Development of a Rapid and Fully Automated Factor VIII-Inhibitor Assay, Insensitive for Emicizumab, and a Lowest Level of Quantification of 0.2BU/mL.

BACKGROUND: Factor VIII inhibitors are measured using labour and resource expensive Nijmegen or Bethesda assays, which lack sensitivity for low-titre-inhibitors, and show high variations in quality surveys, mainly because of manual assay procedures. METHODS: A new rapid, fully automated, factor VIII inhibitor assay is presented, the core of which is use of full-length recombinant FVIII (rFVIII) (Kovaltry®) as inhibitor substrate instead of plasma FVIII, resulting in rapid binding of inhibitors to rFVIII due to absence of VWF. Dramatic shortening of incubation time facilitated full automation on an analyser capable of three subsequent sample dilution steps and three reagent additions. Equal volume mixtures of sample and rFVIII (1.0 U/mL) were incubated 10 minutes/37°C, whereafter remaining FVIII-activity was analysed with a kinetic chromogenic assay, allowing inhibitor-activity calculation without preceding FVIII-activity calibration, using a Ceveron s100 analyser. RESULTS: Mean titre in 60 non-haemophiliacs was 0.0BU/mL (SD 0.1), yielding a Limit-of-Blank of 0.1BU/mL and Lower-Limit-of-Quantification of 0.2BU/mL. Analyses were performed with the new method and a Nijmegen Assay in 28 inhibitor-positive clinical samples, 14 containing emicizumab and 14 without. Correlation coefficient in emicizumab-free type-I-inhibitor samples was r=1.0. Emicizumab dependency of the method was excluded in spiking experiments with inhibitor-positive-samples. Reproducibility was tested by analysing seven samples in three laboratories on five days, twice daily; CV of all samples were <15%. CONCLUSION: We present development data of a sensitive and specific, rapid, automated FVIII inhibitor assay generating results within 20 minutes, is less resource intensive than standard assays, with potential to improve assay variability.

Synthetic extremophiles via species-specific formulations improve microbial therapeutics.

Microorganisms typically used to produce food and pharmaceuticals are now being explored as medicines and agricultural supplements. However, maintaining high viability from manufacturing until use remains an important challenge, requiring sophisticated cold chains and packaging. Here we report synthetic extremophiles of industrially relevant gram-negative bacteria (Escherichia coli Nissle 1917, Ensifer meliloti), gram-positive bacteria (Lactobacillus plantarum) and yeast (Saccharomyces boulardii). We develop a high-throughput pipeline to define species-specific materials that enable survival through drying, elevated temperatures, organic solvents and ionizing radiation. Using this pipeline, we enhance the stability of E. coli Nissle 1917 by more than four orders of magnitude over commercial formulations and demonstrate its capacity to remain viable while undergoing tableting and pharmaceutical processing. We further show, in live animals and plants, that synthetic extremophiles remain functional against enteric pathogens and as nitrogen-fixing plant supplements even after exposure to elevated temperatures. This synthetic, material-based stabilization enhances our capacity to apply microorganisms in extreme environments on Earth and potentially during exploratory space travel.

Alzheimer's disease CSF biomarkers correlate with early pathology and alterations in neuronal and glial gene expression.

INTRODUCTION: Normal pressure hydrocephalus (NPH) patients undergoing cortical shunting frequently show early AD pathology on cortical biopsy, which is predictive of progression to clinical AD. The objective of this study was to use samples from this cohort to identify CSF biomarkers for AD-related CNS pathophysiologic changes using tissue and fluids with early pathology, free of post-mortem artifact. METHODS: We analyzed Simoa, proteomic, and metabolomic CSF data from 81 patients with previously documented pathologic and transcriptomic changes. RESULTS: AD pathology on biopsy correlates with CSF ß-amyloid-40/42, neurofilament light chain (NfL), and phospho-tau-181(p-tau181)/ß-amyloid-42, while several gene expression modules correlate with NfL. Proteomic analysis highlights 7 core proteins that correlate with pathology and gene expression changes on biopsy, and metabolomic analysis of CSF identifies disease-relevant groups that correlate with biopsy data.. DISCUSSION: As additional biomarkers are added to AD diagnostic panels, our work provides insight into the CNS pathophysiology these markers are tracking.

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage.

In the modern "omics" era, measurement of the human exposome is a critical missing link between genetic drivers and disease outcomes. High-resolution mass spectrometry (HRMS), routinely used in proteomics and metabolomics, has emerged as a leading technology to broadly profile chemical exposure agents and related biomolecules for accurate mass measurement, high sensitivity, rapid data acquisition, and increased resolution of chemical space. Non-targeted approaches are increasingly accessible, supporting a shift from conventional hypothesis-driven, quantitation-centric targeted analyses toward data-driven, hypothesis-generating chemical exposome-wide profiling. However, HRMS-based exposomics encounters unique challenges. New analytical and computational infrastructures are needed to expand the analysis coverage through streamlined, scalable, and harmonized workflows and data pipelines that permit longitudinal chemical exposome tracking, retrospective validation, and multi-omics integration for meaningful health-oriented inferences. In this article, we survey the literature on state-of-the-art HRMS-based technologies, review current analytical workflows and informatic pipelines, and provide an up-to-date reference on exposomic approaches for chemists, toxicologists, epidemiologists, care providers, and stakeholders in health sciences and medicine. We propose efforts to benchmark fit-for-purpose platforms for expanding coverage of chemical space, including gas/liquid chromatography-HRMS (GC-HRMS and LC-HRMS), and discuss opportunities, challenges, and strategies to advance the burgeoning field of the exposome.

Extended genome-wide association study employing the African genome resources panel identifies novel susceptibility loci for Alzheimer's disease in individuals of African ancestry.

INTRODUCTION: Despite a two-fold risk, individuals of African ancestry have been underrepresented in Alzheimer's disease (AD) genomics efforts. METHODS: Genome-wide association studies (GWAS) of 2,903 AD cases and 6,265 controls of African ancestry. Within-dataset results were meta-analyzed, followed by functional genomics analyses. RESULTS: A novel AD-risk locus was identified in MPDZ on chromosome (chr) 9p23 (rs141610415, MAF = 0.002, P = 3.68×10-9). Two additional novel common and nine rare loci were identified with suggestive associations (P < 9×10-7). Comparison of association and linkage disequilibrium (LD) patterns between datasets with higher and lower degrees of African ancestry showed differential association patterns at chr12q23.2 (ASCL1), suggesting that this association is modulated by regional origin of local African ancestry. DISCUSSION: These analyses identified novel AD-associated loci in individuals of African ancestry and suggest that degree of African ancestry modulates some associations. Increased sample sets covering as much African genetic diversity as possible will be critical to identify additional loci and deconvolute local genetic ancestry effects. HIGHLIGHTS: Genetic ancestry significantly impacts risk of Alzheimer's Disease (AD). Although individuals of African ancestry are twice as likely to develop AD, they are vastly underrepresented in AD genomics studies. The Alzheimer's Disease Genetics Consortium has previously identified 16 common and rare genetic loci associated with AD in African American individuals. The current analyses significantly expand this effort by increasing the sample size and extending ancestral diversity by including populations from continental Africa. Single variant meta-analysis identified a novel genome-wide significant AD-risk locus in individuals of African ancestry at the MPDZ gene, and 11 additional novel loci with suggestive genome-wide significance at P < 9×10-7. Comparison of African American datasets with samples of higher degree of African ancestry demonstrated differing patterns of association and linkage disequilibrium at one of these loci, suggesting that degree and/or geographic origin of African ancestry modulates the effect at this locus. These findings illustrate the importance of increasing number and ancestral diversity of African ancestry samples in AD genomics studies to fully disentangle the genetic architecture underlying AD, and yield more effective ancestry-informed genetic screening tools and therapeutic interventions.

Tailoring Plasmonic Fields with Shape-Controlled Single-Crystal Gold Metasurfaces.

Geometry and crystallinity play a critical role in the wavelength-dependent optical responses and plasmonic local near-field distributions of metallic nanostructures. Nevertheless, the ability to tailor the shape and position of crystalline metal surface nanostructures has remained a challenge that limits control of their enhanced local fields and represents a barrier to harnessing their individual and collective responses. Here, we describe a solution deposition method in the presence of anionic additives, which yields shape-controlled, single-crystal plasmonic gold nanostructures on Ag(100) and Au(100) substrates. Use of SO42- ions yields smooth Au(111)-faceted square pyramids with large plasmonic Raman enhancements. Halide additives produce textured hillocks comprising edge- and screw-type dislocations (Cl-), or platelets with large-area Au(100) terraces and (110) step edges (Br-), while SO42- and Br- additive combinations provide Au(110)-faceted square pyramids. With lithographic patterning, this chemistry yields metal deposition with precise geometry and location control to provide single-crystal, plasmonic gold metasurfaces with tailored optical response. The appropriately designed metasurfaces can then generate large Raman scattering enhancements, far greater than high density gold square pyramids with random surface disposition. Shape-controlled single-crystal plasmonic metasurfaces will thus offer opportunities to tune the characteristics of nanostructures, providing enhanced optical, photocatalytic, and sensory response.

Pediatric asthma comorbidities: Global impact and unmet needs.

Real-world data on the range and impact of comorbid health conditions that affect pediatric asthma are scant, especially from developing countries. Lack of data hinders effective diagnosis, treatment, and overall management of these complex cases. We, hereby, describe the common pediatric asthma comorbid conditions in terms of evidence for association, potential mechanisms of impact on asthma control, and treatment benefit. Obesity, upper airway allergies, dysfunctional breathing, multiple sensitizations, depressive disorders, food allergy, and gastro-esophageal reflux are common associations with difficult-to-treat asthma. On the other hand, asthma symptoms and/or management may negatively impact the well-being of children through drug adverse effects, worsening of anaphylaxis symptoms, and disturbing mental health. Awareness of these ailments may be crucial for designing the optimum care for each asthmatic child individually and may ultimately improve the quality of life of patients and their families. A multidisciplinary team of physicians is required to identify and manage such comorbidities aiming to mitigate the over-use of asthma pharmacotherapy. Asthma research should target relevant real-world difficulties encountered at clinical practice and focus on interventions that would mitigate the impact of such comorbidities. Finally, policymakers and global healthcare organizations are urged to recognize pediatric asthma control as a healthcare priority and allocate resources for research and clinical interventions. In other words, global asthma control needs support by compassionate scientific partnership.

Developmental pyrethroid exposure in mouse leads to disrupted brain metabolism in adulthood.

Environmental and genetic risk factors, and their interactions, contribute significantly to the etiology of neurodevelopmental disorders (NDDs). Recent epidemiology studies have implicated pyrethroid pesticides as an environmental risk factor for autism and developmental delay. Our previous research showed that low-dose developmental exposure to the pyrethroid pesticide deltamethrin in mice caused male-biased changes in the brain and in NDD-relevant behaviors in adulthood. Here, we used a metabolomics approach to determine the broadest possible set of metabolic changes in the adult male mouse brain caused by low-dose pyrethroid exposure during development. Using a litter-based design, we exposed mouse dams during pregnancy and lactation to deltamethrin (3 mg/kg or vehicle every 3 days) at a concentration well below the EPA-determined benchmark dose used for regulatory guidance. We raised male offspring to adulthood and collected whole brain samples for untargeted high-resolution metabolomics analysis. Developmentally exposed mice had disruptions in 116 metabolites which clustered into pathways for folate biosynthesis, retinol metabolism, and tryptophan metabolism. As a cross-validation, we integrated metabolomics and transcriptomics data from the same samples, which confirmed previous findings of altered dopamine signaling. These results suggest that pyrethroid exposure during development leads to disruptions in metabolism in the adult brain, which may inform both prevention and therapeutic strategies.

Neurotoxicology of dopamine: Victim or assailant?

Since the identification of dopamine as a neurotransmitter in the mid-20th century, investigators have examined the regulation of dopamine homeostasis at a basic biological level and in human disorders. Genetic animal models that manipulate the expression of proteins involved in dopamine homeostasis have provided key insight into the consequences of dysregulated dopamine. As a result, we have come to understand the potential of dopamine to act as an endogenous neurotoxin through the generation of reactive oxygen species and reactive metabolites that can damage cellular macromolecules. Endogenous factors, such as genetic variation and subcellular processes, and exogenous factors, such as environmental exposures, have been identified as contributors to the dysregulation of dopamine homeostasis. Given the variety of dysregulating factors that impact dopamine homeostasis and the potential for dopamine itself to contribute to further cellular dysfunction, dopamine can be viewed as both the victim and an assailant of neurotoxicity. Parkinson's disease has emerged as the exemplar case study of dopamine dysregulation due to the genetic and environmental factors known to contribute to disease risk, and due to the evidence of dysregulated dopamine as a pathologic and pathogenic feature of the disease. This review, inspired by the talk, "Dopamine in Durham: location, location, location" presented by Dr. Miller for the Jacob Hooisma Memorial Lecture at the International Neurotoxicology Association meeting in 2023, offers a primer on dopamine toxicity covering endogenous and exogenous factors that disrupt dopamine homeostasis and the actions of dopamine as an endogenous neurotoxin.

A platform technology for ultra-long acting intratumoral therapy.

Intratumoral (IT) therapy is a powerful method of controlling tumor growth, but a major unsolved problem is the rapidity that injected drugs exit tumors, limiting on-target exposure and efficacy. We have developed a generic long acting IT delivery system in which a drug is covalently tethered to hydrogel microspheres (MS) by a cleavable linker; upon injection the conjugate forms a depot that slowly releases the drug and "bathes" the tumor for long periods. We established technology to measure tissue pharmacokinetics and studied MSs attached to SN-38, a topoisomerase 1 inhibitor. When MS ~ SN-38 was injected locally, tissues showed high levels of SN-38 with a long half-life of ~ 1 week. IT MS ~ SN-38 was ~ tenfold more efficacious as an anti-tumor agent than systemic SN-38. We also propose and provide an example that long-acting IT therapy might enable safe use of two drugs with overlapping toxicities. Here, long-acting IT MS ~ SN-38 is delivered with concurrent systemic PARP inhibitor. The tumor is exposed to both drugs whereas other tissues are exposed only to the systemic drug; synergistic anti-tumor activity supported the validity of this approach. We propose use of this approach to increase efficacy and reduce toxicities of combinations of immune checkpoint inhibitors such as αCTLA-4 and αPD-1.

A revision of the genus Wrangelia (Wrangeliaceae, Ceramiales) in Bermuda resolves six new species including W. ryancraigii from the mesophotic zone.

Four species of the genus Wrangelia are presently known from the western Atlantic Ocean: W. argus, W. bicuspidata, W. penicillata, and W. gordoniae, with the first three historically being reported from Bermuda. Morphological and molecular barcode (COI-5P) and phylogenetic analyses used in this study (SSU, LSU, rbcL) indicated eight species groupings of Wrangelia in Bermuda, excluding two of the historically recognized species, retaining only W. argus while adding seven new species, of which six are formally described. What had been historically reported as W. penicillata from Bermuda was shown to be distinct from Mediterranean Sea specimens (type locality) and was shown to be a mixture of W. hesperia sp. nov. and W. incrassata sp. nov. Along with these two, three other new species (W. laxa sp. nov., W. ryancraigii sp. nov., and W. secundiramea sp. nov.) have complete rhizoidal cortication tightly covering axial cells of indeterminate axes below the apices, distinguishing them from the two local incompletely corticated congeners W. argus and W. abscondita sp. nov., the latter a morphologically cryptic sister species with W. bicuspidata from the Caribbean Sea. Only one of the new species, W. ryancraigii, has thus far been observed in the mesophotic zone off the Bermuda platform, and it is morphologically cryptic with the euphotic zone's W. laxa.

Chemical and Electrochemical Characterization of Hot-Pressed Li6PS5Cl Solid State Electrolyte: Operating Pressure-Invariant High Ionic Conductivity.

Sulfide solid state electrolytes (SSE) are among the most promising materials in the effort to replace liquid electrolytes, largely due to their comparable ionic conductivities. Among the sulfide SSEs, Argyrodites (Li6PS5X, X=Cl, Br, I) further stand out due to their high theoretical ionic conductivity (~1×10-2 S cm-1) and interfacial stability against reactive metal anodes such as lithium. Generally, solid state electrolyte pellets are pressed from powder feedstock at room temperature, however, pellets fabricated by cold pressing consistently result in low bulk density and high porosity, facilitating interfacial degradation reactions and allowing dendrites to propagate through the pores and grain boundaries. Here, we demonstrate the mechanical and electrochemical implications of hot-pressing standalone LPSCl SSE pellets with near-theoretical ionic conductivity, superior cycling performance, and enhanced mechanical stability. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and x-ray diffraction spectroscopy (XRD) analysis reveal no chemical changes to the Argyrodite surface after hot pressing up to 250 °C. Moreover, we use electrochemical impedance spectroscopy (EIS) to understand mechanical stability of Argyrodite SSE pellets as a function of externally applied pressure, demonstrating for the first time pressed standalone Argyrodite pellets with near-theoretical conductivities at external pressures below 14 MPa.

Water Ligands Regulate the Redox Leveling Mechanism of the Oxygen-Evolving Complex of the Photosystem II.

Understanding how water ligands regulate the conformational changes and functionality of the oxygen-evolving complex (OEC) in photosystem II (PSII) throughout the catalytic cycle of oxygen evolution remains a highly intriguing and unresolved challenge. In this study, we investigate the effect of water insertion (WI) on the redox state of the OEC by using the molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) hybrid methods. We find that water binding significantly reduces the free energy change for proton-coupled electron transfer (PCET) from Mn to YZ•, underscoring the important regulatory role of water binding, which is essential for enabling the OEC redox-leveling mechanism along the catalytic cycle. We propose a water binding mechanism in which WI is thermodynamically favored by the closed-cubane form of the OEC, with water delivery mediated by Ca2+ ligand exchange. Isomerization from the closed- to open-cubane conformation at three post-WI states highlights the importance of the location of the MnIII center in the OEC and the orientation of its Jahn-Teller axis to conformational changes of the OEC, which might be critical for the formation of the O-O bond. These findings reveal a complex interplay between conformational changes in the OEC and the ligand environment during the activation of the OEC by YZ•. Analogous regulatory effects due to water ligand binding are expected to be important for a wide range of catalysts activated by redox state transitions in aqueous environments.

Electron Transfer Dynamics at Dye-Sensitized SnO2/TiO2 Core/Shell Electrodes in Aqueous/Nonaqueous Electrolyte Mixtures.

The dynamics of photoinduced electron transfer were measured at dye-sensitized photoanodes in aqueous (acetate buffer), nonaqueous (acetonitrile), and mixed solvent electrolytes by nanosecond transient absorption spectroscopy (TAS) and ultrafast optical-pump terahertz-probe spectroscopy (OPTP). Higher injection efficiencies were found in mixed solvent electrolytes for dye-sensitized SnO2/TiO2 core/shell electrodes, whereas the injection efficiency of dye-sensitized TiO2 electrodes decreased with the increasing acetonitrile concentration. The trend in injection efficiency for the TiO2 electrodes was consistent with the solvent-dependent trend in the semiconductor flat band potential. Photoinduced electron injection in core/shell electrodes has been understood as a two-step process involving ultrafast electron trapping in the TiO2 shell followed by slower electron transfer to the SnO2 core. The driving force for shell-to-core electron transfer increases as the flat band potential of TiO2 shifts negatively with increasing concentrations of acetonitrile. In acetonitrile-rich electrolytes, electron injection is suppressed due to the very negative flat band potential of the TiO2 shell. Interestingly, a net negative photoconductivity in the SnO2 core is observed in mixed solvent electrolytes by OPTP. We hypothesize that an electric field is formed across the TiO2 shell from the oxidized dye molecules after injection. Conduction band electrons in SnO2 are trapped at the core/shell interface by the electric field, resulting in a negative photoconductivity transient. The overall electron injection efficiency of the dye-sensitized SnO2/TiO2 core/shell photoanodes is optimized in mixed solvents. The ultrafast transient conductivity data illustrate the crucial role of the electrolyte in regulating the driving forces for electron injection and charge separation at dye-sensitized semiconductor interfaces.

Mutation-induced shift of the photosystem II active site reveals insight into conserved water channels.

Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a Mn4CaO5 cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the Mn4CaO5 cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.

Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer.

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

Toward harmonized interpretation of anticardiolipin and anti-ß2-glycoprotein I antibody detection for diagnosis of antiphospholipid syndrome using defined level intervals and likelihood ratios: communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies.

BACKGROUND: Improving harmonization of the clinical interpretation of anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) antibodies immunoglobulin G (IgG)/immunoglobulin M (IgM) in the diagnosis of antiphospholipid syndrome (APS) is desirable. Likelihood ratios (LRs) with corresponding test-result intervals can identify the power of a test to discriminate between a diseased and nondiseased patient and may be useful for the semiquantitative interpretation of aCL/aß2GPI results. OBJECTIVES: To determine moderate and high thresholds for aCL and aß2GPI IgG/IgM measured with chemiluminescent immunoassay, enzyme-linked immunosorbent assay, fluorescence enzyme immunoassay, and multiplex flow immunoassay. METHODS: aCL and aß2GPI antibodies IgG/IgM were determined with 4 solid-phase systems in a case-control study population including 381 APS patients and 727 controls. Interval-specific LRs (IS-LR) were calculated for ranges determined by prespecified specificity and sensitivity levels. Three methods were used for determining thresholds that separated low, moderate, and high positive antibody levels. Interassay agreement was checked with Cohen's kappa statistics. RESULTS: Assay- and antibody-specific thresholds demonstrated increasing IS-LR, reflecting different clinical significance for low, moderate, and high levels, especially for IgG aCL and aß2GPI and in thrombotic APS. IS-LRs per antibody and unit range were comparable across solid-phase platforms resulting in enhanced harmonization of result interpretation. Agreement between assays for identifying high levels was improved by semiquantitative interpretation compared with that by quantitative reporting. CONCLUSION: aCL and aß2GPI IgG/IgM moderate and high thresholds were determined for 4 analytical platforms. Thresholds improve harmonized interpretation of aCL/aß2GPI levels across platforms. The proposed thresholds should be verified in an independent case-control study to check interlaboratory transferability.

Clinical and molecular correlates of the Index of Severity for Eosinophilic Esophagitis.

BACKGROUND: The Index of Severity for Eosinophilic Esophagitis (I-SEE) is a new expert-defined clinical tool that classifies disease severity of eosinophilic esophagitis (EoE). OBJECTIVE: We aimed to determine whether I-SEE is associated with patient characteristics, molecular features of EoE, or both. METHODS: We analyzed a prospective cohort of patients with EoE from the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR). Associations between I-SEE and clinical and molecular features (assessed by an EoE diagnostic panel [EDP]) were assessed. RESULTS: In 318 patients with chronic EoE (209 adults, 109 children), median total I-SEE score was 7.0, with a higher symptoms and complications score in children than adults (4.0 vs 1.0; P < .001) and higher inflammatory and fibrostenotic features scores in adults than children (3.0 vs 1.0 and 3.0 vs 0, respectively; both P < .001). Total I-SEE score had a bimodal distribution with the inactive to moderate categories and severe category. EDP score correlated with total I-SEE score (r = -0.352, P < .001) and both inflammatory and fibrostenotic features scores (r = -0.665, P < .001; r = -0.446, P < .001, respectively), but not with symptoms and complications scores (r = 0.047, P = .408). Molecular severity increased from inactive to mild and moderate, but not severe, categories. Longitudinal changes of modified I-SEE scores and inflammatory and fibrostenotic features scores reflected histologic and molecular activity. CONCLUSIONS: I-SEE score is associated with select clinical features across severity categories and with EoE molecular features for nonsevere categories, warranting further validation.

Nonepithelial Gene Expression Correlates With Symptom Severity in Adults With Eosinophilic Esophagitis.

BACKGROUND: The mechanistic basis of the variable symptomatology seen in eosinophilic esophagitis (EoE) remains poorly understood. OBJECTIVE: We examined the correlation of a validated, patient-reported outcome metric with a broad spectrum of esophageal transcripts to uncover potential symptom pathogenesis. METHODS: We extracted data from 146 adults with EoE through the Consortium of Eosinophilic Gastrointestinal Disease Researchers. Patients were subgrouped by esophageal dilation history. We compared a validated patient-reported outcome metric, the EoE Activity Index (EEsAI), with a set of transcripts expressed in the esophagus of patients with EoE, the EoE Diagnostic Panel (EDP). We used single-cell RNA sequencing data to identify the cellular source of EEsAI-related EDP genes and further analyzed patients with mild and severe symptoms. RESULTS: The EEsAI correlated with the EDP total score, especially in patients without recent esophageal dilation (r = -0.31; P = .003). We identified 14 EDP genes that correlated with EEsAI scores (r ≥ 0.3; P < .05). Of these, 11 were expressed in nonepithelial cells and three in epithelial cells. During histologic remission, only four of 11 nonepithelial genes (36%) versus all three epithelial genes (100%) had decreased expression to less than 50% of that in active EoE. Fibroblasts expressed five of 11 nonepithelial EEsAI-associated EDP genes (45%). A subset of nonepithelial genes (eight of 11; 73%), but not EoE-representative genes (none of four; 0%; CCL26, CAPN14, DSG1, and SPINK7), was upregulated in patients with EoE with the highest versus lowest symptom burden. CONCLUSION: The correlation of symptoms and nonepithelial esophageal gene expression substantiates that nonepithelial cells (eg, fibroblasts) likely contribute to symptom severity.

Of sea, rivers and symbiosis: Diversity, systematics, biogeography and evolution of the deeply diverging florideophycean order Hildenbrandiales (Rhodophyta).

The Hildenbrandiales, a typically saxicolous red algal order, is an early diverging florideophycean group with global significance in marine and freshwater ecosystems across diverse temperature zones. To comprehensively elucidate the diversity, phylogeny, biogeography, and evolution of this order, we conducted a thorough re-examination employing molecular data derived from nearly 700 specimens. Employing a species delimitation method, we identified Evolutionary Species Units (ESUs) within the Hildenbrandiales aiming to enhance our understanding of species diversity and generate the first time-calibrated tree and ancestral area reconstruction for this order. Mitochondrial cox1 and chloroplast rbcL markers were used to infer species boundaries, and subsequent phylogenetic reconstructions involved concatenated sequences of cox1, rbcL, and 18S rDNA. Time calibration of the resulting phylogenetic tree used a fossil record from a Triassic purportedly freshwater Hildenbrandia species and three secondary time points from the literature. Our species delimitation analysis revealed an astounding 97 distinct ESUs, quintupling the known diversity within this order. Our time-calibration analysis placed the origin of Hildenbrandiales (crown age) in the Ediacaran period, with freshwater species emerging as a monophyletic group during the later Permian to early Triassic. Phylogenetic reconstructions identified seven major clades, experiencing early diversification during the Silurian to Carboniferous period. Two major evolutionary events-colonization of freshwater habitats and obligate systemic symbiosis with a marine fungus-marked this order, leading to significant morphological alterations without a commensurate increase in species diversification. Despite the remarkable newly discovered diversity, the extant taxon diversity appears relatively constrained when viewed against an evolutionary timeline spanning over 800 million years. This limitation may stem from restricted geographic sampling or the prevalence of asexual reproduction. However, species richness estimation and rarefaction analyses suggest a substantially larger diversity yet to be uncovered-potentially four times greater. These findings drastically reshape our understanding of the deeply diverging florideophycean order Hildenbrandiales species diversity, and contribute valuable insights into this order's evolutionary history and ecological adaptations. Supported by phylogenetic, ecological and morphological evidence, we established the genus Riverina gen. nov. to accommodate freshwater species of Hildenbrandiales, which form a monophyletic clade in our analyses. This marks the first step toward refining the taxonomy of the Hildenbrandiales, an order demanding thorough revisions, notably with the creation of several genera to address the polyphyletic status of Hildenbrandia. However, the limited diagnostic features pose a challenge, necessitating a fresh approach to defining genera. A potential solution lies in embracing a molecular systematic perspective, which can offer precise delineations of taxonomic boundaries.