g., reversal) occurs with the varenicline and nicotine interoceptive stimuli.

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.

A fucosyltransferase inhibition assay using image-analysis and digital microfluidics.

Sialyl-LewisX and LewisX are cell-surface glycans that influence cell-cell adhesion behaviors. These glycans are assembled by α(1,3)-fucosyltransferase enzymes. Their increased expression plays a role in inflammatory disease, viral and microbial infections, and cancer. Efficient screens for specific glycan modifications such as those catalyzed by fucosyltransferases are tended toward costly materials and large instrumentation. We demonstrate for the first time a fucosylation inhibition assay on a digital microfluidic system with the integration of image-based techniques. Specifically, we report a novel lab-on-a-chip approach to perform a fluorescence-based inhibition assay for the fucosylation of a labeled synthetic disaccharide, 4-methylumbelliferyl ß-N-acetyllactosaminide. As a proof-of-concept, guanosine 5'-diphosphate has been used to inhibit Helicobacter pylori α(1,3)-fucosyltransferase. An electrode shape (termed "skewed wave") is designed to minimize electrode density and improve droplet movement compared to conventional square-based electrodes. The device is used to generate a 10 000-fold serial dilution of the inhibitor and to perform fucosylation reactions in aqueous droplets surrounded by an oil shell. Using an image-based method of calculating dilutions, referred to as "pixel count," inhibition curves along with IC50 values are obtained on-device. We propose the combination of integrating image analysis and digital microfluidics is suitable for automating a wide range of enzymatic assays.

A High-Throughput Glycosyltransferase Inhibition Assay for Identifying Molecules Targeting Fucosylation in Cancer Cell-Surface Modification.

In cancers, increased fucosylation (attachment of fucose sugar residues) on cell-surface glycans, resulting from the abnormal upregulation of the expression of specific fucosyltransferase enzymes (FUTs), is one of the most important types of glycan modifications associated with malignancy. Fucosylated glycans on cell surfaces are involved in a multitude of cellular interactions and signal regulation in normal biological processes, as well as in disease. For example, sialyl LewisX is a fucosylated cell-surface glycan that is abnormally abundant in some cancers where it has been implicated in facilitating metastasis, allowing circulating tumor cells to bind to the epithelial tissue within blood vessels and invade into secondary sites by taking advantage of glycan-mediated interactions. To identify inhibitors of FUT enzymes as potential cancer therapeutics, we have developed a novel high-throughput assay that makes use of a fluorogenically labeled oligosaccharide as a probe of fucosylation. This probe, which consists of a 4-methylumbelliferyl glycoside, is recognized and hydrolyzed by specific glycoside hydrolase enzymes to release fluorescent 4-methylumbelliferone, yet when the probe is fucosylated prior to treatment with the glycoside hydrolases, hydrolysis does not occur and no fluorescent signal is produced. We have demonstrated that this assay can be used to measure the inhibition of FUT enzymes by small molecules, because blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. Employing this assay, we have screened a focused library of small molecules for inhibitors of a human FUT enzyme involved in the synthesis of sialyl LewisX and demonstrated that our approach can be used to identify potent FUT inhibitors from compound libraries in microtiter plate format.

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.

Experimental analysis of behavior and tobacco regulatory research on nicotine reduction.

With the signing of H.R. 1256, the Family Smoking Prevention and Tobacco Control Act, the United States Food and Drug Administration (FDA) gained regulatory authority over the tobacco industry. A notable clause in this Act permits the FDA to regulate nicotine yields. However, they cannot completely remove this addictive constituent from tobacco products. This restriction has prompted the FDA to seek research on the threshold dose of nicotine that does not support dependence. This idea of threshold dose has led to an interesting reframing of scientific questions. For example, some researchers studying nicotine from this regulatory perspective translated the notion of an addiction threshold to a construct thought to play a role in addiction but which can be more readily operationalized. Examples include reinforcement threshold, discrimination threshold, and reinforcer-enhancement threshold. In this Perspective Paper, we highlight the importance of behavioral pharmacology and, specifically, the experimental analysis of behavior to help establish a scientific basis for policy decisions regarding nicotine yields. Recent research, including exemplars provided herein, note vast individual differences in the effects of nicotine at a known dose. Unfortunately, the behavioral and biological factors that contribute to such individual variations remain to be understood. We believe that behavior analysts are uniquely well-positioned to contribute to this understanding.

Structure-Guided Directed Evolution of Glycosidases: A Case Study in Engineering a Blood Group Antigen-Cleaving Enzyme.

Directed evolution is an incredibly powerful strategy for engineering enzyme function. Applying this approach to glycosidases offers enormous potential for the development of highly specialized tools in chemical glycobiology. Performing enzyme directed evolution requires the generation, by random mutagenesis, of mutant libraries from which large numbers of variant enzymes must be screened in high-throughput assays. A structure-guided "semirational" method for library creation allows researchers to target specific amino acid positions for mutagenesis, concentrating mutations where they might be most effective in order to produce mutant libraries of a manageable size, minimizing screening effort while maximizing the chances of finding improved mutants. Well-designed assays, which may use specially prepared substrates, enable efficient screening of these mutant libraries. This chapter will detail general methods in the structure-guided directed evolution of glycosidases, which have previously been employed in engineering a blood group antigen-cleaving enzyme.