Carbohydrate-active enzyme (CAZyme) discovery and engineering via (Ultra)high-throughput screening.

Carbohydrate-active enzymes (CAZymes) constitute a diverse set of enzymes that catalyze the assembly, degradation, and modification of carbohydrates. These enzymes have been fashioned into potent, selective catalysts by millennia of evolution, and yet are also highly adaptable and readily evolved in the laboratory. To identify and engineer CAZymes for different purposes, (ultra)high-throughput screening campaigns have been frequently utilized with great success. This review provides an overview of the different approaches taken in screening for CAZymes and how mechanistic understandings of CAZymes can enable new approaches to screening. Within, we also cover how cutting-edge techniques such as microfluidics, advances in computational approaches and synthetic biology, as well as novel assay designs are leading the field towards more informative and effective screening approaches.

Synthesis and glycosidase inhibition of 3,4,5-trihydroxypiperidines using a one-pot amination-cyclisation cascade reaction.

Trihydroxypiperidines are a therapeutically valuable class of iminosugar. We applied a one-pot amination-cyclisation cascade reaction to synthesise 3,4,5-trihydroxypiperidine stereoisomers in three steps from commercially available pentoses and in excellent overall yields. Using our methodology, the yields of the syntheses of meso-1, meso-2 and 3L are the highest reported to date. The synthetic methodology was readily extended to the three-step synthesis of N-alkyl derivatives by replacing the ammonia nitrogen source with a primary amine. The trihydroxypiperidines and N-alkyl analogues were screened for enzyme inhibitory activity using Fabrazyme (Fabry disease), GCase (Gaucher's disease), Agrobacterium sp. ß-glucosidase, and Escherichia coli ß-galactosidase. N-Phenylethyl 3,4,5-trihydroxypiperidine (N-phenylethyl-1-(3R,4R,5S)-piperidine-3,4,5-triol) showed good inhibitory activity of Fabrazyme (Ki = 46 µM). This activity was abolished when the N-phenylethyl group was removed or replaced with a non-aromatic alkyl chain.

Diastereodivergent nucleophile-nucleophile alkene chlorofluorination.

The selective hetero-dihalogenation of alkenes provides useful building blocks for a broad range of chemical applications. Unlike homo-dihalogenation, selective hetero-dihalogenation reactions, especially fluorohalogenation, are underdeveloped. Current approaches combine an electrophilic halogen source with a nucleophilic halogen source, which necessarily leads to anti-addition, and regioselectivity has only been achieved using highly activated alkenes. Here we describe an alternative, nucleophile-nucleophile approach that adds chloride and fluoride ions over unactivated alkenes in a highly regio-, chemo- and diastereoselective manner. A curious switch in the reaction mechanism was discovered, which triggers a complete reversal of the diastereoselectivity to promote either anti- or syn-addition. The conditions are demonstrated on an array of pharmaceutically relevant compounds, and detailed mechanistic studies reveal the selectivity and the switch between the syn- and anti-diastereomers are based on different active iodanes and which of the two halides adds first.

Parametric g-formula for Testing Time-Varying Causal Effects: What It Is, Why It Matters, and How to Implement It in Lavaan.

Psychologists leverage longitudinal designs to examine the causal effects of a focal predictor (i.e., treatment or exposure) over time. But causal inference of naturally observed time-varying treatments is complicated by treatment-dependent confounding in which earlier treatments affect confounders of later treatments. In this tutorial article, we introduce psychologists to an established solution to this problem from the causal inference literature: the parametric g-computation formula. We explain why the g-formula is effective at handling treatment-dependent confounding. We demonstrate that the parametric g-formula is conceptually intuitive, easy to implement, and well-suited for psychological research. We first clarify that the parametric g-formula essentially utilizes a series of statistical models to estimate the joint distribution of all post-treatment variables. These statistical models can be readily specified as standard multiple linear regression functions. We leverage this insight to implement the parametric g-formula using lavaan, a widely adopted R package for structural equation modeling. Moreover, we describe how the parametric g-formula may be used to estimate a marginal structural model whose causal parameters parsimoniously encode time-varying treatment effects. We hope this accessible introduction to the parametric g-formula will equip psychologists with an analytic tool to address their causal inquiries using longitudinal data.

Disordered but effective: short linear motifs as gene therapy targets for hyperexcitability disorders.

Multiple approaches have targeted voltage-gated sodium (Nav) channels for analgesia. In this issue of the JCI, Shin et al. identified a peptide aptamer, NaViPA1, carrying a short polybasic motif flanked by serine residues in a structurally disordered region of loop 1 in tetrodotoxin-sensitive (TTX-S) but not tetrodotoxin-resistant (TTX-R) channels. NaViPA1h inhibited TTX-S NaV channels and attenuated excitability of sensory neurons. Delivery of NaViPA1 in vivo via adeno-associated virions restricted its expression to peripheral sensory neurons and induced analgesia in rats. Targeting of short linear motifs in this manner may provide a gene therapy modality, with minimal side effects due to its peripherally-restricted biodistribution, which opens up a therapeutic strategy for hyperexcitability disorders, including pain.

Tool to Resolve Distortions in Elemental and Isotopic Imaging.

Nanoscale secondary ion mass spectrometry (NanoSIMS) makes it possible to visualize elements and isotopes in a wide range of samples at a high resolution. However, the fidelity and quality of NanoSIMS images often suffer from distortions because of a requirement to acquire and integrate multiple image frames. We developed an optical flow-based algorithm tool, NanoSIMS Stabilizer, for all-channel postacquisition registration of images. The NanoSIMS Stabilizer effectively deals with the distortions and artifacts, resulting in a high-resolution visualization of isotope and element distribution. It is open source with an easy-to-use ImageJ plugin and is accompanied by a Python version with GPU acceleration.

An SN1-Approach to Cross-Coupling: Deoxygenative Arylation Facilitated by the ß-Silicon Effect.

We report a dual metal-catalyzed method for the cross-coupling of unprotected alcohols by exploiting the ß-Si effect. This deoxygenative Suzuki-Miyaura reaction tolerates a range of primary, secondary, and tertiary alcohol substrates along with diverse functional groups and heterocycles. Mechanistic experiments including KIE, VTNA, and Eyring analyses suggest the existence of a carbocation intermediate on the reaction pathway, consistent with a rare SN1 pathway for the activation of an electrophile in cross-coupling reactions. A novel bis-imidazolium N-heterocyclic carbene (NHC) ligand was found to be optimal for reactivity, and nickel(0)-, nickel(I)- and nickel(II)- complexes of this ligand were isolated and characterized. In contrast to more well-established shorter chain ligands, these long-chain NHCs are found to have characteristically large bite angles, which may be critical for enabling the deoxygenative arylation of aliphatic alcohols.

Estimated Financial Impact of 2021 Office-Visit Work Relative Unit Updates on Surgical Global Periods.

This cross-sectional study assesses the impact of changes to medical billing and coding work relative value units in 2021.

Projecting the clinical burden of chronic kidney disease at the patient level (Inside CKD): a microsimulation modelling study.

Background: Chronic kidney disease (CKD) is a global concern that presents significant challenges for disease management. Several factors drive CKD prevalence, including primary risk factors, such as type 2 diabetes and hypertension, and an ageing population. Inside CKD is an international initiative that aims to raise awareness of the substantial burden incurred by CKD. Methods: Using a peer-reviewed microsimulation method, the clinical burden of CKD was estimated from 2022 to 2027. Demographic data from the Americas, Europe, and Asia-Pacific/Middle East were used to generate virtual populations and to project the prevalence of CKD, kidney replacement therapy, associated cardiovascular complications, comorbid conditions, and all-cause mortality in the CKD population over the modelled time frame. Findings: Across the 31 participating countries/regions, the total prevalence of CKD was projected to rise to 436.6 million cases by 2027 (an increase of 5.8% from 2022), with most cases (∼80%) undiagnosed. Inside CKD projected a mean of 8859 cases of heart failure, 10,244 of myocardial infarction, and 7797 of stroke per 100,000 patients with CKD by 2027. Interpretation: The clinical impact of CKD is substantial and likely to increase; the high prevalence of undiagnosed cases and associated complications may benefit from the implementation of health policy interventions that promote screening, earlier diagnosis, and interventions to improve outcomes. Funding: AstraZeneca.

A Snapshot of Lead in Consumer Products Across Four US Jurisdictions.

BACKGROUND: Following the removal of lead from gasoline, paint and pipes were thought to be the main sources of lead exposure in the United States. However, consumer products, such as certain spices, ceramic and metal cookware, traditional health remedies, and cultural powders, are increasingly recognized as important sources of lead exposure across the United States. OBJECTIVE: This paper reviews data from four US jurisdictions that conduct in-home investigations for children with elevated blood lead levels (BLLs) to examine the prevalence of lead exposures associated with consumer products, in comparison with housing-related sources. METHODS: Authors reviewed investigation data (2010-2021) provided by California, Oregon, New York City, and King County, Washington, and compared the extent of lead exposures associated with housing-related vs. consumer products-related sources. DISCUSSION: The proportion of investigations identifying consumer products-related sources of lead exposure varied by jurisdiction (range: 15%-38%). A review of US CDC and US FDA alerts and New York City data indicates that these types of lead-containing products are often sourced internationally, with many hand carried into the United States during travel. Based on surveillance data, we believe that US immigrant and refugee communities are at an increased risk for lead exposures associated with these products. To engage health authorities, there is a need for evidentiary data. We recommend implementing a national product surveillance database systematically tracking data on consumer products tested by childhood lead poisoning prevention programs. The data repository should be centralized and accessible to all global stakeholders, including researchers and governmental and nongovernmental agencies, who can use these data to inform investigations. Effectively identifying and addressing the availability of lead-containing consumer products at their source can focus resources on primary prevention, reducing lead exposures for users abroad and in the United States. https://doi.org/10.1289/EHP14336.

Current and Ongoing Developments in Targeting Clostridioides difficile Infection and Recurrence.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacterial pathogen that causes severe gastrointestinal infection in humans. This review provides background information on C. difficile infection and the pathogenesis and toxigenicity of C. difficile. The risk factors, causes, and the problem of recurrence of disease and current therapeutic treatments are also discussed. Recent therapeutic developments are reviewed including small molecules that inhibit toxin formation, disrupt the cell membrane, inhibit the sporulation process, and activate the host immune system in cells. Other treatments discussed include faecal microbiota treatment, antibody-based immunotherapies, probiotics, vaccines, and violet-blue light disinfection.

Development of the Food-Related Control Scale for Long-Term Care.

OBJECTIVE: Develop and test a Food-Related Control Scale (FRCS) measuring resident-perceived control in long-term care food service. DESIGN: A bank of 15 initial items based on a multidimensional locus of control construct was developed initially. Expert review, cognitive interviews, a pilot study, and factor analysis were used to validate the instrument and assess reliability. SETTING: Individual phone-based cognitive interviews and 16 skilled nursing facilities in the US. PARTICIPANTS: Cognitive interviews included a convenience sample of independently living adults aged ≥ 65 (n = 13), whereas the pilot study included skilled nursing facility-residing adults (n = 166). VARIABLES MEASURED: Perception of food-related control in a long-term care setting. ANALYSIS: Cognitive interviews were analyzed to develop items. Quantitative data from skilled nursing facility residents were analyzed using SAS software for structural equation modeling and factor analysis. RESULTS: A 2-dimensional construct (9 items) of the FRCS demonstrated reliability with factor analysis. Concurrent validity within the locus of control construct was demonstrated with the Multidimensional Health Locus of Control Scale (standardized estimate of 0.430; P < 0.1). CONCLUSIONS AND IMPLICATIONS: The FRCS may be used to determine how residents in long-term care perceive control over their food experiences. Further testing is necessary to determine the appropriateness of the FRCS for different population uses.

Effect of the mGlu2 positive allosteric modulator biphenyl-indanone A as a monotherapy and as adjunct to a low dose of L-DOPA in the MPTP-lesioned marmoset.

Activation of metabotropic glutamate 2 (mGlu2) receptors is a potential novel therapeutic approach for the treatment of parkinsonism. Thus, when administered as monotherapy or as adjunct to a low dose of L-3,4-dihydroxyphenylalanine (L-DOPA), the mGlu2 positive allosteric modulator (PAM) LY-487,379 alleviated parkinsonism in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned primates. Here, we sought to investigate the effect of biphenyl-indanone A (BINA), a highly selective mGlu2 PAM whose chemical scaffold is unrelated to LY-487,379, to determine if a structurally different mGlu2 PAM would also confer anti-parkinsonian benefit. In monotherapy experiments, MPTP-lesioned marmosets were injected with either vehicle, L-DOPA/benserazide (15/3.75 mg/kg, positive control) or BINA (0.1, 1, 10 mg/kg). In adjunct to a low L-DOPA dose experiments, MPTP-lesioned marmosets were injected with L-DOPA/benserazide (7.5/1.875 mg/kg) in combination with vehicle or BINA (0.1, 1, 10 mg/kg). Parkinsonism, dyskinesia and psychosis-like behaviours (PLBs) were then quantified. When administered alone, BINA 1 and 10 mg/kg decreased parkinsonism severity by ~22% (p < 0.01) and ~47% (p < 0.001), when compared with vehicle, which was comparable with the global effect of a high L-DOPA dose. When administered in combination with a low L-DOPA dose, BINA 1 and 10 mg/kg decreased global parkinsonism by ~38% (p < 0.001) and ~53% (p < 0.001). BINA 10 mg/kg decreased global dyskinesia by ~94% (p < 0.01) and global PLBs by ~92% (p < 0.01). Our results provide additional evidence that mGlu2 positive allosteric modulation elicits anti-parkinsonian effects. That this benefit is not related to a particular chemical scaffold suggests that it may be a class effect rather than the effect of a specific molecule.

Positive allosteric mGluR2 modulation with BINA alleviates dyskinesia and psychosis-like behaviours in the MPTP-lesioned marmoset.

There is mounting evidence that positive allosteric modulation of metabotropic glutamate type 2 receptors (mGluR2) is an efficacious approach to reduce the severity of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia, psychosis-like behaviours (PLBs), while conferring additional anti-parkinsonian benefit. However, the mGluR2 positive allosteric modulators (PAMs) tested so far, LY-487,379 and CBiPES, share a similar chemical scaffold. Here, we sought to assess whether similar benefits would be conferred by a structurally-distinct mGluR2 PAM, biphenylindanone A (BINA). Six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmosets exhibiting dyskinesia and PLBs were administered L-DOPA with either vehicle or BINA (0.1, 1, and 10 mg/kg) in a randomised within-subject design and recorded. Behaviour was analysed by a blinded rater who scored the severity of each of parkinsonism, dyskinesia and PLBs. When added to L-DOPA, BINA 0.1 mg/kg, 1 mg/kg, and 10 mg/kg all significantly reduced the severity of global dyskinesia, by 40%, 52% and 53%, (all P < 0.001) respectively. BINA similarly attenuated the severity of global PLBs by 35%, 48%, and 50%, (all P < 0.001) respectively. Meanwhile, BINA did not alter the effect of L-DOPA on parkinsonism exhibited by the marmosets. The results of this study provide incremental evidence of positive allosteric modulation of mGluR2 as an effective therapeutic strategy for alleviating dyskinesia and PLBs, without hindering the anti-parkinsonian action of L-DOPA. Furthermore, this therapeutic benefit does not appear to be confined to a particular chemical scaffold.

Nineteen-Year-Old Woman with Symptomatic Intramuscular Thigh Hemangioma-Radiographic Changes and Management: A Case Report.

CASE: We report a case of an intramuscular thigh hemangioma in a 19-year-old woman with a several year history of atraumatic thigh pain. Radiographs obtained by her primary care physician demonstrated periosteal bone reaction, prompting referral to Orthopaedic Oncology department. The patient had successful symptomatic management with propranolol. CONCLUSION: The case highlights the diagnosis and potential treatments. In a stepwise approach to care for symptomatic benign vascular lesions, propranolol has been a proven therapeutic option and may be a useful first-line therapy for symptomatic hemangiomas.

Real-time imaging of axonal membrane protein life cycles.

The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

Long-Term Prospective Outcomes of Intensity Modulated Radiotherapy for Locally Advanced Lung Cancer: A Secondary Analysis of a Randomized Clinical Trial.

Importance: The optimal radiotherapy technique for unresectable locally advanced non-small cell lung cancer (NSCLC) is controversial, so evaluating long-term prospective outcomes of intensity-modulated radiotherapy (IMRT) is important. Objective: To compare long-term prospective outcomes of patients receiving IMRT and 3-dimensional conformal radiotherapy (3D-CRT) with concurrent carboplatin/paclitaxel for locally advanced NSCLC. Design, Setting, and Participants: A secondary analysis of a prospective phase 3 randomized clinical trial NRG Oncology-RTOG 0617 assessed 483 patients receiving chemoradiotherapy (3D-CRT vs IMRT) for locally advanced NSCLC based on stratification. Main Outcomes and Measures: Long-term outcomes were analyzed, including overall survival (OS), progression-free survival (PFS), time to local failure, development of second cancers, and severe grade 3 or higher adverse events (AEs) per Common Terminology Criteria for Adverse Events, version 3. The percentage of an organ volume (V) receiving a specified amount of radiation in units of Gy is reported as V(radiation dose). Results: Of 483 patients (median [IQR] age, 64 [57-70] years; 194 [40.2%] female), 228 (47.2%) received IMRT, and 255 (52.8%) received 3D-CRT (median [IQR] follow-up, 5.2 [4.8-6.0] years). IMRT was associated with a 2-fold reduction in grade 3 or higher pneumonitis AEs compared with 3D-CRT (8 [3.5%] vs 21 [8.2%]; P = .03). On univariate analysis, heart V20, V40, and V60 were associated with worse OS (hazard ratios, 1.06 [95% CI, 1.04-1.09]; 1.09 [95% CI, 1.05-1.13]; 1.16 [95% CI, 1.09-1.24], respectively; all P < .001). IMRT significantly reduced heart V40 compared to 3D-CRT (16.5% vs 20.5%; P < .001). Heart V40 (<20%) had better OS than V40 (≥20%) (median [IQR], 2.5 [2.1-3.1] years vs 1.7 [1.5-2.0] years; P < .001). On multivariable analysis, heart V40 (≥20%), was associated with worse OS (hazard ratio, 1.34 [95% CI, 1.06-1.70]; P = .01), whereas lung V5 and age had no association with OS. Patients receiving IMRT and 3D-CRT had similar rates of developing secondary cancers (15 [6.6%] vs 14 [5.5%]) with long-term follow-up. Conclusions and Relevance: These findings support the standard use of IMRT for locally advanced NSCLC. IMRT should aim to minimize lung V20 and heart V20 to V60, rather than constraining low-dose radiation bath. Lung V5 and age were not associated with survival and should not be considered a contraindication for chemoradiotherapy. Trial Registration: ClinicalTrials.gov Identifier: NCT00533949.

APOA5 deficiency causes hypertriglyceridemia by reducing amounts of lipoprotein lipase in capillaries.

Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5's function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5-/- mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5-/- mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5-/- and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.

Surgical Management and Long-Term Evaluation of Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PNETs) arise from neuroendocrine cells and are a rare class of heterogenous tumors with increasing incidence. The diagnosis, staging, treatment, and prognosis of PNETs depend heavily on identifying the histologic features and biological mechanisms. Here, the authors provide an overview of the diagnostic workup (biomarkers and imaging), grade, and staging of PNETs. The authors also explore associated genetic mutations and molecular pathways and describe updated guidelines on surgical and systemic treatment modalities.