Engineering the Mechanical Stability of a Therapeutic Affibody/PD-L1 Complex by Anchor Point Selection.

Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical stability of a molecular complex between a therapeutic non-immunoglobulin scaffold called Affibody and the extracellular domain of the immune checkpoint protein PD-L1. We used a combination of single-molecule AFM force spectroscopy (AFM-SMFS) with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) simulations to understand the pulling point dependency of mechanostability of the Affibody:(PD-L1) complex. We observed diverse mechanical responses depending on the anchor point. For example, pulling from residue #22 on Affibody generated an intermediate unfolding event attributed to partial unfolding of PD-L1, while pulling from Affibody's N-terminus generated force-activated catch bond behavior. We found that pulling from residue #22 or #47 on Affibody generated the highest rupture forces, with the complex breaking at up to ~ 190 pN under loading rates of ~104-105 pN/sec, representing a ~4-fold increase in mechanostability as compared with low force N-terminal pulling. SMD simulations provided consistent tendencies in rupture forces, and through visualization of force propagation networks provided mechanistic insights. These results demonstrate how mechanostability of therapeutic protein-protein interfaces can be controlled by informed selection of anchor points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.

Engineering an artificial catch bond using mechanical anisotropy.

Catch bonds are a rare class of protein-protein interactions where the bond lifetime increases under an external pulling force. Here, we report how modification of anchor geometry generates catch bonding behavior for the mechanostable Dockerin G:Cohesin E (DocG:CohE) adhesion complex found on human gut bacteria. Using AFM single-molecule force spectroscopy in combination with bioorthogonal click chemistry, we mechanically dissociate the complex using five precisely controlled anchor geometries. When tension is applied between residue #13 on CohE and the N-terminus of DocG, the complex behaves as a two-state catch bond, while in all other tested pulling geometries, including the native configuration, it behaves as a slip bond. We use a kinetic Monte Carlo model with experimentally derived parameters to simulate rupture force and lifetime distributions, achieving strong agreement with experiments. Single-molecule FRET measurements further demonstrate that the complex does not exhibit dual binding mode behavior at equilibrium but unbinds along multiple pathways under force. Together, these results show how mechanical anisotropy and anchor point selection can be used to engineer artificial catch bonds.

Protocol to measure validity and reliability of colorectal, breast, cervical and lung cancer screening questions from the 2021 National Health Interview Survey: Methodology and design.

Previous studies demonstrate that self-reports of mammography screening for breast cancer and colonoscopy screening for colorectal cancer demonstrate concordance, based on adherence to screening guidelines, with electronic medical records (EMRs) in over 90% of those interviewed, as well as high sensitivity and specificity, and can be used for monitoring our Healthy People goals. However, for screening tests for cervical and lung cancers, and for various sub-populations, concordance between self-report and EMRs has been noticeably lower with poor sensitivity or specificity. This study aims to test the validity and reliability of lung, colorectal, cervical, and breast cancer screening questions from the 2021 and 2022 National Health Interview Survey (NHIS). We present the protocol for a study designed to measure the validity and reliability of the NHIS cancer screening questions compared to EMRs from four US-based healthcare systems. We planned a randomized trial of a phone- vs web-based survey with NHIS questions that were previously revised based on extensive cognitive interviewing. Our planned sample size will be 1576 validity interviews, and 1260 interviews randomly assigned at 1 or 3 months after the initial interview. We are enrolling people eligible for cancer screening based on age, sex, and smoking history per US Preventive Services Task Force recommendations. We will evaluate question validity using concordance, sensitivity, specificity, positive predictive value, negative predictive value, and report-to-records ratio. We further are randomizing participants to complete a second survey 1 vs 3 months later to assess question reliability. We suggest that typical measures of concordance may need to be reconsidered in evaluating cancer screening questions.

Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing.

Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we present the development of enzyme proximity sequencing, a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment. We use enzyme proximity sequencing to analyze how 6399 missense mutations influence folding stability and catalytic activity in a D-amino acid oxidase from Rhodotorula gracilis. The resulting datasets demonstrate activity-based constraints that limit folding stability during natural evolution, and identify hotspots distant from the active site as candidates for mutations that improve catalytic activity without sacrificing stability. Enzyme proximity sequencing can be extended to other enzyme classes and provides valuable insights into biophysical principles governing enzyme structure and function.

Integrating Dynamic Network Analysis with AI for Enhanced Epitope Prediction in PD-L1:Affibody Interactions.

Understanding binding epitopes involved in protein-protein interactions and accurately determining their structure is a long standing goal with broad applicability in industry and biomedicine. Although various experimental methods for binding epitope determination exist, these approaches are typically low throughput and cost intensive. Computational methods have potential to accelerate epitope predictions, however, recently developed artificial intelligence (AI)-based methods frequently fail to predict epitopes of synthetic binding domains with few natural homologs. Here we have developed an integrated method employing generalized-correlation-based dynamic network analysis on multiple molecular dynamics (MD) trajectories, initiated from AlphaFold2 Multimer structures, to unravel the structure and binding epitope of the therapeutic PD-L1:Affibody complex. Both AlphaFold2 and conventional molecular dynamics trajectory analysis alone each proved ineffectual in differentiating between two putative binding models referred to as parallel and perpendicular. However, our integrated approach based on dynamic network analysis showed that the perpendicular mode was significantly more stable. These predictions were validated using a suite of experimental epitope mapping protocols including cross linking mass spectrometry and next-generation sequencing-based deep mutational scanning. Our research highlights the potential of deploying dynamic network analysis to refine AI-based structure predictions for precise predictions of protein-protein interaction interfaces.

Isolating mononuclear cells from fetal bone and liver for metabolic, functional, and immunophenotypic analyses in nonhuman primates.

Studying fetal hematopoiesis is challenging as hematopoiesis transitions from the liver to bone marrow. Obtaining human samples is not possible, and small animal models may not provide sufficient biological material. Here, we present a protocol for isolating hematopoietic cells from the nonhuman primate fetal liver and bone. We describe steps for using cells from the same fetus for fluorescence lifetime imaging microscopy to measure metabolism, assessing cellular function, and flow cytometry for immunophenotyping at the single-cell level. For complete details on the use and execution of this protocol, please refer to Nash et al. (2023).1.

Distance functions of carabids in crop fields depend on functional traits, crop type and adjacent habitat: a synthesis.

Natural pest and weed regulation are essential for agricultural production, but the spatial distribution of natural enemies within crop fields and its drivers are mostly unknown. Using 28 datasets comprising 1204 study sites across eight Western and Central European countries, we performed a quantitative synthesis of carabid richness, activity densities and functional traits in relation to field edges (i.e. distance functions). We show that distance functions of carabids strongly depend on carabid functional traits, crop type and, to a lesser extent, adjacent non-crop habitats. Richness of both carnivores and granivores, and activity densities of small and granivorous species decreased towards field interiors, whereas the densities of large species increased. We found strong distance decays in maize and vegetables whereas richness and densities remained more stable in cereals, oilseed crops and legumes. We conclude that carabid assemblages in agricultural landscapes are driven by the complex interplay of crop types, adjacent non-crop habitats and further landscape parameters with great potential for targeted agroecological management. In particular, our synthesis indicates that a higher edge-interior ratio can counter the distance decay of carabid richness per field and thus likely benefits natural pest and weed regulation, hence contributing to agricultural sustainability.

Interruptions in bladder cancer care during the COVID-19 public health emergency.

BACKGROUND: Academic and community urology centers participating in a pragmatic clinical trial in non-muscle-invasive bladder cancer completed monthly surveys assessing restrictions in aspects of bladder cancer care due to the COVID-19 Public Health Emergency. Our objective was to describe pandemic-related restrictions on bladder cancer care. METHODS: We invited 32 sites participating in a multicenter pragmatic bladder cancer trial to complete monthly surveys distributed through REDCap beginning in May 2020. These surveys queried sites on whether they were experiencing restrictions in the use of elective surgery, transurethral resection of bladder tumors (TURBT), radical cystectomy, office cystoscopy, and intravesical bacillus Calmette-Guerin (BCG) availability. Responses were collated with descriptive statistics. RESULTS: Of 32 eligible sites, 21 sites had at least a 50% monthly response rate over the study period and were included in the analysis. Elective surgery was paused at 76% of sites in May 2020, 48% of sites in January 2021, and 52% of sites in January 2022. Over those same periods, coinciding with COVID-19 incidence waves, TURBT was restricted at 10%, 14%, and 14% of sites, respectively, radical cystectomy was restricted at 10%, 14%, and 19% of sites, respectively, and cystoscopy was restricted at 33%, 0%, and 10% of sites, respectively. CONCLUSIONS: Bladder cancer care was minimally restricted compared with more pronounced restrictions seen in general elective surgeries during the COVID-19 pandemic.

Operative vs Nonoperative Management of Acute Cholecystitis During the Different Trimesters of Pregnancy.

Importance: Acute cholecystitis (AC) management during pregnancy requires balancing the risk of pregnancy loss or preterm delivery (adverse pregnancy outcomes [APOs]) with or without surgery. Guidelines recommend cholecystectomy across trimesters; however, trimester-specific evidence on the risks of AC and its management is lacking. Objective: To assess cholecystectomy frequency in pregnant people with AC, compare the rates of APOs in pregnant people with or without AC, and compare the rates of APOs in people with AC who did or did not undergo cholecystectomy. Design, Setting, and Participants: This retrospective, population-based cohort study used data for pregnant people with AC from the IBM MarketScan Commercial Claims and Encounters Database from January 1, 2007, to December 31, 2019, and a propensity score-matched cohort of pregnant people without AC. Trimester status (first [T1], second [T2], and third [T3]), APOs, and cholecystectomy were defined by administrative claims. Data were analyzed from October 2021 to July 2022. Exposures: Pregnant patients with or without AC. Pregnant patients with AC who did or did not receive cholecystectomy. Main Outcomes and Measures: The main outcomes were cholecystectomy during pregnancy and APOs (ie, preterm delivery and pregnancy loss). Pregnant patients with and without AC were compared to assess the association of AC with risk of APOs. Propensity score inverse-probability weighting was used to calculate treatment-associated APO risk among patients with 1-year follow-up. Results: The study included 5759 pregnant patients with AC (mean [SD] age, 30.1 [6.6] years) and 23 036 controls (mean [SD] age, 29.9 [6.7] years) after propensity score matching. Among 3426 pregnant patients with AC and 1-year follow-up, 1182 (34.5%) underwent cholecystectomy during the pregnancy (684 [41.7%] presenting with AC in T1, 404 [40.4%] in T2, and 94 [12.0%] in T3). Acute cholecystitis during pregnancy, irrespective of treatment, was associated with higher odds of APO compared with no AC during pregnancy across all trimesters (odds ratio [OR], 1.69 [95% CI, 1.54-1.85]). Compared with nonoperative management, receipt of surgery was associated with lower odds of APOs across all trimesters (OR, 0.75 [95% CI, 0.63-0.87]), in T1 (OR, 0.81 [95% CI, 0.66-1.00]), in T2 (OR, 0.71 [95% CI, 0.50-1.00]), and in T3 (OR, 0.45 [95% CI, 0.28-0.70]). Conclusions and Relevance: In this study, cholecystectomy was associated with lower risk of APO in patients with AC across all trimesters, with the greatest benefit in T3. However, only 34.5% overall and 12.0% of patients in T3 had a cholecystectomy. These findings support guidelines recommending cholecystectomy during pregnancy and should inform decision-making discussions. Greater guideline adherence and surgery use, especially in T3, may represent an opportunity to improve outcomes for pregnant people with AC.

Protocol of the Comparison of Intravesical Therapy and Surgery as Treatment Options (CISTO) study: a pragmatic, prospective multicenter observational cohort study of recurrent high-grade non-muscle invasive bladder cancer.

BACKGROUND: Bladder cancer poses a significant public health burden, with high recurrence and progression rates in patients with non-muscle-invasive bladder cancer (NMIBC). Current treatment options include bladder-sparing therapies (BST) and radical cystectomy, both with associated risks and benefits. However, evidence supporting optimal management decisions for patients with recurrent high-grade NMIBC remains limited, leading to uncertainty for patients and clinicians. The CISTO (Comparison of Intravesical Therapy and Surgery as Treatment Options) Study aims to address this critical knowledge gap by comparing outcomes between patients undergoing BST and radical cystectomy. METHODS: The CISTO Study is a pragmatic, prospective observational cohort trial across 36 academic and community urology practices in the US. The study will enroll 572 patients with a diagnosis of recurrent high-grade NMIBC who select management with either BST or radical cystectomy. The primary outcome is health-related quality of life (QOL) at 12 months as measured with the EORTC-QLQ-C30. Secondary outcomes include bladder cancer-specific QOL, progression-free survival, cancer-specific survival, and financial toxicity. The study will also assess patient preferences for treatment outcomes. Statistical analyses will employ targeted maximum likelihood estimation (TMLE) to address treatment selection bias and confounding by indication. DISCUSSION: The CISTO Study is powered to detect clinically important differences in QOL and cancer-specific survival between the two treatment approaches. By including a diverse patient population, the study also aims to assess outcomes across the following patient characteristics: age, gender, race, burden of comorbid health conditions, cancer severity, caregiver status, social determinants of health, and rurality. Treatment outcomes may also vary by patient preferences, health literacy, and baseline QOL. The CISTO Study will fill a crucial evidence gap in the management of recurrent high-grade NMIBC, providing evidence-based guidance for patients and clinicians in choosing between BST and radical cystectomy. The CISTO study will provide an evidence-based approach to identifying the right treatment for the right patient at the right time in the challenging clinical setting of recurrent high-grade NMIBC. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03933826. Registered on May 1, 2019.

Iterative Machine Learning for Classification and Discovery of Single-Molecule Unfolding Trajectories from Force Spectroscopy Data.

We report the application of machine learning techniques to expedite classification and analysis of protein unfolding trajectories from force spectroscopy data. Using kernel methods, logistic regression, and triplet loss, we developed a workflow called Forced Unfolding and Supervised Iterative Online (FUSION) learning where a user classifies a small number of repeatable unfolding patterns encoded as images, and a machine is tasked with identifying similar images to classify the remaining data. We tested the workflow using two case studies on a multidomain XMod-Dockerin/Cohesin complex, validating the approach first using synthetic data generated with a Monte Carlo algorithm and then deploying the method on experimental atomic force spectroscopy data. FUSION efficiently separated traces that passed quality filters from unusable ones, classified curves with high accuracy, and identified unfolding pathways that were undetected by the user. This study demonstrates the potential of machine learning to accelerate data analysis and generate new insights in protein biophysics.

Mental health knowledge and classroom experiences of school teachers in Aragon, Spain.

Background: Research shows that many mental disorders begin in childhood but are sometimes not diagnosed until later years. School-age children spend much of their time in schools and have daily interactions with school teachers. Aim: Examine school teachers' experiences of mental disorders in school going children and adolescents and their associated mental health training needs. Method and sample: A descriptive cross-sectional study was carried out with teachers in Infant-Primary and Secondary Education-Baccalaureate schools. Results: A convenience sample of 685 teachers responded to the online survey. Participants worked in both urban and rural areas and in Infant-Primary and Secondary Education-Baccalaureate schools. Over half of participants reported classroom experiences of learner mental disorders such as ADHD, anxiety, conduct disorders or autism. Most participants acknowledged a training need, both in recognition of symptoms of mental disorders and in care resources and processes. However, 80% of respondents reported having not received any training in this regard. Participant preferences for training included face-to-face or hybrid - combined online learning. Participants also considered the management of their own mental health to be deficient, therefore any training should incorporate personal mental health awareness and self-help strategies. Conclusion: In Aragón (Spain), teachers of children and adolescents with mental disorders, recognize a need for training in the identification of symptoms and other aspects of mental healthcare, such as availability and access to services. Protocols for early identification and referral would promote mentally healthy school environments and reduce stigma which could be a barrier to timely intervention. In addition, any training should include mental health self-care for teachers.

Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening.

Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of d-Amino acid oxidase from Rhodotorula gracilis (RgDAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between RgDAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled library screening by fluorescence activated cell sorting (FACS). We screened a RgDAAOx variant library containing ∼106 clones while lowering the d-Ala substrate concentration over three sorting rounds in order to isolate variants with low Km. After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower Km than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the RgDAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes.

Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays.

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.

Non-Canonical Activin A Signaling Stimulates Context-Dependent and Cellular-Specific Outcomes in CRC to Promote Tumor Cell Migration and Immune Tolerance.

We have shown that activin A (activin), a TGF-ß superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4-/-) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4-/- mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-ß-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.

Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.

Multiplexed on-yeast serological assay for immune escape screening of SARS-CoV-2 variants.

The emergence of the SARS-CoV-2 Omicron variant altered patient risk profiles and shifted the trajectory of the COVID-19 pandemic. Therefore, sensitive serological tests capable of analyzing patient IgG responses to multiple variants in parallel are highly desirable. Here, we present an adaptable serological test based on yeast surface display and serum biopanning that characterizes immune profiles against SARS-CoV-2 Wuhan (B lineage), Delta (B.1.617.2 lineage), and Omicron (B.1.1.529 lineage) receptor-binding domain (RBD) variants. We examined IgG titers from 30 serum samples from COVID-19-convalescent and vaccinated cohorts in Switzerland, and assessed the relative affinity of polyclonal serum IgG for RBD domains. We demonstrate that serum IgGs from patients recovered from severe COVID-19 between March-June 2021 bound tightly to both original Wuhan and Delta RBD variants, but failed to recognize Omicron RBDs, representing an affinity loss of >10- to 20-fold. Our yeast immunoassay is easily tailored, expandable and parallelized with newly emerging RBD variants.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.

Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1.

We report an enzyme cascade with horseradish peroxidase-based readout for screening human arginase-1 (hArg1) activity. We combined the four enzymes hArg1, ornithine decarboxylase, putrescine oxidase, and horseradish peroxidase in a reaction cascade that generated colorimetric or fluorescent signals in response to hArg1 activity and used this cascade to assay wild-type and variant hArg1 sequences as soluble enzymes and displayed on the surface of Escherichia coli. We screened a curated 13-member hArg1 library covering mutations that modified the electrostatic environment surrounding catalytic residues D128 and H141, and identified the R21E variant with a 13% enhanced catalytic turnover rate compared to wild type. Our scalable one-pot single-step arginase assay with continuous kinetic readout is amenable to high-throughput screening and directed evolution of arginase libraries and testing drug candidates for arginase inhibition.

Defining the Performance of Clinician's Ability to Screen for Laryngeal Mass From Voice.

OBJECTIVE: Defining a clinician's ability to perceptually identify mass from voice will inform the feasibility, design priorities, and performance standards for tools developed to screen for laryngeal mass from voice. This study defined clinician ability of and examined the impact of expertise on screening for laryngeal mass from voice. STUDY DESIGN: Task comparison study between experts and nonexperts rating voices for the probability of a laryngeal mass. SETTING: Online, remote. METHODS: Experts (voice-focused speech-language pathologists and otolaryngologists) and nonexperts (general medicine providers) rated 5-s/i/voice samples (with pathology defined by laryngoscopy) for the probability of laryngeal mass via an online survey. The intraclass correlation coefficient (ICC) estimated interrater and intrarater reliability. Diagnostic performance metrics were calculated. A linear mixed effects model examined the impact of expertise and pathology on ratings. RESULTS: Forty clinicians (21 experts and 19 nonexperts) evaluated 344 voice samples. Experts outperformed nonexperts, with a higher area under the curve (70% vs 61%), sensitivity (49% vs 36%), and specificity (83% vs 77%) (all comparisons p < .05). Interrater reliability was fair for experts and poor for nonexperts (ICC: 0.48 vs 0.34), while intrarater reliability was excellent and good, respectively (ICC: 0.9 and 0.6). The main effects of expertise and underlying pathology were significant in the linear model (p < .001). CONCLUSION: Clinicians demonstrate inadequate performance screening for laryngeal mass from voice to use auditory perception for dysphonia triage. Experts' superior performance indicates that there is acoustic information in a voice that may be utilized to detect laryngeal mass based on voice.