Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome.

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5 mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5 mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5 mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5 mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development.

The Current State of Advanced Pediatric Emergency Medicine Point-of-Care Ultrasound (POCUS) Training: Exploring Recent POCUS Fellowship Application Trends and Alternate Training Models.

OBJECTIVES: This study aims to assess the current state of advanced pediatric emergency medicine (PEM) point-of-care ultrasound (POCUS) training in North America, including trends in dedicated PEM POCUS fellowships and alternative advanced POCUS training pathways, to better guide future educational efforts within the field. METHODS: We identified and surveyed 22 PEM POCUS fellowship directors across the United States and Canada regarding PEM POCUS fellowship application trends, potential barriers to pursuing additional POCUS training, and novel training models that meet the needs of the PEM POCUS workforce. RESULTS: The past 5 years have seen a growth in both PEM POCUS fellowship program number and trainee positions available, with a general impression by fellowship directors of a high demand for faculty who have these training credentials. However, there was a discordant drop in fellowship applicants and corresponding match rate in 2022, the cause of which is not clear. A number of programs are offering alternative advanced training options including combined PEM/POCUS fellowships and POCUS tracks within PEM fellowship. CONCLUSION: As POCUS use within PEM evolves, a growing number of advanced training options are being developed. Understanding the motivations and barriers for pursuing advanced POCUS training can help to shape these options going forward, to ensure the experience incorporated within each model meets the needs of trainees, the needs of PEM divisions, and the future needs of our field.

Assessing utility of thyroid in vitro screening assays through comparisons to observed impacts in vivo.

To better understand endocrine disruption, the U.S. Environmental Protection Agency's (USEPA) Endocrine Disruptor Screening Program (EDSP) utilizes a two-tiered approach to investigate the potential of a chemical to interact with the estrogen, androgen, or thyroid systems. As in vivo testing lacks the throughput to address data gaps on endocrine bioactivity for thousands of chemicals, in vitro high-throughput screening (HTS) methods are being developed to screen larger chemical libraries. The primary objective of this work was to investigate for how many of the 52 chemicals with weight-of-evidence (WoE) determinations from EDSP Tier 1 screening there are available in vitro HTS data supporting a thyroid impact. HTS data from the USEPA ToxCast program and the EDSP WoE were collected for this analysis. Considering the complexity of endocrine disruption and interpreting HTS data, concordance between in vitro activity and in vivo effects ranges from 58 to 78%. Based on this evaluation, we conclude that the current suite of HTS assays is beneficial for prioritizing chemicals for further inquiry; however, without a more detailed analysis, one cannot conclude whether HTS results are the primary mode-of-action. Furthermore, development of in vitro assays for additional thyroid-relevant molecular initiating events is required to effectively predict in vivo thyroid impacts.

Bacteroides ovatus Promotes IL-22 Production and Reduces Trinitrobenzene Sulfonic Acid-Driven Colonic Inflammation.

The intestinal microbiota influences the development and function of the mucosal immune system. However, the exact mechanisms by which commensal microbes modulate immunity is not clear. We previously demonstrated that commensal Bacteroides ovatus ATCC 8384 reduces mucosal inflammation. Herein, we aimed to identify immunomodulatory pathways employed by B. ovatus. In germ-free mice, mono-association with B. ovatus shifted the CD11b+/CD11c+ and CD103+/CD11c+ dendritic cell populations. Because indole compounds are known to modulate dendritic cells, B. ovatus cell-free supernatant was screened for tryptophan metabolites by liquid chromatography-tandem mass spectrometry and larger quantities of indole-3-acetic acid were detected. Analysis of cecal and fecal samples from germ-free and B. ovatus mono-associated mice confirmed that B. ovatus could elevate indole-3-acetic acid concentrations in vivo. Indole metabolites have previously been shown to stimulate immune cells to secrete the reparative cytokine IL-22. Addition of B. ovatus cell-free supernatant to immature bone marrow-derived dendritic cells stimulated IL-22 secretion. The ability of IL-22 to drive repair in the intestinal epithelium was confirmed using a physiologically relevant human intestinal enteroid model. Finally, B. ovatus shifted the immune cell populations in trinitrobenzene sulfonic acid-treated mice and up-regulated colonic IL-22 expression, effects that correlated with decreased inflammation. Our data suggest that B. ovatus-produced indole-3-acetic acid promotes IL-22 production by immune cells, yielding beneficial effects on colitis.

The metabolic profile of Bifidobacterium dentium reflects its status as a human gut commensal.

BACKGROUND: Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium. RESULTS: B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment. CONCLUSIONS: Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.

Detecting Ventilator-Induced Diaphragmatic Dysfunction Using Point-of-Care Ultrasound in Children With Long-term Mechanical Ventilation.

Long-term mechanical ventilation (MV) is defined as the use of MV for more than 6 hours per day for at least 3 weeks. Children requiring long-term MV include those with neuromuscular disease, central dysregulation, or lung dysfunction. Such children with medical complexity may be at risk for ventilator-induced diaphragmatic dysfunction. Ventilator-induced diaphragmatic dysfunction has been described in adult patients requiring acute MV with ultrasound (US). At this time, diaphragmatic US has not been evaluated in the pediatric post-acute care setting or incorporated into weaning strategies. We present 24 cases of children requiring long-term MV who underwent diaphragmatic US examinations to evaluate for ventilator-induced diaphragmatic dysfunction.

The Use of Point-of-Care Ultrasound to Evaluate Pulsus Paradoxus in Children With Asthma.

Pulsus paradoxus (PP) is defined as a fall of systolic blood pressure of greater than 10 mm Hg during the inspiratory phase of respiration. Measurement of PP is recommended by national and international asthma guidelines as an objective measure of asthma severity but is rarely used in clinical practice. Cardiac point-of-care ultrasound with pulsed wave Doppler imaging measuring respiratory-phasic changes of mitral valve inflow velocities is well described in cardiac tamponade as "sonographic" PP. We present 10 cases of acute asthma presenting to an emergency department showing the finding of sonographic determined PP in the apical 4-chamber view of the heart on pulsed wave Doppler imaging.

Implementing the chick embryo model to study vestibular developmental disorders.

Children with congenital vestibular disorders show delayed motor development and challenges in maintaining posture and balance. Computed tomography images reveal that these children have abnormal inner ears in the form of a sac, with the semicircular canals missing or truncated. Little is known about how this inner ear abnormality affects central vestibular development. At present, mice with the chromodomain helicase DNA-binding protein 7 mutation are the most common model for studying congenital vestibular disorders, despite forming multiple diverse inner ear phenotypes and inducing abnormal cerebellar and visual system development. To identify the effects of a sac-like inner ear on central vestibular development, we have designed and implemented a new model, the anterior-posterior axis rotated otocyst (ARO) chick, which forms a sac-like inner ear in 85% of cases. The ARO chick is produced by anterior-posterior rotation of the otocyst at embryonic day 2. Here, we describe for the first time the 15% of ARO chicks that form three small semicircular canals and rename the ARO chicks forming sacs (ARO/s chicks). The basic features of the vestibular sensory organs in ARO/s chicks are similar to those found in patients' sacs, and ARO/s hatchlings experience balance and walking problems like patients. Thus, ARO/s chicks have a reproducible inner ear phenotype without abnormalities in vestibular-related structures, making the model a relatively simple one to evaluate the relationship between the sac-like inner ear pathology and formation of the central vestibular neural circuitry. Here, we describe unpublished details on the surgical approaches to produce ARO chicks, including pitfalls and difficulties to avoid.NEW & NOTEWORTHY This paper describes simple techniques for chick otocyst rotation resulting in a sac-like inner ear (85%), the common phenotype in congenital vestibular disorders. We now describe anterior-posterior axis rotated otocyst chicks, which form three small canals (15%), and rename chicks forming a sac (ARO/s chicks). Basic protocols and potential complications of otocyst rotation are described. With the use of ARO/s chicks, it will be possible to determine how the vestibular neural circuit is modified by sac-like inner ear formation.

Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results.

High-throughput screening (HTS) and computational technologies have emerged as important tools for chemical hazard identification. The US Environmental Protection Agency (EPA) launched the Toxicity ForeCaster (ToxCast) Program, which has screened thousands of chemicals in hundreds of mammalian-based HTS assays for biological activity. The data are being used to prioritize toxicity testing on those chemicals likely to lead to adverse effects. To use HTS assays in predicting hazard to both humans and wildlife, it is necessary to understand how broadly these data may be extrapolated across species. The US EPA Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/ ) tool was used to assess conservation of the 484 protein targets represented in the suite of ToxCast assays and other HTS assays. To demonstrate the utility of the SeqAPASS data for guiding extrapolation, case studies were developed which focused on targets of interest to the US Endocrine Disruptor Screening Program and the Organisation for Economic Cooperation and Development. These case studies provide a line of evidence for conservation of endocrine targets across vertebrate species, with few exceptions, and demonstrate the utility of SeqAPASS for defining the taxonomic domain of applicability for HTS results and identifying organisms for suitable follow-up toxicity tests.

Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis.

Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis' sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis' sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology.

Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4-tert-octylphenol or 17ß-trenbolone exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid-mediated amphibian metamorphosis and reproductive development. During the validation phase, two well-characterized endocrine-disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4-tert-octylphenol (tOP) and xenoandrogen 17ß-trenbolone (TB). Xenopus laevis embryos were exposed, in flow-through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l-1 ) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l-1 ) until 8 weeks post-metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment-related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l-1 ) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose-dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l-1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine-active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

Cognitive enhancing treatment with a PPARγ agonist normalizes dentate granule cell presynaptic function in Tg2576 APP mice.

Hippocampal network hyperexcitability is considered an early indicator of Alzheimer's disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I-O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I-O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues.

African clawed frog Xenopus sp. is used extensively for developmental biology and toxicology research. Amid concerns of environmental pollutants disrupting endocrine systems and causing altered reproductive development in wildlife, eco-toxicology research has led to a focus on linking molecular initiating events to population-level effects. As such, efforts to better understand reproductive development at the molecular level in these model species are warranted. To that end, transcriptomes were characterized in differentiating Xenopus tropicalis gonad tissues at Nieuwkoop and Faber (NF) stage 58 (pro-metamorphosis), NF66 (completion of metamorphosis), 1week post-metamorphosis (1WPM), and 2weeks post-metamorphosis (2WPM). Differential expression analysis between tissue types at each developmental stage revealed a substantial divergence of ovary and testis transcriptomes starting between NF58 and NF66; transcriptomes continued to diverge through 2WPM. Generally, testis-enriched transcripts were expressed at relatively constant levels, while ovary-enriched transcripts were up-regulated within this developmental period. Functional analyses of differentially expressed transcripts allowed linkages to be made between their putative human orthologues and specific cellular processes associated with differentiating gonad tissues. In ovary tissue, genetic programs direct germ cells through meiosis to the diplotene stage when maternal mRNAs are transcribed and trafficked to oocytes for translation following fertilization. In the testis, gene expression is consistent with connective tissue development, tubule formation, and germ cell support (Leydig and Sertoli cells). This dataset exhibited remarkable consistency with transcript profiles previously described in gonad tissues across species, and emphasizes the universal importance of certain transcripts for germ cell development and preparation of these tissues for reproduction.

Biomarker discovery for early detection of hepatocellular carcinoma in hepatitis C-infected patients.

Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of (18)O/(16)O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using (18)O/(16)O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.

Pediatric Emergency Medicine Ultrasound Fellowship Programs.

Point of care ultrasound (POCUS) has undergone important growth in the field of Pediatric Emergency Medicine (PEM) in the last 14 years and is recognized as a critical diagnostic tool in the care of ill and injured children. The first PEM POCUS fellowship was established in 2010. Now, there are currently 30 ultrasound fellowships that offer training to PEM physicians. In 2014, 46 PEM POCUS leaders established the P2 (PEM POCUS) Network (www.P2network.org). This serves as a platform for sharing expertise, building research collaborations, and offering mentorship in the use of POCUS in PEM. In 2019, a multinational group of experts in PEM POCUS published the first consensus guidelines for prioritizing core applications of POCUS, which are fundamental to PEM fellowship training 1. In 2022, the international research priorities for PEM POCUS were published 2. In the same year, the development of a consensus-based definition of focused assessment with sonography for trauma (FAST) in children was established 3.