Autism-related dietary preferences mediate autism-gut microbiome associations.

There is increasing interest in the potential contribution of the gut microbiome to autism spectrum disorder (ASD). However, previous studies have been underpowered and have not been designed to address potential confounding factors in a comprehensive way. We performed a large autism stool metagenomics study (n = 247) based on participants from the Australian Autism Biobank and the Queensland Twin Adolescent Brain project. We found negligible direct associations between ASD diagnosis and the gut microbiome. Instead, our data support a model whereby ASD-related restricted interests are associated with less-diverse diet, and in turn reduced microbial taxonomic diversity and looser stool consistency. In contrast to ASD diagnosis, our dataset was well powered to detect microbiome associations with traits such as age, dietary intake, and stool consistency. Overall, microbiome differences in ASD may reflect dietary preferences that relate to diagnostic features, and we caution against claims that the microbiome has a driving role in ASD.

The adaptor TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating signaling via the receptor for IL-2.

The number of Foxp3+ regulatory T cells (Treg cells) must be tightly controlled for efficient suppression of autoimmunity with no impairment of normal immune responses. Here we found that the adaptor TRAF3 was intrinsically required for restraining the lineage determination of thymic Treg cells. T cell-specific deficiency in TRAF3 resulted in a two- to threefold greater frequency of Treg cells, due to the more efficient transition of precursors of Treg cells into Foxp3+ Treg cells. TRAF3 dampened interleukin 2 (IL-2) signaling by facilitating recruitment of the tyrosine phosphatase TCPTP to the IL-2 receptor complex, which resulted in dephosphorylation of the signaling molecules Jak1 and Jak3 and negative regulation of signaling via Jak and the transcription factor STAT5. Our results identify a role for TRAF3 as an important negative regulator of signaling via the IL-2 receptor that affects the development of Treg cells.

Pairwise biosynthesis of ion channels stabilizes excitability and mitigates arrhythmias.

Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.

TRAF3 regulates STAT6 activation and T-helper cell differentiation by modulating the phosphatase PTP1B.

The adaptor protein tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifaceted regulator of lymphocyte biology that plays key roles in modulation of the molecular signals required for T-cell activation and function. TRAF3 regulates signals mediated by the T-cell receptor (TCR), costimulatory molecules, and cytokine receptors, which each drive activation of the serine/threonine kinase Akt. The impact of TRAF3 upon TCR-CD28-mediated activation of Akt, and thus on the diverse cellular processes regulated by Akt, including CD4 T-cell fate decisions, remains poorly understood. We show here that TRAF3 deficiency led to impaired Akt activation and thus to impaired in vitro skewing of CD4 T cells into the TH1 and TH2 fates. We investigated the role of TRAF3 in regulation of signaling pathways that drive TH1 and TH2 differentiation and found that TRAF3 enhanced activation of signal transducer and activator of transcription 6 (STAT6), thus promoting skewing toward the TH2 fate. TRAF3 promoted STAT6 activation by regulating recruitment of the inhibitory molecule protein tyrosine phosphatase 1B to the IL-4R signaling complex, in a manner that required integration of TCR-CD28- and IL-4R-mediated signals. This work reveals a new mechanism for TRAF3-mediated regulation of STAT6 activation in CD4 T cells and adds to our understanding of the diverse roles played by TRAF3 as an important regulator of T-cell biology.

Sepsis leads to lasting changes in phenotype and function of naïve CD8 T cells.

Sepsis, an amplified immune response to systemic infection, is characterized by a transient cytokine storm followed by chronic immune dysfunction. Consequently, sepsis survivors are highly susceptible to newly introduced infections, suggesting sepsis can influence the function and composition of the naïve CD8 T cell pool and resulting pathogen-induced primary CD8 T cell responses. Here, we explored the extent to which sepsis induces phenotypic and functional changes within the naïve CD8 T cell pool. To interrogate this, the cecal ligation and puncture (CLP) mouse model of polymicrobial sepsis was used. In normal, non-septic mice, we show type-I interferon (IFN I)-mediated signaling plays an important role in driving the phenotypic and functional heterogeneity in the naïve CD8 T cell compartment leading to increased representation of Ly6C+ naïve CD8 T cells. In response to viral infection after sepsis resolution, naïve Ly6C+ CD8 T cells generated more primary effector and memory CD8 T cells with slower conversion to a central memory CD8 T cell phenotype (Tcm) than Ly6C- naïve CD8 T cells. Importantly, as a potent inducer of cytokine storm and IFN I production, sepsis leads to increased representation of Ly6C+ naïve CD8 T cells that maintained their heightened ability to respond (i.e., effector and memory CD8 T cell accumulation and cytokine production) to primary LCMV infection. Lastly, longitudinal analyses of peripheral blood samples obtained from septic patients revealed profound changes in CD8 T cell subset composition and frequency compared to healthy controls. Thus, sepsis has the capacity to alter the composition of naïve CD8 T cells, directly influencing primary CD8 T cell responses to newly introduced infections.

CDC Laboratory Recommendations for Syphilis Testing, United States, 2024.

This report provides new CDC recommendations for tests that can support a diagnosis of syphilis, including serologic testing and methods for the identification of the causative agent Treponema pallidum. These comprehensive recommendations are the first published by CDC on laboratory testing for syphilis, which has traditionally been based on serologic algorithms to detect a humoral immune response to T. pallidum. These tests can be divided into nontreponemal and treponemal tests depending on whether they detect antibodies that are broadly reactive to lipoidal antigens shared by both host and T. pallidum or antibodies specific to T. pallidum, respectively. Both types of tests must be used in conjunction to help distinguish between an untreated infection or a past infection that has been successfully treated. Newer serologic tests allow for laboratory automation but must be used in an algorithm, which also can involve older manual serologic tests. Direct detection of T. pallidum continues to evolve from microscopic examination of material from lesions for visualization of T. pallidum to molecular detection of the organism. Limited point-of-care tests for syphilis are available in the United States; increased availability of point-of-care tests that are sensitive and specific could facilitate expansion of screening programs and reduce the time from test result to treatment. These recommendations are intended for use by clinical laboratory directors, laboratory staff, clinicians, and disease control personnel who must choose among the multiple available testing methods, establish standard operating procedures for collecting and processing specimens, interpret test results for laboratory reporting, and counsel and treat patients. Future revisions to these recommendations will be based on new research or technologic advancements for syphilis clinical laboratory science.

Direct Bottom-Up In Situ Growth: A Paradigm Shift for Studies in Wet-Chemical Synthesis of Gold Nanoparticles.

Plasmonic gold nanoparticles have been used increasingly in solid-state systems because of their applicability in fabricating novel sensors, heterogeneous catalysts, metamaterials, and thermoplasmonic substrates. While bottom-up colloidal syntheses take advantage of the chemical environment to control size, shape, composition, surface chemistry, and crystallography of the nanostructures precisely, it can be challenging to assemble nanoparticles rationally from suspension onto solid supports or within devices. In this Review, we discuss a powerful recent synthetic methodology, bottom-up in situ substrate growth, which circumvents time-consuming batch presynthesis, ligand exchange, and self-assembly steps by applying wet-chemical synthesis to form morphologically controlled nanostructures on supporting materials. First, we briefly introduce the properties of plasmonic nanostructures. Then we comprehensively summarize recent work that adds to the synthetic understanding of in situ geometrical and spatial control (patterning). Next, we briefly discuss applications of plasmonic hybrid materials prepared by in situ growth. Overall, despite the vast potential advantages of in situ growth, the mechanistic understanding of these methodologies remains far from established, providing opportunities and challenges for future research.

SERS in 3D cell models: a powerful tool in cancer research.

Unraveling the cellular and molecular mechanisms underlying tumoral processes is fundamental for the diagnosis and treatment of cancer. In this regard, three-dimensional (3D) cancer cell models more realistically mimic tumors compared to conventional 2D cell cultures and are more attractive for performing such studies. Nonetheless, the analysis of such architectures is challenging because most available techniques are destructive, resulting in the loss of biochemical information. On the contrary, surface-enhanced Raman spectroscopy (SERS) is a non-invasive analytical tool that can record the structural fingerprint of molecules present in complex biological environments. The implementation of SERS in 3D cancer models can be leveraged to track therapeutics, the production of cancer-related metabolites, different signaling and communication pathways, and to image the different cellular components and structural features. In this review, we highlight recent progress in the use of SERS for the evaluation of cancer diagnosis and therapy in 3D tumoral models. We outline strategies for the delivery and design of SERS tags and shed light on the possibilities this technique offers for studying different cellular processes, through either biosensing or bioimaging modalities. Finally, we address current challenges and future directions, such as overcoming the limitations of SERS and the need for the development of user-friendly and robust data analysis methods. Continued development of SERS 3D bioimaging and biosensing systems, techniques, and analytical strategies, can provide significant contributions for early disease detection, novel cancer therapies, and the realization of patient-tailored medicine.

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation.

Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

Severe CNS involvement in a subset of long-term treated children with infantile-onset Pompe disease.

INTRODUCTION: The standard of care for patients with infantile-onset Pompe disease (IOPD) is enzyme replacement therapy (ERT), which does not cross the blood brain barrier. While neuromuscular manifestations of IOPD are well-described, central nervous system (CNS) manifestations of this disorder are far less characterized. Here we describe severe CNS-related neurological manifestations including seizures and encephalopathy in six individuals with IOPD. METHOD: We identified six children with IOPD who developed CNS manifestations such as seizures and/or encephalopathy. We studied their brain magnetic resonance imaging scans (MRIs) and graded the severity of white matter hyperintensities (WMHI) using the Fazekas scale scoring system as previously published. Longitudinal cognitive measures were available from 4/6 children. RESULTS: All six IOPD patients (4 males/2 females) had been treated with ERT for 12-15 years. Seizures and/or encephalopathy were noted at a median age at onset of 11.9 years (range 9-15 years). All were noted to have extensive WMHI in the brain MRIs and very high Fazekas scores which preceded the onset of neurological symptoms. Longitudinal IQ scores from four of these children suggested developmental plateauing. DISCUSSION: Among a subset of IOPD patients on long-term ERT, CNS manifestations including hyperreflexia, encephalopathy and seizures may become prominent, and there is likely an association between these symptoms and significant WMHI on MRI. Further study is needed to identify risk factors for CNS deterioration among children with IOPD and develop interventions to prevent neurological decline.

Clinical and inflammatory features of traffic-related diesel exposure in children with asthma.

BACKGROUND: Epidemiologic studies have revealed associations between traffic-related pollutants such as diesel particulate matter (PM) and asthma outcomes in children, but the inflammatory features associated with diesel PM exposure in children with asthma are not understood. OBJECTIVE: To evaluate symptoms, exacerbations, and lung function measures in children with uncontrolled asthma and their associations with residential proximity to major roadways and to determine associations between diesel PM exposure and systemic inflammatory cytokines, circulating markers of T-cell activation and exhaustion, and metabolomic features using biomarker studies. METHODS: Children 5 to 17 years of age with physician-diagnosed, uncontrolled asthma despite treatment with an asthma controller medication completed a research visit involving questionnaires, lung function testing, and venipuncture for biomarker studies. Geocoding was performed to quantify residential proximity to major roadways and pollutant exposure. RESULTS: A total of 447 children with uncontrolled asthma were enrolled. Children living closer to highly trafficked roadways were more disadvantaged and had more exposure to diesel PM, more exacerbations prompting an emergency department visit, and lower lung function measures. Children with the highest diesel PM exposure, compared with children with the lowest diesel PM exposure, also had blunted cytokine secretion and evidence of T-cell exhaustion, including disturbances in several metabolites associated with glutathione formation and oxidative stress. CONCLUSION: Traffic-related diesel PM exposure in children with poorly controlled asthma is associated with poorer clinical outcomes and unique patterns of inflammation and oxidative stress. These findings argue for continued mitigation efforts to improve traffic-related air quality and health equity in children with asthma.

Ethical Principles Do Not Support Mandatory Preanesthesia Pregnancy Screening Tests: A Narrative Review.

Respect for patient autonomy is a pillar of medical ethics, manifested predominantly through informed consent. Mandatory (routine) nonconsented preoperative urine pregnancy testing does not adequately respect patient autonomy, is potentially coercive, and has the potential to cause harm medically, psychologically, socially, and financially. Inaccuracies in pregnancy testing can result in false-positive and false-negative results, especially in early pregnancy. There is substantial scientific evidence that anesthesia is not harmful to the fetus, raising the question of whether pregnancy testing provides substantial benefit to the patient. Not performing a preanesthesia pregnancy test has not been associated with significant medicolegal consequences. We review the ethical implications of mandatory preanesthesia pregnancy testing in light of these facts.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating.

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Unreliable Continuous Treatment Indicators in Propensity Score Analysis.

Propensity score analyses (PSA) of continuous treatments often operationalize the treatment as a multi-indicator composite, and its composite reliability is unreported. Latent variables or factor scores accounting for this unreliability are seldom used as alternatives to composites. This study examines the effects of the unreliability of indicators of a latent treatment in PSA using the generalized propensity score (GPS). A Monte Carlo simulation study was conducted varying composite reliability, continuous treatment representation, variability of factor loadings, sample size, and number of treatment indicators to assess whether Average Treatment Effect (ATE) estimates differed in their relative bias, Root Mean Squared Error, and coverage rates. Results indicate that low composite reliability leads to underestimation of the ATE of latent continuous treatments, while the number of treatment indicators and variability of factor loadings show little effect on ATE estimates, after controlling for overall composite reliability. The results also show that, in correctly specified GPS models, the effects of low composite reliability can be somewhat ameliorated by using factor scores that were estimated including covariates. An illustrative example is provided using survey data to estimate the effect of teacher adoption of a workbook related to a virtual learning environment in the classroom.

The Postbiotic Butyrate Mitigates Gut Mucosal Disruption Caused by Acute Ethanol Exposure.

We aimed to test how the postbiotic butyrate impacts select gut bacteria, small intestinal epithelial integrity, and microvascular endothelial activation during acute ethanol exposure in mice and primary human intestinal microvascular endothelial cells (HIMECs). Supplementation during an acute ethanol challenge with or without tributyrin, a butyrate prodrug, was delivered to C57BL/6 mice. A separate group of mice received 3 days of clindamycin prior to the acute ethanol challenge. Upon euthanasia, blood endotoxin, cecal bacteria, jejunal barrier integrity, and small intestinal lamina propria dendritic cells were assessed. HIMECs were tested for activation following exposure to ethanol ± lipopolysaccharide (LPS) and sodium butyrate. Tributyrin supplementation protected a butyrate-generating microbe during ethanol and antibiotic exposure. Tributyrin rescued ethanol-induced disruption in jejunal epithelial barrier, elevated plasma endotoxin, and increased mucosal vascular addressin cell-adhesion molecule-1 (MAdCAM-1) expression in intestinal microvascular endothelium. These protective effects of tributyrin coincided with a tolerogenic dendritic response in the intestinal lamina propria. Lastly, sodium butyrate pre- and co-treatment attenuated the direct effects of ethanol and LPS on MAdCAM-1 induction in the HIMECs from a patient with ulcerative colitis. Tributyrin supplementation protects small intestinal epithelial and microvascular barrier integrity and modulates microvascular endothelial activation and dendritic tolerizing function during a state of gut dysbiosis and acute ethanol challenge.

Teaching Acid-Base Fundamentals and Introducing pH using Butterfly Pea Flower Tea.

Stimulating interest in science at an early age is important for STEM education. This work details an educational activity utilizing the anthocyanins found in the butterfly pea flower (Clitoria ternatea). This activity was developed for use in official classroom settings, online, and/or at-home with parental or educator guidance. Primary and high school students aged 7 to 14 performed a straightforward extraction of anthocyanin pH indicators from Clitoria ternatea with hot water. Students were able to use this indicator and its vast range of colors to compare the acidity and basicity of different household solutions. Most responses recorded show that students used reasoning from the indicator and a subsequent chemical reaction to correctly differentiate acids from bases and compare their strengths. Overall, this activity's application of non-toxic and easily accessible indicators from the butterfly pea flower assisted in introducing young students to various concepts in acid-base chemistry, including acid/base strength and pH, solute dissolution, neutralization reactions, and qualitative analysis.

Mechanisms of Change in Nature-Based Pediatric Occupational Therapy: The Ecology of Change in Outdoor Therapy Model.

The purpose of this qualitative constructivist grounded theory study was to develop a model to explain potential mechanisms of change in the nature-based pediatric occupational therapy process, based on analysis of the perspectives of occupational therapy practitioners currently engaging in nature-based practice with children. Using 27 photos and interview data from 22 participants the Ecology of Change in Outdoor Therapy (ECO-Therapy) Model was developed. The model suggests mechanisms of change in nature-based pediatric occupational therapy may involve six iterative phases: Longing for Freedom, Embarking on Adventure, Dancing with Nature, Claiming Self-Agency, Braving Real-Life Challenges, and Growing Adaptive Capacity. The practitioner and child experiencing these phases throughout the nature-based occupational therapy process may lead to improved participation and performance in the child's daily life.

Functional re-organization of hippocampal-cortical gradients during naturalistic memory processes.

The functional organization of the hippocampus mirrors that of the cortex, changing smoothly along connectivity gradients and abruptly at inter-areal boundaries. Hippocampal-dependent cognitive processes require flexible integration of these hippocampal gradients into functionally related cortical networks. To understand the cognitive relevance of this functional embedding, we acquired fMRI data while participants viewed brief news clips, either containing or lacking recently familiarized cues. Participants were 188 healthy mid-life adults and 31 adults with mild cognitive impairment (MCI) or Alzheimer's disease (AD). We employed a recently developed technique - connectivity gradientography - to study gradually changing patterns of voxel to whole brain functional connectivity and their sudden transitions. We observed that functional connectivity gradients of the anterior hippocampus map onto connectivity gradients across the default mode network during these naturalistic stimuli. The presence of familiar cues in the news clips accentuates a stepwise transition across the boundary from the anterior to the posterior hippocampus. This functional transition is shifted in the posterior direction in the left hippocampus of individuals with MCI or AD. These findings shed new light on the functional integration of hippocampal connectivity gradients into large-scale cortical networks, how these adapt with memory context and how these change in the presence of neurodegenerative disease.