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Environmental exposure data are a key component of chemical and ecological assessments, supporting and guiding environmental management decisions and regulations. Measures taken to protect the environment based on exposure data can have social and economic implications. Flawed information may lead to measures being taken in the wrong place or to important action not being taken. Although the advantages of harmonizing evaluation methods have been demonstrated for hazard information, no comparable approach is established for exposure data evaluation. The goal of Criteria for Reporting and Evaluating Exposure Datasets (CREED) is to improve the transparency and consistency with which exposure data are evaluated regarding usability in environmental assessments. Here, we describe the synthesis of the CREED process, and propose methods and tools to summarize and interpret the outcomes of the data usability evaluation in support of decision-making and communication. The CREED outcome includes a summary that reports any key gaps or shortcomings in the reliability (data quality) and relevance (fitness for purpose) of the data being considered. The approach has been implemented in a workbook template (provided as Supporting Information), for assessors to readily follow the workflow and create a report card for any given dataset. The report card communicates the outcome of the CREED evaluation and summarizes important dataset attributes, providing a concise reference pertaining to the dataset usability for a specified purpose and documenting data limitations that may restrict data use or increase environmental assessment uncertainty. The application of CREED is demonstrated through three case studies, which also were used during beta testing of the methodology. As experience with the CREED approach application develops, further improvements may be identified and incorporated into the framework. Such development is to be encouraged in the interest of better science and decision-making, and to make environmental monitoring and assessment more cost-effective. Integr Environ Assess Manag 2024;20:1019-1034. © 2024 SETAC., (© 2024 SETAC.)

Cancer cells take up amino acids from the extracellular space to drive cell proliferation and viability. Similar mechanisms are applied by immune cells, resulting in the competition between conventional T cells, or indeed chimeric antigen receptor (CAR) T cells and tumor cells, for the limited availability of amino acids within the environment. We demonstrate that T cells can be re-engineered to express SLC7A5 or SLC7A11 transmembrane amino acid transporters alongside CARs. Transporter modifications increase CAR T-cell proliferation under low tryptophan or cystine conditions with no loss of CAR cytotoxicity or increased exhaustion. Transcriptomic and phenotypic analysis reveals that downstream, SLC7A5/SLC7A11-modified CAR T cells upregulate intracellular arginase expression and activity. In turn, we engineer and phenotype a further generation of CAR T cells that express functional arginase 1/arginase 2 enzymes and have enhanced CAR T-cell proliferation and antitumor activity. Thus, CAR T cells can be adapted to the amino acid metabolic microenvironment of cancer, a hitherto recognized but unaddressed barrier for successful CAR T-cell therapy., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In anamniote embryos, the major wave of zygotic genome activation starts during the mid-blastula transition. However, some genes escape global genome repression, are activated substantially earlier, and contribute to the minor wave of genome activation. The mechanisms underlying the minor wave of genome activation are little understood. We explored the genomic organization and cis-regulatory mechanisms of a transcription body, in which the minor wave of genome activation is first detected in zebrafish. We identified the miR-430 cluster as having excessive copy number and the highest density of Pol-II-transcribed promoters in the genome, and this is required for forming the transcription body. However, this transcription body is not essential for, nor does it encompasse, minor wave transcription globally. Instead, distinct minor-wave-specific promoter architecture suggests that promoter-autonomous mechanisms regulate the minor wave of genome activation. The minor-wave-specific features also suggest distinct transcription initiation mechanisms between the minor and major waves of genome activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Micropollutants (MPs) in wastewater pose a growing concern for their potential adverse effects on the receiving aquatic environment, and some countries have started requiring that wastewater treatment plants remove them to a certain extent. Broad spectrum advanced treatment processes, such as ozonation, activated carbon or their combination, are expected to yield a significant reduction in the toxicity of effluents. Here we quantify the reduction of effluent toxicity potentially achieved by implementing these advanced treatment solutions in a selection of European wastewater treatment plants. To this end, we refer to a list of "total pollution proxy substances" (TPPS) composed of 1337 chemicals commonly found in wastewater effluents according to a compilation of datasets of measured concentrations. We consider these substances as an approximation of the "chemical universe" impinging on the European wastewater system. We evaluate the fate of the TPPS in conventional and advanced treatment plants using a compilation of experimental physicochemical properties that describe their sorption, volatilization and biodegradation during activated sludge treatment, as well as known removal efficiency in ozonation and activated carbon treatment, while filling the gaps through in silico prediction models. We estimate that the discharge of micropollutants with wastewater effluents in the European Union has a cumulative MP toxicity to the environment equal to the discharge of untreated wastewater of ca. 160 million population equivalents (PE), i.e. about 30 % of the generated wastewater in the EU. If all plants above a capacity of 100,000 PE were equipped with advanced treatment, we show that this load would be reduced to about 95 million PE. In addition, implementing advanced treatment in wastewater plants above 10,000 PE discharging to water bodies with an average dilution ratio smaller than 10 would yield a widespread improvement in terms of exposure of freshwater ecosystems to micropollutants, almost halving the part of the stream network exposed to the highest toxic risks. Our analysis provides background for a cost-effectiveness appraisal of advanced treatment "at the end of the pipe", which could lead to optimized interventions. This should not be regarded as a stand-alone solution, but as a complement to policies for the control of emissions at the source for the most problematic MPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previous studies have described reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal/oropharyngeal swab and saliva samples. This multisite clinical evaluation describes the validation of an improved sample preparation method for extraction-free RT-LAMP and reports clinical performance of four RT-LAMP assay formats for SARS-CoV-2 detection. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) was 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva, compared with quantitative RT-PCR (RT-qPCR); analyzing samples with RT-qPCR ORF1ab C T values of ≤25 and ≤33, DSe values were 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88%-98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based direct RT-LAMP of saliva from asymptomatic individuals who may otherwise be missed using symptomatic testing alone., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations so as to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as quantitative RT-PCR (RT-qPCR), particularly throughout the first months of the coronavirus disease 2019 pandemic. We investigated the use of LamPORE, where loop-mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations., Methods: In an asymptomatic prospective cohort, for 3 weeks in September 2020, health-care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza-like illness from March 2020 to June 2020 were similarly tested from nasopharyngeal swabs., Results: In the asymptomatic cohort a total of 1200 participants supplied 23 427 samples (3966 swab, 19 461 saliva) over a 3-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (decreasing to approximately 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared with the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%., Conclusions: LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Pathological complete response (pathCR) in rectal cancer is beneficial, as up to 75% of patients do not experience regrowth of the primary tumour, but it is poorly understood. We hypothesised that the changes seen in the pre-treatment biopsies of pathCR but not seen in residual tumour after chemoradiotherapy were the determinants of responsiveness., Methods: Two groups of patients with either complete response (pathCR group, N = 24) or no response (poor response group, N = 24) were retrieved. Pre-treatment biopsies of cancers from these patients underwent high read depth amplicon sequencing for a targeted panel, exome sequencing, methylation profiling and immunohistochemistry for DNA repair pathway proteins., Results: Twenty four patients who underwent pathCR and twenty-four who underwent poor response underwent molecular characterisation. Patients in the pathCR group had significantly higher tumour mutational burden and neoantigen load, frequent copy number alterations but fewer structural variants and enrichment for driver mutations in the PI3K/AKT/mTOR signalling pathway. There were no significant differences in tumour heterogeneity as measured by MATH score. Methylation analysis demonstrated enrichment for hypomethyation in the PI3K/AKT/mTOR signalling pathway., Discussion: The phenomenon of pathCR in rectal cancer may be related to immunovisibility caused by a high tumour mutational burden phenotype. Potential therapy resistance mechanisms involve the PI3K/AKT/mTOR signalling pathway, but tumour heterogeneity does not seem to play a role in resistance., (© 2021. The Author(s).)

Background: Faecal transplantation is an evidence-based treatment for Clostridioides difficile . Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for faecal microbiota transplantation (FMT), spiked samples and asymptomatic inpatients in an acute surgical unit.  Methods: Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10 -1 to 10 -5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. Results: In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10 -2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10 -1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. Conclusion: RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool., Competing Interests: Competing interests: ADB – has received travel funding to the Oxford Nanopore Community Meeting 2019 from Oxford Nanopore. The rest of the authors declare no competing interests., (Copyright: © 2021 Manzoor SE et al.)

Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples., Methods: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls., Results: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot., Conclusions: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types., Competing Interests: Conflicts of Interest: A.D.B. has received travel funding and speaker fees from Illumina Inc.

Objective: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers., Design: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020., Setting: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK., Participants: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded., Intervention: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked., Main Outcome Measure: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology., Results: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ 2 =21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02)., Conclusions and Relevance: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices., Competing Interests: Competing interests: MTD reports personal fees from Abingdon Health, outside the submitted work. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Colorectal Peritoneal metastases (CPM) develop in 15% of colorectal cancers. Cytoreductive surgery and heated intraperitoneal chemotherapy (CRS & HIPEC) is the current standard of care in selected patients with limited resectable CPM. Despite selection using known prognostic factors survival is varied and morbidity and mortality are relatively high. There is a need to improve patient selection and a paucity of research concerning the biology of isolated CPM. We aimed to determine the biology associated with transition from primary CRC to CPM and of patients with CPM not responding to treatment with CRS & HIPEC, to identify those suitable for treatment with CRS & HIPEC and to identify targets for existing repurposed or novel treatment strategies. A cohort of patients with CPM treated with CRS & HIPEC was recruited and divided according to prognosis. Molecular profiling of the transcriptome (n = 25), epigenome (n = 24) and genome (n = 21) of CPM and matched primary CRC was performed. CPM were characterised by frequent Wnt/ β catenin negative regulator mutations, TET2 mutations, mismatch repair mutations and high tumour mutational burden. Here we show the molecular features associated with CPM development and associated with not responding to CRS & HIPEC. Potential applications include improving patient selection for treatment with CRS & HIPEC and in future research into novel and personalised treatments targeting the molecular features identified here.

Background: In January 2020 reports of unidentified severe respiratory illness were described in Wuhan, China. A rapid expansion in cases affecting most countries around the globe led to major changes in the way people live their daily lives. In the United Kingdom, the Department of Health and Social Care directed healthcare providers to establish additional resources to manage the anticipated surge in cases that could overwhelm the health services. A priority area was testing for SARS-CoV-2 RNA and its detection by qualitative RT-PCR., Design: A laboratory workflow twinning research environment with clinical laboratory capabilities was implemented and validated in the University of Birmingham within 4 days of the project initiation. The diagnostic capability was centred on an IVD CE-marked RT-PCR kit and designed to provide surge capacity to the nearby Queen Elizabeth Hospital. The service was initially tasked with testing healthcare workers (HCW) using throat swabs, and subsequently the process investigated the utility of using saliva as an alternative sample type., Results: Between the 8th April 2020 and the 30th April 2020, the laboratory tested a total of 1282 HCW for SARS-CoV-2 RNA in throat swabs. RNA was detected in 54 % of those who reported symptoms compatible with COVID-19, but in only 4% who were asymptomatic., Conclusion: This capability was established rapidly and utilised a cold-chain free methodology, applicable to a wide range of settings, and which can provide surge capacity and support to clinical laboratories facing increasing pressure during periods of national crisis., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Our objective was to evaluate the protective efficacy of the neurosteroid pregnanolone (3α-hydroxy-5β pregnan-20-one), a GABA A receptor-positive allosteric modulator, as an adjunct to benzodiazepine therapy against the chemical warfare nerve agent (CWNA) sarin (GB), using whole-body exposure, an operationally relevant route of exposure to volatile GB., Methods: Rats implanted with telemetry transmitters for the continuous measurement of cortical electroencephalographic (EEG) activity were exposed for 60 minutes to 3.0 LCt 50 of GB via whole-body exposure. At the onset of toxic signs, rats were administered an intramuscular injection of atropine sulfate (2 mg/kg) and the oxime HI-6 (93.6 mg/kg) to increase survival rate and, 30 minutes after seizure onset, treated subcutaneously with diazepam (10 mg/kg) and intravenously with pregnanolone (4 mg/kg) or vehicle. Animals were evaluated for GB-induced status epilepticus (SE), spontaneous recurrent seizures (SRS), impairment in spatial memory acquisition, and brain pathology, and treatment groups were compared., Results: Delayed dual therapy with pregnanolone and diazepam reduced time in SE in GB-exposed rats compared to those treated with delayed diazepam monotherapy. The combination therapy of pregnanolone with diazepam also prevented impairment in the Morris water maze and reduced the neuronal loss and neuronal degeneration, evaluated at one and three months after exposure., Significance: Neurosteroid administration as an adjunct to benzodiazepine therapy offers an effective means to treat benzodiazepine-refractory SE, such as occurs following delayed treatment of GB exposure. This study is the first to present data on the efficacy of delayed pregnanolone and diazepam dual therapy in reducing seizure activity, performance deficits and brain pathology following an operationally relevant route of exposure to GB and supports the use of a neurosteroid as an adjunct to standard anticonvulsant therapy for the treatment of CWNA-induced SE., Competing Interests: Author 5 was paid under a contract to process and analyze EEG data while blinded to experimental conditions. All other authors have no conflicts of interest.

The first excited electronic state of molecular oxygen, O(2)(a(1)Δ(g)), is formed in the upper atmosphere by the photolysis of O(3). Its lifetime is over 70 min above 75 km, so that during the day its concentration is about 30 times greater than that of O(3). In order to explore its potential reactivity with atmospheric constituents produced by meteoric ablation, the reactions of Mg, Fe, and Ca with O(2)(a) were studied in a fast flow tube, where the metal atoms were produced either by thermal evaporation (Ca and Mg) or by pulsed laser ablation of a metal target (Fe), and detected by laser induced fluorescence spectroscopy. O(2)(a) was produced by bubbling a flow of Cl(2) through chilled alkaline H(2)O(2), and its absolute concentration determined from its optical emission at 1270 nm (O(2)(a(1)Δ(g) - X(3)Σ(g) (-)). The following results were obtained at 296 K: k(Mg + O(2)(a) + N(2) → MgO(2) + N(2)) = (1.8 ± 0.2) × 10(-30) cm(6) molecule(-2) s(-1); k(Fe + O(2)(a) → FeO + O) = (1.1 ± 0.1) × 10(-13) cm(3) molecule(-1) s(-1); k(Ca + O(2)(a) + N(2) → CaO(2) + N(2)) = (2.9 ± 0.2) × 10(-28) cm(6) molecule(-2) s(-1); and k(Ca + O(2)(a) → CaO + O) = (2.7 ± 1.0) × 10(-12) cm(3) molecule(-1) s(-1). The total uncertainty in these rate coefficients, which mostly arises from the systematic uncertainty in the O(2)(a) concentration, is estimated to be ±40%. Mg + O(2)(a) occurs exclusively by association on the singlet surface, producing MgO(2)((1)A(1)), with a pressure dependent rate coefficient. Fe + O(2)(a), on the other hand, shows pressure independent kinetics. FeO + O is produced with a probability of only ∼0.1%. There is no evidence for an association complex, suggesting that this reaction proceeds mostly by near-resonant electronic energy transfer to Fe(a(5)F) + O(2)(X). The reaction of Ca + O(2)(a) occurs in an intermediate regime with two competing pressure dependent channels: (1) a recombination to produce CaO(2)((1)A(1)), and (2) a singlet∕triplet non-adiabatic hopping channel leading to CaO + O((3)P). In order to interpret the Ca + O(2)(a) results, we utilized density functional theory along with multireference and explicitly correlated CCSD(T)-F12 electronic structure calculations to examine the lowest lying singlet and triplet surfaces. In addition to mapping stationary points, we used a genetic algorithm to locate minimum energy crossing points between the two surfaces. Simulations of the Ca + O(2)(a) kinetics were then carried out using a combination of both standard and non-adiabatic Rice-Ramsperger-Kassel-Marcus (RRKM) theory implemented within a weak collision, multiwell master equation model. In terms of atmospheric significance, only in the case of Ca does reaction with O(2)(a) compete with O(3) during the daytime between 85 and 110 km.

This research examined the use of a single facultative pond for treatment of an intermittent discharge from a UK campsite. The system was monitored over an 11-month period to determine the optimum time for discharge in terms of quality standards. The results showed that based on organic strength, discharge was possible in winter between November and March but February was the optimum to meet nutrient and suspended solids requirements. The pond showed rapid acclimatisation to the influent wastewater, with biochemical oxygen demand removal rates during the filling period of around 60 kg ha(-1) day(-1) and removal efficiencies of ∼95% after maturation. The system proved simple to operate. A major design factor is the requirement for storage of net incoming precipitation, which may provide dilution of residual pollutants but requires additional system capacity.

Summer annuals overwinter as seeds in the soil seed bank. This is facilitated by a cold-induced increase in dormancy during seed maturation followed by a switch to a state during seed imbibition in which cold instead promotes germination. Here, we show that the seed maturation transcriptome in Arabidopsis thaliana is highly temperature sensitive and reveal that low temperature during seed maturation induces several genes associated with dormancy, including DELAY OF GERMINATION1 (DOG1), and influences gibberellin and abscisic acid levels in mature seeds. Mutants lacking DOG1, or with altered gibberellin or abscisic acid synthesis or signaling, in turn show reduced ability to enter the deeply dormant states in response to low seed maturation temperatures. In addition, we find that DOG1 promotes gibberellin catabolism during maturation. We show that C-REPEAT BINDING FACTORS (CBFs) are necessary for regulation of dormancy and of GA2OX6 and DOG1 expression caused by low temperatures. However, the temperature sensitivity of CBF transcription is markedly reduced in seeds and is absent in imbibed seeds. Our data demonstrate that inhibition of CBF expression is likely a critical feature allowing cold to promote rather than inhibit germination and support a model in which CBFs act in parallel to a low-temperature signaling pathway in the regulation of dormancy.

The research investigated some factors influencing the rate of stabilisation of wastewater in the spring period in continental climate waste stabilisation ponds, and in particular the potential for bringing forward the discharge date by optimising storage capacity and dilution. Experiments using pilot and modelscale ponds were set up in Almaty, Kazakhstan. These simulated operating regimes for a facultative and storage/maturation pond system subject to ice cover from late November until late March. Two pilot-scale facultative ponds were operated at hydraulic retention times (HRT) of 20 and 30 days, with surface loading rates of 100 and 67 kg BOD ha(-1) day(-1). Effluent from the 20-day HRT facultative pond was then fed to two pilot-scale storage/maturation ponds which had been partially emptied and allowed to refill over the winter period with no removal of effluent. The paper discusses the results of the experiments with respect to selection of an operating regime to make treated wastewater available early in the spring. Preliminary results indicate that there may be potential for alternative operating protocols designed to maximise their performance and economic potential.

The fluoride reactivation process was evaluated for measuring the level of sarin or soman nerve agents reactivated from substrates in plasma and tissue from in vivo exposed guinea pigs (Cava porcellus), in blood from in vivo exposed rhesus monkeys (Macaca mulatta), and in spiked human plasma and purified human albumin. Guinea pig exposures ranged from 0.05 to 44 LD50, and reactivated nerve agent levels ranged from 1.0 ng/mL in plasma obtained from 0.05 LD50 sarin-exposed guinea pigs to an average of 147 ng/g in kidney tissue obtained from two 2.0 LD50 soman-exposed guinea pigs. Positive dose-response relationships were observed in all low-level, 0.05 to 0.4 LD50, exposure studies. An average value of 2.4 ng/mL for reactivated soman was determined in plasma obtained from two rhesus monkeys three days after a 2 LD50 exposure. Of the five types of guinea pig tissue studied, plasma, heart, liver, kidney and lung, the lung and kidney tissue yielded the highest amounts of reactivated agent. In similar tissue and with similar exposure procedures, reactivated soman levels were greater than reactivated sarin levels. Levels of reactivated agents decreased rapidly with time while the guinea pig was alive, but decreased much more slowly after death. This latter chemical stability should facilitate forensic retrospective identification. The high level of reactivated agents in guinea pig samples led to the hypothesis that the principal source of reactivated agent came from the agent-carboxylesterase adduct. However, there could be contributions from adducts of the cholinesterases, albumin and fibrous tissue, as well. Quantitative analysis was performed with a GC-MS system using selected ion monitoring of the 99 and 125 ions for sarin and the 99 and 126 ions for soman. Detection levels were as low as 0.5 ng/mL. The assay was precise and easy to perform, and has potential for exposure analysis from organophosphate nerve agents and pesticides in other animal species.

Intramuscular (i.m.) diazepam is included by the US military as an anticonvulsant in the standard therapeutic regimen for organophosphorus nerve agent intoxication. In this study we investigated the pharmacokinetics of diazepam after i.m. administration while monitoring pharmacodynamic (electroencephalogram, EEG) data in soman-exposed guinea pigs. Prior to experiments the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered pyridostigmine (0.026 mg x kg(-1) i.m.) 30 min prior to soman (56 microg x kg(-1), 2 x LD50; subcutaneously, s.c.), which was followed in 1 min by atropine sulfate (2 mg x kg(-1) i.m.) and pralidoxime chloride (25 mg x kg(-1) i.m.). All animals receiving this regimen developed seizure activity. Diazepam (10 mg x kg(-1) i.m.) was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure terminated or not terminated at 30 min after diazepam. Serial blood samples were obtained from each animal. Diazepam (10 mg x kg(-1) i.m.) terminated seizure activity in 52% of the animals within 30 min. The pharmacokinetics were characterized by a one-compartment model with first-order absorption and elimination. The maximum plasma concentrations (Cmax) were 991 and 839 ng x ml(-1) for seizure terminated and not terminated, respectively. Mean plasma concentrations of diazepam were significantly different (P < 0.05) for seizure terminated vs not terminated groups at 30 min. The plasma Cmax in seizure-terminated animals in this study is similar to the minimum range of plasma diazepam (200-800 ng x ml(-1)) reported to suppress seizure activity in humans. It has been reported in an earlier study that the minimum effective i.m. dose (0.1 mg x kg(-1)) required to prevent soman-induced convulsions in Rhesus monkeys produces a mean Cmax of 50 ng x ml(-1) for diazepam. The data from our current study suggest that a higher dose (and corresponding Cmax) is necessary to terminate ongoing seizure activity., (Copyright 2001 John Wiley & Sons, Ltd.)

Myricetin and gossypetin, two hexahydroxylated flavonoids, are capable of modifying low density lipoprotein (LDL) to increase greatly its uptake by macrophages. When human 125I-labelled LDL was incubated with 100-1000 microM myricetin or gossypetin, it was subsequently endocytosed much faster by mouse peritoneal macrophages. This modification did not occur at a concentration of 10 microM. Nine other flavonoids containing up to five hydroxyl substituents did not modify LDL to any great extent at 100 microM. The modification of LDL by 100 microM myricetin was time-dependent and complete by 6 hr. Flavonoids can sometimes act as pro-oxidants but myricetin did not act by oxidizing the LDL, as the LDL lipid hydroperoxide content was not increased by myricetin, nor did it promote the depletion of the endogenous antioxidant alpha-tocopherol in the LDL. High concentrations of myricetin caused the aggregation of LDL particles, as judged by light microscopy, agarose gel electrophoresis, retention by a membrane filter and sedimentability by centrifugation. SDS-PAGE indicated that the apolipoprotein B-100 molecules of LDL particles were covalently crosslinked. The uptake and degradation by macrophages of myricetin-modified 125I-labelled LDL reached saturation at about 10 micrograms protein/mL, suggesting the existence of a high affinity uptake process for the modified LDL. The uptake of myricetin-modified 125I-labelled LDL was not competed for by a large excess of non-labelled native LDL or acetylated LDL. We conclude that myricetin and gossypetin at high concentrations are capable of modifying LDL by a novel non-oxidative mechanism to a form taken up by macrophages by a high affinity process.

The effects of HI-6 and pralidoxime chloride (2-PAM) on soman-induced lethality, time to death and several cholinergic parameters in rats were compared to understand the beneficial action of HI-6. Treatment with atropine sulfate (ATS) or HI-6 alone protected against 1.2 and 2.5 LD50s of soman respectively, whereas 2-PAM or methylated atropine (AMN) alone afforded no protection. Addition of ATS, but not AMN, to HI-6-treated rats enhanced the protection from 2.5 to 5.5 LD50s. HI-6 increased the time-to-death, while 2-PAM had no effect; a combination of HI-6 and ATS provided the most significant increase in time-to-death. Cholinesterase (ChE) activity was not altered in any tissue by ATS, HI-6 or 2-PAM treatment individually, but was markedly inhibited in all tissues by 100 micrograms/kg of soman. In soman-poisoned rats, the HI-6, but not the 2-PAM, group had significantly higher levels of ChE in blood and other peripheral tissues than did the group given soman alone. Neither HI-6 nor 2-PAM affected soman-inhibited ChE in the brain. Additional ATS treatment had no effect on ChE activity. HI-6 and 2-PAM neither modified baseline brain acetylcholine (ACh) or choline (Ch) levels nor protected against soman-induced ACh or Ch elevation. 2-PAM exhibited a 4-fold more potent in vitro inhibition of 3H-quinuclidinyl benzilate (3H-QNB) binding and sodium-dependent high-affinity Ch uptake (HACU) than did HI-6 in brain tissues. The findings that 2-PAM is a more potent in vitro inhibitor of muscarinic receptor binding and HACU than HI-6, and yet neither elevates ChE activity in the periphery nor protects rats against soman poisoning, indicate the importance of higher ChE activity in the periphery of HI-6-treated rats. Maintenance by HI-6 of a certain amount of active ChE in the periphery appears to be important for survival after soman exposure.

The appearance of lipid-laden macrophages is a characteristic feature in the development of the atherosclerotic plaque. The functional status of macrophages located within the intima of atherosclerotic lesions is as yet unknown; nevertheless, macrophages are known to be exceedingly responsive to their environment and can differentiate to different functional states. The objective of this study was to determine the influence of two definable macrophage functional states, namely the IFN-primed state and the cytocidal state, on the capacity of macrophages to bind and degrade lipoproteins. We report that priming of macrophages with IFN-beta or IFN-gamma failed to influence the ability of macrophages to degrade native low density lipoprotein or acetylated low density lipoprotein (AcLDL). However, challenge with stimuli that induce expression of the cytocidal state (poly[I:C] and LPS) resulted in a marked inhibition of the capacity of the cells to degrade both lipoproteins. The poly[I:C]-induced inhibition of 125I-AcLDL degradation was accompanied by a proportional decrease in the binding of the ligand to its receptor which Scatchard analysis revealed was due to a decrease in receptor number rather than a change in receptor affinity for 125I-AcLDL. However, in addition to the down-regulation of receptor activity, the degradation of endocytosed 125I-AcLDL was also suppressed in macrophages that had been exposed to poly[I:C]. This latter observation suggests that the degradation of endocytosed lipid is also regulated at a second, previously unidentified level, independent of the availability of cell surface ligand receptors. We speculate that this down-regulation in the intracellular hydrolysis of endocytosed lipid may account for the observed accumulation of 125I-AcLDL in these cells.

Low density lipoproteins (LDL) can be oxidatively modified in vitro by macrophages and certain other cell types so that macrophages will take them up much faster. This process may be important in the formation of cholesterol-laden foam cells derived from macrophages in atherosclerotic lesions. In this study, we have shown that certain flavonoids, plant constituents found in the diet, are potent inhibitors of the modification of 125I-labelled LDL by macrophages, with IC50 values in the micromolar range (e.g. morin and fisetin 1 microM; quercetin and gossypetin 2 microM). The potencies of individual flavonoids in inhibiting LDL modification did not correlate with their previously determined potencies as inhibitors of 5-lipoxygenase and cyclo-oxygenase. The modification of LDL by macrophages exhibits a lag period of about 4-6 hr before enhanced uptake is detected. During this time, there is a rapid depletion in its content of alpha-tocopherol (an endogenous antioxidant found in lipoproteins) followed by a large increase in the level of hydroperoxides. The flavonoids conserved the alpha-tocopherol content of LDL and delayed the onset of detectable lipid peroxidation. Flavonoids also inhibited the cell-free oxidation of LDL mediated by CuSO4. These findings raise the possibility that flavonoids may protect LDL against oxidation in atherosclerotic lesions and may therefore be natural anti-atherosclerotic components of the diet, although this will depend to a large extent on their pharmacokinetics.

1. The kinetics of the depletion of alpha-tocopherol in human low-density lipoprotein (LDL) were measured during macrophage-mediated and cell-free oxidation. The formation of oxidatively modified, high-uptake species of LDL in these systems was not detectable until all of the endogenous alpha-tocopherol had been consumed. 2. Supplementation of the alpha-tocopherol content of LDL by loading in vivo extended the duration of the lag period during which no detectable oxidative modification occurred. 3. The addition of a flavonoid (morin) prevented both alpha-tocopherol consumption and oxidative modification of LDL. 4. The alpha-tocopherol contents of LDLs from a range of individual donors could not be used to predict their relative resistance to oxidation, indicating that other endogenous antioxidants may also be present, and quantitatively significant, in human LDL.

Sub-acute exposure to anti-cholinesterase organophosphorous compounds induces, in humans, cognitive and emotional deficits which include depression, anxiety, emotional lability, and schizophrenic-like symptoms. Neuroleptic drugs used to treat similar clinical conditions bind to serotonergic (S2) and dopaminergic (D2) receptors, suggesting that these sites are involved in the psychiatric consequences of organophosphate exposure. Rats were given saline or soman (50 micrograms/kg, SC) on a sub-acute regimen (three times weekly for four weeks) and killed 1 hr, 1 day or 3 days after the final injection. Response of regional neuroleptic receptors to soman intoxication was assessed using 3H-spiperone as ligand. Initial high affinity binding experiments using mianserine, haloperidol, or both to identify specific cortical binding revealed that mianserine displaceable binding sites showed the greatest down-regulation in response to soman. Subsequent kinetic analyses of mianserine displaceable 3H-spiperone binding indicated a dramatic decrease in the number of hippocampal binding sites and a decrease in the affinity of cortical binding sites. These S2 sites, considered to be involved with neural excitation, have the ability to self-regulate and appear to be involved in the expression of soman neurotoxicity.

The intracarotid artery quick injection technique of Oldendorf was utilized to determine the Brain Uptake Index (BUI) of radio-labeled peptides in comparison with 3H2O or 14C-antipyrine as counterlabels. The normalized BUI values for 3H-MIF-I, 3H-alpha-MSH and 14C-AVP were 13.7, 9.6 and 13.0 respectively at 15 sec after injection consistent with their having readily penetrated the blood-brain barrier. The BUI values were similar, though somewhat increased, at 10 min postinjection consistent with their ready exit across the blood-brain barrier. At 15 sec after injection 0.5+/-0.1%/g brain of the originally injected peptide label was recovered; and 0.1+/-0.2%/g brain was recovered after 10 min. The label was distributed uniformly in the major brain regions at both times. However, the percentage of the originally injected label/g of pineal and pituitary gland tissue was 10-20 X increased as compared with the major brain regions as would be expected by their location outside the blood-brain barrier. The in vitro uptake of the radio-labeled peptides by synaptosomes prepared from the whole brain and the major brain regions was passive; it was not temperature dependent, nor was it Na+ dependent. However, the binding of the three peptides by the synaptosomes varied considerably: AVP greater than MSH greater MIF: 50 greater than 5 greater 1. The penetratin of the blood-brain barrier by the three peptides is consistent with their having CNS effects.

Indomethacin is currently used for the pharmacologic closure of PDA in preterm infants with respiratory distress syndrome. However, the response to the drug has been variable and the disposition of the drug in preterm infants is not well understood. We studied the pharmacokinetics of indomethacin in nine preterm infants with birth weights ranging from 800 to 1,960 gm and gestational ages of 28 to 36 weeks. Three different dose schedules (0.1, 0.25, 0.3 mg/kg dose) were used. The plasma half-life of indomethacin ranged from 11 to 20 hours. Peak levels were achieved within four hours and ranged from 0.027 to 0.310 microgram/ml. The half-life in infants less than 32 weeks' gestation was significantly prolonged compared to that in infants greater than 32 weeks. Protein-binding studies with 14C indomethacin showed that 98% of indomethacin was protein bound. Absorption of orally administered indomethacin appears to be poor and incomplete. No immediate major complications could be correlated to indomethacin therapy in this study.

Synaptosomes were incubated at various time intervals following injection of 120 micrograms/kg SC of soman or sarin or with various concentrations (10(-8) to 10(-2) M) of soman or sarin in vitro. Total cholinesterase (ChE) activities in each brain region were also measured. Following soman injection, sodium-dependent, high affinity choline uptake (SDHACU) was decreased from 1 to 4 hr in the cortex and from 1 to 2 hr in the hippocampus, but increased from 2 to 24 hr in the striatum. Similarly, following sarin injection SDHACU was decreased at 0.5 hr in the cortex and from 1 to 4 hr in the hippocampus, but increased at 1 hr in the striatum. Injection of soman severely inhibited (83-99%) total ChE activity in the cortex, hippocampus and striatum from 1 to 24 hr. In contrast, sarin did not severely inhibit ChE activity in these regions and maximal inhibition (40-60%) did not occur until 24 hr after injection. With both compounds, by 168 hr ChE activity in all regions had partially recovered. Incubation of synaptosomes with soman or sarin in vitro at concentrations below 10(-4) M did not affect SDHACU in any of the brain regions. These data demonstrated that acute soman and sarin injection produced similar effects upon SDHACU in different brain regions, although the time-course of these effects was different for the two compounds. These effects were probably neither due to a direct action of these compounds on the uptake process nor dependent on ChE inhibition.