Dynamic Foxp3-chromatin interaction controls tunable Treg cell function.

Nuclear factor Foxp3 determines regulatory T (Treg) cell fate and function via mechanisms that remain unclear. Here, we investigate the nature of Foxp3-mediated gene regulation in suppressing autoimmunity and antitumor immune response. Contrasting with previous models, we find that Foxp3-chromatin binding is regulated by Treg activation states, tumor microenvironment, and antigen and cytokine stimulations. Proteomics studies uncover dynamic proteins within Foxp3 proximity upon TCR or IL-2 receptor signaling in vitro, reflecting intricate interactions among Foxp3, signal transducers, and chromatin. Pharmacological inhibition and genetic knockdown experiments indicate that NFAT and AP-1 protein Batf are required for enhanced Foxp3-chromatin binding in activated Treg cells and tumor-infiltrating Treg cells to modulate target gene expression. Furthermore, mutations at the Foxp3 DNA-binding domain destabilize the Foxp3-chromatin association. These representative settings delineate context-dependent Foxp3-chromatin interaction, suggesting that Foxp3 associates with chromatin by hijacking DNA-binding proteins resulting from Treg activation or differentiation, which is stabilized by direct Foxp3-DNA binding, to dynamically regulate Treg cell function according to immunological contexts.

Fate and behaviour of Microplastics (> 25µm) within the water distribution network, from water treatment works to service reservoirs and customer taps.

Water treatment works have previously shown high efficiency in removing microplastics > 25 µm from raw source water. However, what is less well known is the extent to which microplastics of this size class are generated or lost within the water distribution network, particularly whether there is a greater presence in the customer tap than in the water treatment works outlet. This study focused on the presence of 21 different types of synthetic polymer particles with sizes larger than 25 µm examined through multiple rounds of sampling at outlets of water treatment works (WTW), service reservoirs (SR), and customer taps (CT) managed by seven different water companies in Britain. Nineteen different types of polymers were detected; their signature and concentration varied based on the round of sampling, the location within the water supply network, and the water company responsible for managing the supply. Among the polymers examined, polyamide (PA), polyethene terephthalate (PET), polypropylene (PP), and polystyrene (PS) were the most commonly found. Apart from PET having its highest concentration of 0.0189 microplastic per litre (MP/L) in the SR, the concentrations of the other three most frequent polymers (PS = 0.017 MP/L, PA = 0.0752 MP/L, PP= 0.1513 MP/L) were highest in the CT. The overall prevalence of this size of microplastics in the network is low, but there was a high variability of polymer types and occurrences. These spatial and temporal variations suggested that the MP in the distribution network may exist as a series of pulses. Given the presence and polymer types, the potential for some of the microplastics to originate from materials used in the water network and domestic plumbing systems cannot be ruled out. As found before, the absolute number of microplastics in the water distribution network remained extremely low.

Exploring the Use of the Picture Exchange Communication System (PECS) in Special Education Settings.

The Picture Exchange Communication System (PECS) is an Augmentative and Alternative Communication (AAC) system which is widely used to support children with developmental disabilities. In the present study, we surveyed individuals responsible for implementing PECS in special educational settings in the United Kingdom (N=283). We explored knowledge of and adherence to the  intervention, with a view to identifying training and support needs. Specifically, we examined participants' knowledge, implementation accuracy, training experiences, access to resources, and attitudes towards PECS. We developed hierarchical logistic regression models to explore the association between training experience and both knowledge and use of PECS. We pre-registered our methods, predictions and the analysis plan on the Open Science Framework (OSF).We found considerable variation in practitioner knowledge and implementation of PECS. Formal training predicted greater knowledge and more accurate implementation when practitioner role and the degree of setting support were accounted for. While PECS was rated by a large majority to be effective and practical, many participants identified that time and the availability of resources were barriers to implementation. We also found that the purpose of PECS was not always fully understood by practitioners, and we identified some consistent gaps in knowledge and implementation.  This study contributes new information regarding the real-world  use of PECS in educational settings and offers new insights for supporting practitioners.

Electrochemical Performance of Biocompatible TiC Films Deposited through Nonreactive RF Magnetron Sputtering for Neural Interfacing.

The efficacy of neural electrode stimulation and recording hinges significantly on the choice of a neural electrode interface material. Transition metal carbides (TMCs), particularly titanium carbide (TiC), have demonstrated exceptional chemical stability and high electrical conductivity. Yet, the fabrication of TiC thin films and their potential application as neural electrode interfaces remains relatively unexplored. Herein, we present a systematic examination of TiC thin films synthesized through nonreactive radio frequency (RF) magnetron sputtering. TiC films were optimized toward high areal capacitance, low impedance, and stable electrochemical cyclability. We varied the RF power and deposition pressure to pinpoint the optimal properties, focusing on the deposition rate, surface roughness, crystallinity, and elemental composition to achieve high areal capacitance and low impedance. The best-performing TiC film showed an areal capacitance of 475 µF/cm2 with a capacitance retention of 93% after 5000 cycles. In addition, the electrochemical performance of the optimum film under varying scanning rates demonstrated a stable electrochemical performance even under dynamic and fast-changing stimulation conditions. Furthermore, the in vitro cell culture for 3 weeks revealed excellent biocompatibility, promoting cell growth compared with a control substrate. This work presents a novel contribution, highlighting the potential of sputtered TiC thin films as robust neural electrode interface materials.

Introducing Routine Assessment of Adverse Childhood Experiences For Looked-After Children: The Use and Properties of the Trauma and Adverse Life Events (TALE) Screening Tool.

The present study aims to illustrate the process of developing, implementing, and clinically validating a new assessment measure, the Trauma and Adverse Life Events (TALE) screening tool, to assess Adverse Childhood Experiences (ACEs) among looked-after children. The TALE was developed by adapting existing ACEs measures to reflect the experiences of looked-after children. The TALE was completed by the local authority social worker for 218 children placed with Five Rivers Child Care (a UK fostering agency, residential, and educational care provider). Reliability was examined and exploratory factor analysis was conducted. Correlations between TALE scores, background variables, and psychosocial wellbeing using the carer-report Strengths and Difficulties Questionnaire (SDQ) and Child Dissociative Checklist (CDC) were also explored. The TALE was found to have acceptable reliability (α = .71). A three-factor solution was found which explained 46.24% of the variance, with factors labelled 'Direct Experience of Abuse', 'Witnessing Harm', and 'Household Dysfunction'. Exposure score was significantly associated with total difficulties score on the SDQ (rs = .24, p < .001) and Impact score was associated with the SDQ's impact score (rs = .33, p < .001). Exposure and Impact scores were both positively correlated with CDC scores (rs = .16, p = .021 and rs = .22, p = .002). This paper presents evidence of the importance of screening looked-after children for ACEs and demonstrates that the TALE is a valid and reliable tool for this purpose. Adverse and traumatic experiences were highly prevalent in this population and appeared to be closely related with children's psychosocial wellbeing. Results emphasise the importance of routine assessment of past experiences within trauma-informed psychological care and intervention planning for looked-after children.

Co-occurrence of macroplastics, microplastics, and legacy and emerging plasticisers in UK soils.

Despite a theoretical link between plastic and plasticiser occurrence in the terrestrial environment, there are few empirical studies of the relationship between these contaminants in soils. We carried out a field study to assess the co-occurrence of plastic waste, and legacy and emerging plasticisers in UK soils (n = 19) from various land uses (woodlands, urban roadsides, urban parklands, landfill-associated). Surface plastics and soil microplastics were quantified and characterised using ATR-FTIR and µ-FTIR. Eight legacy (phthalate) and three emerging (adipate, citrate, trimellitate) plasticisers were quantified using GC-MS. Surface plastics were found at higher prevalence at landfill-associated and urban roadside sites, with levels significantly (2 orders of magnitude) greater than in woodlands. Microplastics were detected in landfill-associated (mean 12.3 particles g-1 dw), urban roadside (17.3 particles g-1 dw) and urban parkland (15.7 particles g-1 dw) soils, but not in woodland soils. The most commonly detected polymers were polyethene, polypropene and polystyrene. Mean ∑plasticiser concentration in urban roadside soils (3111 ng g-1 dw) was significantly higher than in woodlands (134 ng g-1 dw). No significant difference was found between landfill-associated (318 ng g-1 dw) and urban parkland (193 ng g-1 dw) soils and woodlands. Di-n-butyl phthalate (94.7% detection frequency) and the emerging plasticiser trioctyl trimellitate (89.5%) were the most commonly detected plasticisers, with diethylhexyl phthalate (493 ng g-1 dw) and di-iso-decyl phthalate (96.7 ng g-1 dw) present at the highest concentrations. ∑plasticiser concentrations were significantly correlated with surface plastic (R2 = 0.23), but not with soil microplastic concentrations. Whilst plastic litter seems a fundamental source of plasticisers in soils, mechanisms such as airborne transport from source areas may be as important. Based on the data from this study, phthalates remain the dominant plasticisers in soils, but emerging plasticisers are already widespread, as reflected by their presence in all land uses studied.

From the environment into the biomass: microplastic uptake in a protected lamprey species.

The relationship between the ubiquitous presence of microplastics in the environment and exposure of biota needs to be better understood, particularly for vulnerable species and their habitats. In this study, we address the presence of microplastics in the riverine habitat of a threatened lamprey species (Lampetra sp.), both in habitats with protective interventions in place (designated as Special Areas of Conservation), and those without these protective interventions. By sampling both riverbed sediments and larval lamprey, we provide a direct comparison of the microplastic loadings in both, and insights into how knowledge of sediment loadings might predict biological uptake. Microplastic particles, analysed using micro-Fourier transform infrared (µFTIR) spectroscopy, were detected in all samples of lamprey larvae and paired sediment, ranging in abundance from 1.00 to 27.47 particles g-1 in dry lamprey gastrointestinal tract (GIT) tissue, and 0.40 to 105.41 particles g-1 in dry sediment. The most urbanised catchment exhibited the highest average microplastic particle count in both lamprey and sediment. Across sites, the microplastic abundance in lamprey GIT tissue was not correlated with that of the surrounding sediment, suggesting that either specific polymer types are retained or other factors such as larvae residence time within sediment patches may influence biological uptake. The most encountered polymer types in lamprey from their immediate habitat were polyurethane, polyamide, and cellulose acetate. To the best of our knowledge, this is the first study to document microplastic contamination of larval lamprey in-situ, contributing another potential stressor to the population status of a vulnerable species. This highlights where further research on the impacts of plastic contamination of freshwater environments is needed to aid conservation management of this ecologically important species.

The anterior gradient homologue 2 (AGR2) co-localises with the glucose-regulated protein 78 (GRP78) in cancer stem cells, and is critical for the survival and drug resistance of recurrent glioblastoma: in situ and in vitro analyses.

BACKGROUND: Glioblastomas (GBs) are characterised as one of the most aggressive primary central nervous system tumours (CNSTs). Single-cell sequencing analysis identified the presence of a highly heterogeneous population of cancer stem cells (CSCs). The proteins anterior gradient homologue 2 (AGR2) and glucose-regulated protein 78 (GRP78) are known to play critical roles in regulating unfolded protein response (UPR) machinery. The UPR machinery influences cell survival, migration, invasion and drug resistance. Hence, we investigated the role of AGR2 in drug-resistant recurrent glioblastoma cells. METHODS: Immunofluorescence, biological assessments and whole exome sequencing analyses were completed under in situ and in vitro conditions. Cells were treated with CNSTs clinical/preclinical drugs taxol, cisplatin, irinotecan, MCK8866, etoposide, and temozolomide, then resistant cells were analysed for the expression of AGR2. AGR2 was repressed using single and double siRNA transfections and combined with either temozolomide or irinotecan. RESULTS: Genomic and biological characterisations of the AGR2-expressed Jed66_GB and Jed41_GB recurrent glioblastoma tissues and cell lines showed features consistent with glioblastoma. Immunofluorescence data indicated that AGR2 co-localised with the UPR marker GRP78 in both the tissue and their corresponding primary cell lines. AGR2 and GRP78 were highly expressed in glioblastoma CSCs. Following treatment with the aforementioned drugs, all drug-surviving cells showed high expression of AGR2. Prolonged siRNA repression of a particular region in AGR2 exon 2 reduced AGR2 protein expression and led to lower cell densities in both cell lines. Co-treatments using AGR2 exon 2B siRNA in conjunction with temozolomide or irinotecan had partially synergistic effects. The slight reduction of AGR2 expression increased nuclear Caspase-3 activation in both cell lines and caused multinucleation in the Jed66_GB cell line. CONCLUSIONS: AGR2 is highly expressed in UPR-active CSCs and drug-resistant GB cells, and its repression leads to apoptosis, via multiple pathways.

Caddisfly Larvae are a Driver of Plastic Litter Breakdown and Microplastic Formation in Freshwater Environments.

Plastic litter is now pervasive in the aquatic environment. Several marine and terrestrial organisms can fragment plastic with their feeding appendages, facilitating its breakdown and generating microplastics. However, similar studies with freshwater organisms are extremely limited. We explored the interactions between the caddisfly larvae Agrypnia sp. and polylactic acid (PLA) film. The use of plastic by larvae to build their protective cases was investigated, along with their ability to fragment the plastic film as they do with leaf litter. Caddisfly consistently incorporated PLA into their cases alongside leaf material. They also used their feeding appendages to rapidly fragment PLA-forming hundreds of submillimeter-sized microplastics. Although larvae showed a preference for leaf material when constructing cases, plastic use and fragmentation still occurred when leaf material was replete, indicating that this behavior is likely to occur in natural environments that are polluted with plastics. This is thought to be the first documented evidence of active plastic modification by a freshwater invertebrate and therefore reveals a previously unidentified mechanism of plastic fragmentation and microplastic formation in freshwater. Further work is now needed to determine the extent of this behavior across freshwater taxa and the potential implications for the wider ecosystem. Environ Toxicol Chem 2022;41:3058-3069. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Reproducibility of methods required to identify and characterize nanoforms of substances.

Nanoforms (NFs) of a substance may be distinguished from one another through differences in their physicochemical properties. When registering nanoforms of a substance for assessment under the EU REACH framework, five basic descriptors are required for their identification: composition, surface chemistry, size, specific surface area and shape. To make the risk assessment of similar NFs efficient, a number of grouping frameworks have been proposed, which often require assessment of similarity on individual physicochemical properties as part of the group justification. Similarity assessment requires an understanding of the achievable accuracy of the available methods. It must be demonstrated that measured differences between NFs are greater than the achievable accuracy of the method, to have confidence that the measured differences are indeed real. To estimate the achievable accuracy of a method, we assess the reproducibility of six analytical techniques routinely used to measure these five basic descriptors of nanoforms: inductively coupled plasma mass spectrometry (ICP-MS), Thermogravimetric analysis (TGA), Electrophoretic light scattering (ELS), Brunauer-Emmett-Teller (BET) specific surface area and transmission and scanning electron microscopy (TEM and SEM). Assessment was performed on representative test materials to evaluate the reproducibility of methods on single NFs of substances. The achievable accuracy was defined as the relative standard deviation of reproducibility (RSDR) for each method. Well established methods such as ICP-MS quantification of metal impurities, BET measurements of specific surface area, TEM and SEM for size and shape and ELS for surface potential and isoelectric point, all performed well, with low RSDR, generally between 5 and 20%, with maximal fold differences usually <1.5 fold between laboratories. Applications of technologies such as TGA for measuring water content and putative organic impurities, additives or surface treatments (through loss on ignition), which have a lower technology readiness level, demonstrated poorer reproducibility, but still within 5-fold differences. The expected achievable accuracy of ICP-MS may be estimated for untested analytes using established relationships between concentration and reproducibility, but this is not yet the case for TGA measurements of loss on ignition or water content. The results here demonstrate an approach to estimate the achievable accuracy of a method that should be employed when interpreting differences between NFs on individual physicochemical properties.

How can we justify grouping of nanoforms for hazard assessment? Concepts and tools to quantify similarity.

The risk of each nanoform (NF) of the same substance cannot be assumed to be the same, as they may vary in their physicochemical characteristics, exposure and hazard. However, neither can we justify a need for more animal testing and resources to test every NF individually. To reduce the need to test all NFs, (regulatory) information requirements may be fulfilled by grouping approaches. For such grouping to be acceptable, it is important to demonstrate similarities in physicochemical properties, toxicokinetic behaviour, and (eco)toxicological behaviour. The GRACIOUS Framework supports the grouping of NFs, by identifying suitable grouping hypotheses that describe the key similarities between different NFs. The Framework then supports the user to gather the evidence required to test these hypotheses and to subsequently assess the similarity of the NFs within the proposed group. The evidence needed to support a hypothesis is gathered by an Integrated Approach to Testing and Assessment (IATA), designed as decision trees constructed of decision nodes. Each decision node asks the questions and provides the methods needed to obtain the most relevant information. This White paper outlines existing and novel methods to assess similarity of the data generated for each decision node, either via a pairwise analysis conducted property-by-property, or by assessing multiple decision nodes simultaneously via a multidimensional analysis. For the pairwise comparison conducted property-by-property we included in this White paper: The x-fold, Bayesian and Arsinh-OWA distance algorithms performed comparably in the scoring of similarity between NF pairs. The Euclidean distance was also useful, but only with proper data transformation. The x-fold method does not standardize data, and thus produces skewed histograms, but has the advantage that it can be implemented without programming knowhow. A range of multidimensional evaluations, using for example dendrogram clustering approaches, were also investigated. Multidimensional distance metrics were demonstrated to be difficult to use in a regulatory context, but from a scientific perspective were found to offer unexpected insights into the overall similarity of very different materials. In conclusion, for regulatory purposes, a property-by-property evaluation of the data matrix is recommended to substantiate grouping, while the multidimensional approaches are considered to be tools of discovery rather than regulatory methods.

Refinement of the selection of physicochemical properties for grouping and read-across of nanoforms.

Before placing a new nanoform (NF) on the market, its potential adverse effects must be evaluated. This may e.g. be done via hazard and risk assessment. Grouping and read-across of NFs is a possible strategy to reduce resource consumption, maximising the use of existing data for assessment of NFs. The GRACIOUS project provides a framework in which possible grouping and read-across for NFs is mainly based on an evaluation of their similarity. The impact of NFs on human health and the environment depends strongly on the concentration of the NF and its physicochemical properties, such as chemical composition, size distribution, shape, etc. Hence, knowledge of the most relevant physicochemical properties is essential information for comparing similarity. The presented work aims to refine existing proposals for sets of descriptors (descriptor array) that are needed to describe distinct NFs of a material to identify the most relevant ones for grouping and read-across. The selection criteria for refining this descriptor array are explained and demonstrated. Relevant protocols and methods are proposed for each physicochemical property. The required and achievable measurement accuracies of the refined descriptor array are reviewed, as this information is necessary for similarity assessment of NFs based on individual physicochemical properties.

Assessing the similarity of nanoforms based on the biodegradation of organic surface treatment chemicals.

A substance may have one or more nanoforms, defined for regulatory purposes under EU chemicals legislation REACH based on differences in physicochemical properties such as size, shape, specific surface area and surface chemistry including coatings. To reduce the burden of testing each unique nanoform for the environmental risk assessment of nanomaterials, grouping approaches allow simultaneous assessment of multiple nanoforms. Nanoforms with initially different intrinsic properties, could still be considered similar if their environmental fate and effects can be demonstrated to be similar. One hypothesis to group nanoforms with different organic surface modifications is to use parameters linked to biodegradation of the organic surface. The hypothesis contends that nanoforms with a similar core chemistry, but different organic surface treatments may be grouped, if the surface treatment is likely to be lost through biodegradation rapidly upon entering an environmental compartment, such that it no longer modulates fate, exposure and toxicity of the nanoform. To implement grouping according to surface treatment biodegradability, a robust approach to measure the breakdown of particle surface treatments is needed. We present a tiered testing strategy to assess the biodegradation of organic surface treatments used with nanomaterials that can be implemented as part of an Integrated Approach to Testing and Assessment (IATA) for grouping based on surface treatment stability. The tiered approach consists of an initial pre-screening MT2 colorimetric carbon substrate utilisation assay, to provide a rapid assessment of coating degradation, and a second tier of testing using OECD Test Guideline 301F for assessing organic chemical biodegradability. Six common surface treatment substances are assessed using the tiered testing strategy to refine rules for escalating between tiers. Similarity assessment using absolute Euclidean distances and x-fold difference concluded that the Tier 1 assessment can be used as conservative binary screening for biodegradability (no false positive results in Tier 1), whilst for substances showing intermediate biodegradation (10-60% in OECD 301F, Tier 2), similarity assessments can be informative for grouping surface treatments not considered readily biodegradable. Further validation using higher tier tests (e.g., mesocosms) is needed to define acceptable limits of similarity between intermediately biodegradable substances, where differences in biodegradability of the surface coating lead to negligible differences in fate, behaviour and toxicity of the nanoforms, and this is critically discussed.

Similarity assessment of metallic nanoparticles within a risk assessment framework: A case study on metallic nanoparticles and lettuce.

Similarity assessment is one of the means of optimally using scarcely available experimental data on the fate and hazards of nanoforms (NFs) for regulatory purposes. For a set of NFs that are shown to be similar it is allowed in a regulatory context to apply the information available on any of the NFs within the group to the whole set of NFs. Obviously, a proper justification for such a similarity assessment is to be provided. Within the context of exemplifying such a justification, a case study was performed aimed at assessing the similarity of a set of spherical metallic NFs that different with regard to chemical composition (three metals) and particle size (three different sizes). The endpoints of assessment were root elongation and biomass increase of lettuce (Lactuca sativa L.) seedlings and exposure assessment was performed in order to express the actual exposure concentration in terms of time-weighted average particle concentrations. The results of the study show that for the specific endpoints assessed, chemical composition is driving NF toxicity and this is mostly due to impacts on the fate of the NFs. On the other hand, particle size of Cu NFs had a negligible impact on the dose-response relationships for the specific endpoints assessed. It is thus concluded that hazard data available on spherical Cu NF tested in our case can be used to inform on the hazards of any spherical Cu NF within the size range of 25-100 nm, but only applies for the certain endpoints. Also, toxicity data for the Cu2+-ion are suited for such a similarity assessment.

Porous surfaces: stability and recovery of coronaviruses.

The role of indirect contact in the transmission of SARS-CoV-2 is not clear. SARS-CoV-2 persists on dry surfaces for hours to days; published studies have largely focused on hard surfaces with less research being conducted on different porous surfaces, such as textiles. Understanding the potential risks of indirect transmission of COVID-19 is useful for settings where there is close contact with textiles, including healthcare, manufacturing and retail environments. This article aims to review current research on porous surfaces in relation to their potential as fomites of coronaviruses compared to non-porous surfaces. Current methodologies for assessing the stability and recovery of coronaviruses from surfaces are also explored. Coronaviruses are often less stable on porous surfaces than non-porous surfaces, for example, SARS-CoV-2 persists for 0.5 h-5 days on paper and 3-21 days on plastic; however, stability is dependent on the type of surface. In particular, the surface properties of textiles differ widely depending on their construction, leading to variation in the stability of coronaviruses, with longer persistence on more hydrophobic materials such as polyester (1-3 days) compared to highly absorbent cotton (2 h-4 days). These findings should be considered where there is close contact with potentially contaminated textiles.

Earthworms ingest microplastic fibres and nanoplastics with effects on egestion rate and long-term retention.

Microplastic fibres (MPFs) and nanoplastics (NPs) have the potential to be hazardous to soil organisms. Understanding uptake into organisms is key in assessing these effects, but this is often limited by the analytical challenges to quantify smaller-sized plastics in complex matrices. This study used MPFs and NPs containing inorganic tracers (In, Pd) to quantify uptake in the earthworm Lumbricus terrestris. Following seven days exposure, tracer concentrations were measured in earthworms and faeces. Earthworms exposed to 500 µg MPFs/g soil retained an estimated 32 MPFs in their tissues, while at 5000 µg MPFs/g earthworms retained between 2 and 593 MPFs. High variation in body burdens of MPFs was linked to soil retention in earthworms and reduced faeces production, suggesting egestion was being affected by MPFs. NPs uptake and elimination was also assessed over a more extended time-period of 42 days. After 1 day, NPs were no longer detectable in faeces during the elimination phase. However, some retention of NPs in the earthworms was estimated, not linked to retained soil, indicating not all NPs were eliminated. MPFs and NPs uptake can be quantified in earthworms and both particle types can be retained beyond the depuration period, suggesting the potential for longer-term accumulation.

Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation.

Regulatory T (Treg) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how Treg cells are mechanistically induced in vitro (iTreg) and stabilized via transcriptional regulation of Treg lineage-specifying factor Foxp3. We find that acetylation of histone tails at the Foxp3 promoter is required for inducing Foxp3 transcription. Upon induction, histone acetylation signals via bromodomain-containing proteins, particularly targets of inhibitor JQ1, and sustains Foxp3 transcription via a global or trans effect. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements, mainly enhancer CNS2, increases chromatin accessibility and protein binding, stabilizing Foxp3 transcription and obviating the need for the histone acetylation signal. These processes transform stochastic iTreg induction into a stable cell fate, with the former sensitive and the latter resistant to genetic and environmental perturbations. Thus, sequential histone acetylation and DNA demethylation in Foxp3 induction and maintenance reflect stepwise mechanical switches governing iTreg cell lineage specification.

Implementation of an enhanced recovery program for lower extremity bypass.

OBJECTIVE: Enhanced recovery programs (ERPs) have gained wide acceptance across multiple surgical disciplines to improve postoperative outcomes and to decrease hospital length of stay (LOS). However, there is limited information in the existing literature for vascular patients. We describe the implementation and early results of an ERP and barriers to its implementation for lower extremity bypass surgery. Our intention is to provide a framework to assist with implementation of similar ERPs. METHODS: Using the plan, do, check, adjust methodology, a multidisciplinary team was assembled. A database was used to collect information on patient-, procedure-, and ERP-specific metrics. We then retrospectively analyzed patients' demographics and outcomes. RESULTS: During 9 months, an ERP (n = 57) was successfully developed and implemented spanning preoperative, intraoperative, and postoperative phases. ERP and non-ERP patient demographics were statistically similar. Early successes include 97% use of fascia iliaca block and multimodal analgesia administration in 81%. Barriers included only 47% of patients achieving day of surgery mobilization and 19% receiving celecoxib preoperatively. ERP patients had decreased total and postoperative LOS compared with non-ERP patients (n = 190) with a mean (standard deviation) total LOS of 8.32 (8.4) days vs 11.14 (10.1) days (P = .056) and postoperative LOS of 6.12 (6.02) days vs 7.98 (7.52) days (P = .089). There was significant decrease in observed to expected postoperative LOS (1.28 [0.66] vs 1.82 [1.38]; P = .005). Variable and total costs for ERP patients were significantly reduced ($13,208 [$9930] vs $18,777 [$19,118; P < .01] and $29,865 [$22,110] vs $40,328 [$37,820; P = .01], respectively). CONCLUSIONS: Successful implementation of ERP for lower extremity bypass carries notable challenges but can have a significant impact on practice patterns. Further adjustment of our current protocol is anticipated, but early results are promising. Implementation of a vascular surgery ERP reduced variable and total costs and decreased total and postoperative LOS. We believe this protocol can easily be implemented at other institutions using the pathway outlined.

Semi-automated analysis of microplastics in complex wastewater samples.

In order to assess risks to the natural environment from microplastics, it is necessary to have reliable information on all potential inputs and discharges. This relies on stringent quality control measures to ensure accurate reporting. Here we focus on wastewater treatment works (WwTWs) and the complex sample matrices these provide. Composite samples of both influent and effluent were collected over a 24 h period on two separate occasions from eight different WwTWs across the UK. Sludge samples were taken on five occasions from five WwTWs. The WwTW treatments included activated sludge, trickling filter and biological aerated flooded filter with or without tertiary treatment. Using micro-FTIR analysis, microplastics ≥25 µm were identified and quantified. Procedural blanks were used to derive limits of detection (LOD) and limits of quantification (LOQ). Where values were above the LOQ, microplastics in the influent ranged from 955 to 17,214 microplastic particles/L and in the effluent from 2 to 54 microplastic particles/L, giving an average removal rate of 99.8%. Microplastics could be quantified in sludge at concentrations of 301-10,380 microplastics/g dry weight, this analytical method therefore revealing higher concentrations than reported in previous studies. The most common polymers present overall were polyethylene (PE), polypropylene (PP) and polyethylene terephthalate (PET). We also report on critical considerations for blank corrections and quality control measures to ensure reliable microplastic analysis across different sample types.

Identification and Quantification of Microplastics in Potable Water and Their Sources within Water Treatment Works in England and Wales.

Microplastics were characterized in eight water treatment works (WTWs) in England and Wales (UK). Sources included river water, groundwater, and an upland reservoir. Water treatment varied from disinfection, filtration, sedimentation, and activated carbon techniques. At each WTW, five repeat samples of raw and potable water and two repeat sludge samples were taken over 5 months. Microplastics in water were captured on 10 µm filters and nonplastic materials digested in the laboratory. Microplastics ≥25 µm were analyzed using Fourier-transform infrared microscopy. Blanks revealed consistent polyethylene (PE), poly(ethylene terephthalate) (PET), and polypropylene (PP) contamination. Spike recoveries for 63-90 µm polyamide microplastics demonstrated 101% (standard deviation, SD 27%) and 113% (SD 15%) recovery for raw and potable waters and 52% (SD 13%) for sludge. Only four of the six WTWs sampled for raw water and only two of eight WTWs in their potable water had microplastics above the limit of quantification. Considering only the WTWs with quantifiable microplastics, then on average, 4.9 microplastic particles/L were present in raw water and only 0.00011 microplastic particles/L were present in potable water (99.99% removal). Values in waste sludge were highly variable. PE, PET, and PP were the most common polymers quantified in raw water and sludge, and polystyrene and acrylonitrile butadiene styrene were the most common polymers quantified in potable water.