An immuno-northern technique to measure the size of dsRNA byproducts in in vitro transcribed RNA.

Double-stranded RNA is an immunogenic byproduct present in RNA synthesized with in vitro transcription. dsRNA byproducts engage virus-sensing innate immunity receptors and cause inflammation. Removing dsRNA from in vitro transcribed messenger RNA (mRNA) reduces immunogenicity and improves protein translation. Levels of dsRNA are typically 0.1%-0.5% of total transcribed RNA. Because they form such a minor fraction of the total RNA in transcription reactions, it is difficult to confidently identify discrete bands on agarose gels that correspond to the dsRNA byproducts. Thus, the sizes of dsRNA byproducts are largely unknown. Total levels of dsRNA are typically assayed with dsRNA-specific antibodies in ELISA and immuno dot-blot assays. Here we report a dsRNA-specific immuno-northern blot technique that provides a clear picture of the dsRNA size distributions in transcribed RNA. This technique could complement existing dsRNA analytical methods in studies of dsRNA byproduct synthesis, dsRNA removal, and characterization of therapeutic RNA drug substances.

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

Reproductive and whole-body toxicity of Ag-doped and -undoped ZIF-8 nanoparticles and the building blocks: An Artemia-based comparative bioassay.

The present research assessed, for the first time, toxicity of ZIF-8 (1 mg/L) and the building blocks (0.1 mg/L Zn2+ and 0.4 mg/L 2-methylimidazole (2-MIm)), besides that of AgNPs@ZIF-8 (0.25, 0.5, and 1 mg/L) and AgNO3 (0.1 mg/L) to aquatic organisms. Two consecutive generations (F0 & F1) of Artemia salina were exposed to these chemicals. All of the chemical treatments considerably caused mortality in F0, especially AgNPs@ZIF-8 and AgNO3, whereas F1 displayed notable tolerance and survived comparable to the control group, except in the case of AgNO3 treatment. Similarly, growth indices (weight, mainly in ZIF-8, Zn2+, and 2-MIm; length, in Ag-doped ZIF-8 and AgNO3) were significantly retarded in F0 and especially F1 of all treatments, and 2-MIm caused the greatest length retardation in F0. AgNPs@ZIF-8 (0.5 and 1 mg/L), 2-MIm, and AgNO3 postponed the ovary emergence in about 40%-60% of the exposed F0, and ZIF-8 delayed this phenomenon in some individuals of F0 and F1 up to 6 days. This temporal disturbance was also observed in time to first brood of almost all experimental F0 and F1 groups, with being over 80% of F1 exposed to ZIF-8, 2-MIm, and Zn2+, as well as about 50% of F0 treated with 2-MIm, and Zn2+. The highest neonate number was recorded for F0 and F1 exposed to AgNO3 and Zn2+, while ZIF-8 and, importantly, 2-MIm decreased the reproductivity to the lowest levels in both generations. Generally, the reproductive frequency was significantly decreased in all F0 and F1 treatments, especially 2-MIm, ZIF-8, AgNPs@ZIF-8 (0.25 & 1 mg/L). This study highlighted the neglected importance of 2-MIm in assessing overall toxicity of ZIF-8, and even other organic ligands of MOFs, and also filled a gap in the literature by investigating the potential effect of additives such as AgNPs on the toxicity of ZIF-8 and other MOFs.

Zinc oxide nanoparticles disrupt development and function of the olfactory sensory system impairing olfaction-mediated behaviour in zebrafish.

Zinc (Zn) is an essential metal present in numerous enzymes throughout the body, playing a vital role in animal and human health. However, the increasing use of zinc oxide nanomaterials (ZnONPs) in a diverse range of products has raised concerns regarding their potential impacts on health and the environment. Despite these concerns, the toxicity of ZnONP exposure on animal health remain poorly understood. To help address this knowledge gap, we have developed a highly sensitive oxidative stress (OS) biosensor zebrafish capable of detecting cell/tissue-specific OS responses to low doses of various oxidative stressors, including Zn, in a live fish embryo. Using live-imaging analysis with this biosensor zebrafish embryo, we discovered that the olfactory sensory neurons in the brain are especially sensitive to ZnOP exposure. Furthermore, through studies monitoring neutrophil migration and neuronal activation in the embryonic brain and via behaviour analysis, we have found that sub-lethal doses of ZnONPs (ranging from 0.033 to 1 mg/L nominal concentrations), which had no visible effect on embryo growth or morphology, cause significant localised inflammation, disrupting the neurophysiology of olfactory brain tissues and ultimately impaired olfaction-mediated behaviour. Collectively, these findings establish a potent and important effect mechanism for ZnONP toxicity, indicating the olfactory sensory system as the primary target for ZnONPs as an environmental toxicant in aquatic environments. Our result also highlights that even low doses of ZnONPs can have detrimental effects on the olfactory sensory system, surpassing previous expectations. The importance of olfaction in environment sensing, sex behaviours and overall fitness across species raises concerns about the potential impact of ZnONPs on olfaction-mediated brain function and behaviour in animals and humans. Our study emphasises the need for greater consideration of the potential risks associated with these nanomaterials.

Translocation of 14C-polystyrene nanoplastics into fish during a very-low concentration dietary exposure.

Assessing the dietary accumulation of nanoplastics in animals following very-low exposure concentrations is restricted due to analytical limitations. This study adapted a method for synthesising semi-stable 14C-PS NPs (through styrene polymerisation) in small volumes for deployment in environmental studies. The method was developed with non-labelled material where the final polystyrene product had a primary particle size of 35 ± 8 nm (as measured by transmission electron microscopy). This method was then applied to 14C-labelled styrene to produce radiolabelled polystyrene nanoplastics (14C-PS NPs). The 14C-PS NPs were added (top-dressed) to a commercially available fish feed, with a measured concentration of 27.9 ± 2.1 kBq kg-1 (n = 5), equating to 5.9 µg polystyrene kg-1 feed. Fish (rainbow trout; Oncorhynchus mykiss) were fed this diet at a ration of 2% body weight per day for a period of two weeks. On day 3, 7 and 14, the fish were sampled for the mid intestine, hind intestine, kidney and liver, and measured for tissue radioactivity (determined by liquid scintillation counting). Some background activity was detected in the control samples (e.g., 1-16 and 4-11 Bq g-1 in the hind intestine and liver, respectively) which is due to natural background fluorescence. By the end of the experiment, the hind intestine and liver had significantly elevated radioactivity (25.3 and 15.0 Bq g-1, respectively) compared to the control, indicating the accumulation of nano polystyrene. In the liver, this equated to 1.8 µg polystyrene g-1 dry weight. This study confirms the accumulation of nano particles in vertebrates at low, environmentally relevant concentration, and highlights radiolabelling as a methodological approach suitable for exploring the bioaccumulation of nanoplastics and potential impacts.

Toward Understanding the Environmental Risks of Combined Microplastics/Nanomaterials Exposures: Unveiling ZnO Transformations after Adsorption onto Polystyrene Microplastics in Environmental Solutions.

Over recent decades, there has been a dramatic increase in the manufacture of engineered nanomaterials, which has inevitably led to their environmental release. Zinc oxide (ZnO) is among the more abundant nanomaterial manufactured due to its advantageous properties, used for piezoelectric, semiconducting, and antibacterial purposes. Plastic waste is ubiquitous and may break down or delaminate into smaller microplastics, leaving open the question of whether these small polymers may alter the fate of ZnO through adsorption within aquatic media (tap-water and seawater). Here, scanning electron microscopy analysis confirms the effective Zn nano/microstructures adsorption onto polystyrene surfaces after only 24-h incubation in the aquatic media. After pre-aging the nanomaterials for 7-days in different environmental media, nanoprobe X-ray absorption near-edge spectroscopy analysis reveals significant ZnO transformation toward Zn-sulfide and Zn-phosphate. The interaction between a commercial ZnO-based sunscreen with polystyrene and a cleanser consumer containing microbeads with ZnO nanomaterials is also studied, revealing the adsorption of transformed Zn-species in the microplastics surfaces, highlighting the environmental relevancy of this work. Understanding the structural and functional impacts of the microplastics/ZnO complexes, and how they evolve, will provide insights into their chemical nature, stability, transformations, and fate, which is key to predicting their bioreactivity in the environment.

The debranching enzyme Dbr1 regulates lariat turnover and intron splicing.

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of the first viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectroscopy. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

Toxicokinetics and bioaccumulation of silver sulfide nanoparticles in benthic invertebrates in an indoor stream mesocosm.

Mesocosms allow the simulation of environmentally relevant conditions and can be used to establish more realistic scenarios of organism exposure to nanoparticles. An indoor mesocosm experiment simulating an aquatic stream ecosystem was conducted to assess the toxicokinetics and bioaccumulation of silver sulfide nanoparticles (Ag2S NPs) and AgNO3 in the freshwater invertebrates Girardia tigrina, Physa acuta and Chironomus riparius, and determine if previous single-species tests can predict bioaccumulation in the mesocosm. Water was daily spiked at 10 µg Ag L-1. Ag concentrations in water and sediment reached values of 13.4 µg Ag L-1 and 0.30 µg Ag g-1 in the Ag2S NP exposure, and 12.8 µg Ag L-1 and 0.20 µg Ag g-1 in the AgNO3. Silver was bioaccumulated by the species from both treatments, but with approximately 1.5, 3 and 11 times higher body Ag concentrations in AgNO3 compared to Ag2S NP exposures in snails, chironomids and planarians, respectively. In the Ag2S NP exposures, the observed uptake was probably of the particulate form. This demonstrates that this more environmentally relevant Ag nanoform may be bioavailable for uptake by benthic organisms. Interspecies interactions likely occurred, namely predation (planarians fed on chironomids and snails), which somehow influenced Ag uptake/bioaccumulation, possibly by altering organisms´ foraging behaviour. Higher Ag uptake rate constants were determined for AgNO3 (0.64, 80.4 and 1.12 Lwater g-1organism day-1) than for Ag2S NPs (0.05, 2.65 and 0.32 Lwater g-1organism day-1) for planarians, snails and chironomids, respectively. Biomagnification under environmentally realistic exposure seemed to be low, although it was likely to occur in the food chain P. acuta to G. tigrina exposed to AgNO3. Single-species tests generally could not reliably predict Ag bioaccumulation in the more complex mesocosm scenario. This study provides methodologies/data to better understand exposure, toxicokinetics and bioaccumulation of Ag in complex systems, reinforcing the need to use mesocosm studies to improve the risk assessment of environmental contaminants, specifically NPs, in aquatic environments.

Uptake, distribution and elimination of palladium-doped polystyrene nanoplastics in rainbow trout (Oncorhynchus mykiss) following dietary exposure.

The ingestion of nanoplastics (NPs) by fish has led to concerns regarding fish health and food chain transfer, but analytical constraints have hindered quantitative data collection on their uptake and depuration. We used palladium-doped polystyrene nanoplastics (PS-Pd NPs, ~200 nm) to track particle fate in rainbow trout (Oncorhynchus mykiss) during a week-long dietary exposure and subsequent 7-day depuration period on a control diet (no added PS-Pd NPs). At Day 3 and 7 of the exposure, and after depuration, the mid intestine, hind intestine, liver, gallbladder, kidney, gill and carcass were sampled. All organs and the carcass were analysed for total Pd content by inductively couple plasma mass spectrometry. After 3 days of exposure, the mid (32.5 ± 8.3 ng g-1) and hind (42.3 ± 8.2 ng g-1) intestine had significantly higher total Pd concentrations compared to the liver and carcass (1.3 ± 0.4 and 3.4 ± 1.1 ng g-1, respectively). At Day 7, there was no time-related difference in any organ (or the carcass) total Pd concentrations compared to Day 3. When the total Pd content was expressed as a body distribution based on mass of tissue, the carcass contained the highest fraction with 72.5 ± 5.2 % at Day 7, which could raise concerns over transfer to higher trophic levels. The total number of particles that entered the fish over the 7 days was 94.5 ± 13.5 × 106 particles, representing 0.07 ± 0.01 % of the Pd the fish had been fed. Following depuration, there was no detectable Pd in any organ or the carcass, indicating clearance from the fish. These data indicate that these NPs are taken into the internal organs and carcass of fish, yet removal of the exposure results in substantial excretion to below the limit of detection.

Plastic pollution: the science we need for the planet we want.

Plastics are incredibly versatile materials that can bring diverse societal and environmental benefit, yet current practices of production, use and disposal have negative effects on wildlife, the environment and human health leading to growing concern across public, policy makers and industry. This Special Issue in Emerging Topics in Life Sciences describes recent advances in our understanding of the consequences of plastic pollution. In particular, it examines their potential to act as vectors for chemicals and pathogens in the environment; evaluates the effects of plastic pollution on biogeochemical cycling, ecosystem functioning and highlights the potential for enhanced effects in environments that are already subject to substantive changes in their climate. The impacts plastics pose to terrestrial ecosystems including soil communities are described and evaluated, along with evidence of potential issues for human health. With an increase in the production of plastics labelled as 'biodegradable' their context and ecological impacts are reviewed. Finally, we discuss the need to take an integrative, system approach when developing and evaluating solutions to plastic pollution, to achieve the ambitious yet necessary aims of the UN Plastics Treaty.

Plastic pollution requires an integrative systems approach to understand and mitigate risk.

To date, much effort has been placed on quantifying plastic pollution and understanding its negative environmental effects, arguably to the detriment of research and evaluation of potential interventions. This has led to piecemeal progress in interventions to reduce plastic pollution, which do not correspond to the pace of emissions. For substances that are used on a global scale and identified as hazardous, there is a need to act before irreversible damage is done. For example, the history of dichlorodiphenyltrichloethane's (DDT) use has demonstrated that legacy chemicals with properties of persistence can still be found in the environment despite being first prohibited 50 years ago. Despite the growing evidence of harm, evidence to inform actions to abate plastic pollution lag behind. In part, this is because of the multifaceted nature of plastic pollution and understanding the connections between social, economic and environmental dimensions are complex. As such we highlight the utility of integrative systems approaches for addressing such complex issues, which unites a diversity of stakeholders (including policy, industry, academia and society), and provides a framework to identify to develop specific, measurable and time-bound international policies on plastic pollution and meet the ambitious yet necessary goals of the UN Plastic Treaty.

Crystal Structure of the RNA Lariat Debranching Enzyme Dbr1 with Hydrolyzed Phosphorothioate RNA Product.

The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2'-5' phosphodiester bond in RNA lariats produced by the spliceosome. Here, we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) that contain a 2'-5'-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to cocrystallize a phosphorothioate-branched RNA (PS-bRNA) with wild-type Entamoeba histolytica Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond. The crystal structure revealed EhDbr1 in a product-bound state, with the hydrolyzed 2'-5' fragment of the PS-bRNA mimicking the binding mode of the native bRNA substrate. These findings suggest that product inhibition may contribute to the kinetic mechanism of Dbr1. We show that Dbr1 enzymes cleave phosphorothioate linkages at rates ∼10,000-fold more slowly than native phosphate linkages. This new product-bound crystal structure offers atomic details, which can aid inhibitor design. Dbr1 inhibitors could be therapeutic or investigative compounds for human diseases such as human immunodeficiency virus (HIV), amyotrophic lateral sclerosis (ALS), cancer, and viral encephalitis.

Metal transfer to sediments, invertebrates and fish following waterborne exposure to silver nitrate or silver sulfide nanoparticles in an indoor stream mesocosm.

The fate of engineered nanomaterials in ecosystems is unclear. An aquatic stream mesocosm explored the fate and bioaccumulation of silver sulfide nanoparticles (Ag2S NPs) compared to silver nitrate (AgNO3). The aims were to determine the total Ag in water, sediment and biota, and to evaluate the bioavailable fractions of silver in the sediment using a serial extraction method. The total Ag in the water column from a nominal daily dose of 10 µg L-1 of Ag for the AgNO3 or Ag2S NP treatments reached a plateau of around 13 and 12 µg L-1, respectively, by the end of the study. Similarly, the sediment of both Ag-treatments reached ~380 µg Ag kg-1, and with most of it being acid-extractable/labile. The biota accumulated 4-59 µg Ag g-1 dw, depending on the type of Ag-treatment and organism. The oligochaete worm, Lumbriculus variegatus, accumulated Ag from the Ag2S exposure over time, which was similar to the AgNO3 treatment by the end of the experiment. The planarian, Girardia tigrina, and the chironomid larva, Chironomus riparius, showed much higher Ag concentrations than the oligochaete worms; and with a clearer time-dependent statistically significant Ag accumulation relative to the untreated controls. For the pulmonate snail, Physa acuta, bioaccumulation of Ag from AgNO3 and Ag2S NP exposures was observed, but was lower from the nano treatment. The AgNO3 exposure caused appreciable Ag accumulation in the water flea, Daphnia magna, but accumulation was higher in the Ag2S NP treatment (reaching 59 µg g-1 dw). In the rainbow trout, Oncorhynchus mykiss, AgNO3, but not Ag2S NPs, caused total Ag concentrations to increase in the tissues. Overall, the study showed transfer of total Ag from the water column to the sediment, and Ag bioaccumulation in the biota, with Ag from Ag2S NP exposure generally being less bioavailable than that from AgNO3.

The ecotoxicological consequences of microplastics and co-contaminants in aquatic organisms: a mini-review.

Microplastics (MPs, <5 mm in size) are a grave environmental concern. They are a ubiquitous persistent pollutant group that has reached into all parts of the environment - from the highest mountain tops to the depths of the ocean. During their production, plastics have added to them numerous chemicals in the form of plasticizers, colorants, fillers and stabilizers, some of which have known toxicity to biota. When released into the environments, MPs are also likely to encounter chemical contaminants, including hydrophobic organic contaminants, trace metals and pharmaceuticals, which can sorb to plastic surfaces. Additionally, MPs have been shown to be ingested by a wide range of organisms and it is this combination of ingestion and chemical association that gives weight to the notion that MPs may impact the bioavailability and toxicity of both endogenous and exogenous co-contaminants. In this mini-review, we set the recent literature within what has been previously published about MPs as chemical carriers to biota, with particular focus on aquatic invertebrates and fish. We then present a critical viewpoint on the validity of laboratory-to-field extrapolations in this area. Lastly, we highlight the expanding 'microplastic universe' with the addition of anthropogenic particles that have gained recent attention, namely, tire wear particles, nanoplastics and, bio-based or biodegradable MPs, and highlight the need for future research in their potential roles as vehicles of co-contaminant transfer.

Metal content and kinetic properties of yeast RNA lariat debranching enzyme Dbr1.

In eukaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from Entamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from Saccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression E. coli We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec-1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec-1 We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.

Determination of metallic nanoparticles in biological samples by single particle ICP-MS: a systematic review from sample collection to analysis.

A systematic review of the use of single particle ICP-MS to analyse engineered nanomaterials (ENMs) in biological samples (plants, animals, body fluids) has highlighted that efforts have focused on a select few types of ENMs (e.g., Ag and TiO2) and there is a lack of information for some important tissues (e.g., reproductive organs, skin and fatty endocrine organs). The importance of sample storage is often overlooked but plays a critical role. Careful consideration of the ENM and matrix composition is required to select an appropriate protocol to liberate ENMs from a tissue whilst not promoting the transformation of them, or genesis of new particulates. A 'one size fits all' protocol, applicable to all possible types of ENM and biological matrices, does not seem practical. However, alkaline-based extractions would appear to show greater promise for wide applicability to animal tissues, although enzymatic approaches have a role, especially for plant tissues. There is a lack of consistency in metrics reported and how they are determined (e.g. size limit of detection, and proportions of recovery), making comparison between some studies more difficult. In order to establish standardised protocols for regulatory use, effort is needed to: develop certified reference materials, achieve international agree on nomenclature and the use of control samples, and to create a decision tree to help select the best sample preparation for the type of tissue matrix.

Demonstrating the translocation of nanoplastics across the fish intestine using palladium-doped polystyrene in a salmon gut-sac.

Fish are widely reported to ingest microplastics with low levels accumulating in the tissues, but owing to analytical constraints, much less is known about the potential accumulation of nanoplastics via the gut. Recently, the labelling of plastics with inorganic metals (e.g., palladium) has allowed measurements of nanoplastic uptake. The aim of the current study was to quantitatively assess the uptake of nanoplastics by the fish gut using palladium-doped nanoplastics (with a mean hydrodynamic radius of 202 ±â€¯7 nm). By using an ex vivo gut sac exposure system, we show that in 4 h between 200 and 700 million nanoplastics (representing 2.5-9.4% of the administered nanoplastics dose) can enter the mucosa and muscularis layers of the intestine of salmon. Of the particles taken up, up to 700,000 (representing 0.6% of that taken into the tissue) of the nanoplastics passed across the gut epithelium of the anterior intestine and exit into the serosal saline. These data, generated in highly controlled conditions provide a proof-of-concept study, suggesting the potential for nanoplastics to distribute throughout the body, indicating the potential for systemic exposure in fish.

Prospective Validation of an Electronic Health Record-Based, Real-Time Suicide Risk Model.

Importance: Numerous prognostic models of suicide risk have been published, but few have been implemented outside of integrated managed care systems. Objective: To evaluate performance of a suicide attempt risk prediction model implemented in a vendor-supplied electronic health record to predict subsequent (1) suicidal ideation and (2) suicide attempt. Design, Setting, and Participants: This observational cohort study evaluated implementation of a suicide attempt prediction model in live clinical systems without alerting. The cohort comprised patients seen for any reason in adult inpatient, emergency department, and ambulatory surgery settings at an academic medical center in the mid-South from June 2019 to April 2020. Main Outcomes and Measures: Primary measures assessed external, prospective, and concurrent validity. Manual medical record validation of coded suicide attempts confirmed incident behaviors with intent to die. Subgroup analyses were performed based on demographic characteristics, relevant clinical context/setting, and presence or absence of universal screening. Performance was evaluated using discrimination (number needed to screen, C statistics, positive/negative predictive values) and calibration (Spiegelhalter z statistic). Recalibration was performed with logistic calibration. Results: The system generated 115 905 predictions for 77 973 patients (42 490 [54%] men, 35 404 [45%] women, 60 586 [78%] White, 12 620 [16%] Black). Numbers needed to screen in highest risk quantiles were 23 and 271 for suicidal ideation and attempt, respectively. Performance was maintained across demographic subgroups. Numbers needed to screen for suicide attempt by sex were 256 for men and 323 for women; and by race: 373, 176, and 407 for White, Black, and non-White/non-Black patients, respectively. Model C statistics were, across the health system: 0.836 (95% CI, 0.836-0.837); adult hospital: 0.77 (95% CI, 0.77-0.772); emergency department: 0.778 (95% CI, 0.777-0.778); psychiatry inpatient settings: 0.634 (95% CI, 0.633-0.636). Predictions were initially miscalibrated (Spiegelhalter z = -3.1; P = .001) with improvement after recalibration (Spiegelhalter z = 1.1; P = .26). Conclusions and Relevance: In this study, this real-time predictive model of suicide attempt risk showed reasonable numbers needed to screen in nonpsychiatric specialty settings in a large clinical system. Assuming that research-valid models will translate without performing this type of analysis risks inaccuracy in clinical practice, misclassification of risk, wasted effort, and missed opportunity to correct and prevent such problems. The next step is careful pairing with low-cost, low-harm preventive strategies in a pragmatic trial of effectiveness in preventing future suicidality.

Harmful chemicals emitted from electronic cigarettes and potential deleterious effects in the oral cavity.

Use of electronic nicotine delivery systems (ENDS), such as electronic cigarettes (e-cigs), is increasing across the US population and is particularly troubling due to their adoption by adolescents, teens, and young adults. The industry's marketing approach for these instruments of addiction has been to promote them as a safer alternative to tobacco, a behavioral choice supporting smoking cessation, and as the 'cool' appearance of vaping with flavored products (e.g. tutti frutti, bubble gum, and buttered popcorn etc.). Thus, there is a clear need to better document the health outcomes of e-cig use in the oral cavity of the addicted chronic user. There appears to be an array of environmental toxins in the vapors, including reactive aldehydes and carbonyls resulting from the heating elements action on fluid components, as well as from the composition of chemical flavoring agents. The chemistry of these systems shows that the released vapors from the e-cigs frequently contain levels of environmental toxins that considerably exceed federal occupational exposure limits. Additionally, the toxicants in the vapors appear to be retained in the host fluids/tissues at levels often approximating 90% of the levels in the e-cig vapors. These water-soluble reactive toxins can challenge the oral cavity constituents, potentially contributing to alterations in the autochthonous microbiome and host cells critical for maintaining oral homeostasis. This review updates the existing chemistry/environmental aspects of e-cigs, as well as providing an overview of the somewhat limited data on potential oral health effects that could occur across the lifetime of daily e-cig users.