A hemizygous supergene controls homomorphic and heteromorphic self-incompatibility systems in Oleaceae.

Self-incompatibility (SI) has evolved independently multiple times and prevents self-fertilization in hermaphrodite angiosperms. Several groups of Oleaceae such as jasmines exhibit distylous flowers, with two compatibility groups each associated with a specific floral morph.1 Other Oleaceae species in the olive tribe have two compatibility groups without associated morphological variation.2,3,4,5 The genetic basis of both homomorphic and dimorphic SI systems in Oleaceae is unknown. By comparing genomic sequences of three olive subspecies (Olea europaea) belonging to the two compatibility groups, we first locate the genetic determinants of SI within a 700-kb hemizygous region present only in one compatibility group. We then demonstrate that the homologous hemizygous region also controls distyly in jasmine. Phylogenetic analyses support a common origin of both systems, following a segmental genomic duplication in a common ancestor. Examination of the gene content of the hemizygous region in different jasmine and olive species suggests that the mechanisms determining compatibility groups and floral phenotypes (whether homomorphic or dimorphic) in Oleaceae rely on the presence/absence of two genes involved in gibberellin and brassinosteroid regulation.

Two Cases of Viral Re-suppression After M184V + R263K Selection on Dolutegravir/Lamivudine Without Treatment Modification.

Dolutegravir/lamivudine (DTG/3TC) has a high genetic barrier against the development of human immunodeficiency virus drug resistance. We report 2 cases of R263K + M184V mutations during DTG/3TC failure followed by viral suppression after adherence intervention without treatment change that we attribute to residual drug activity, reduced viral fitness, and robust immune competence.

Comparison of HIV-1 DNA load measurements in blood and in relation to successful proviral sequencing.

OBJECTIVE: HIV DNA sequencing is now routinely used for HIV-infected individuals on antiretroviral therapy (ART) with or without partial genotypic history. Successful amplification of HIV pol gene has yet to be correlated with HIV DNA levels. Here, we assessed the relationship between HIV DNA load and sequencing results. METHODS: We analyzed three different qPCR measurements of total (LTR and LTR-gag) and integrated (Alu-LTR) HIV DNA in blood samples collected from viremic as well as virally suppressed HIV-infected individuals on ART. HIV DNA levels were compared to HIV DNA Sanger sequencing and clinical and therapeutic parameters. RESULTS: Among the 135 individuals analyzed for HIV DNA measurements and sequencing, all three HIV DNA measurements were associated with HIV DNA Sanger sequencing results. A threshold of around 2 and 1.5 log copies/million leukocytes of total HIV DNA was identified for LTR and LTR-gag qPCRs, respectively. Integrated HIV DNA positivity was also associated with successful sequencing. We further compared HIV DNA measurement techniques in an extended cohort of 312 individuals and showed that all measurements correlated between the different techniques, regardless of the HIV-1 subtypes analyzed. However, higher detection rates were observed with LTR (96%) compared to LTR-gag (86%) and Alu-LTR (59%) qPCRs. Duration of virological control on ART and CD4 nadir were the main determinants of HIV reservoir size. CONCLUSIONS: HIV DNA measurement is associated with Sanger sequencing success, regardless of the technique used. In a clinical setting, Application of HIV DNA quantification before sequencing should be further evaluated.

Development of a non-target strategy for evaluation of potential biological effects of inhalable aerosols generated during purposeful room conditioning using an in vitro inhalation model.

OBJECTIVES: An integrated in vitro inhalation approach was outlined to estimate potential adverse acute inhalation effects of aerosols from commercial nebulizer applications used for purposeful room conditioning such as disinfection, scenting or others. Aerosol characterization, exposure estimation and evaluation of acute biological effects by in vitro inhalation were included to generate dose-response data, allowing for determination of in vitro lowest observable adverse effect levels (LOAELs). Correlation of these to estimates of human lung deposition was included for quantitative in vitro to in vivo extrapolation approach (QIVIVE) for acute effects during human exposure. METHODS: To test the proposed approach, a case study was undertaken using two realistic test materials. An acute in vitro inhalation setup with air-liquid interface A549-cells in an optimized exposure situation (P.R.I.T.® ExpoCube®) was used to expose cells and analysis of relevant biological effects (viability, mitochondrial membrane potential, stress, IL-8 release) was carried out. RESULTS: The observed dose-responsive effects in a sub-toxic dose-range could be attributed to the main component of one test material and its presence in the aerosol phase of the nebulized material. QIVIVE resulted in a factor of at least 256 between the in vitro LOAEL and the estimated acute human lung exposure for this test material. CONCLUSIONS: The case-study shows the value of the non-target in vitro inhalation testing approach especially in case of a lack of knowledge on complex product composition. It is expected that approaches like this will be of high value for product safety and environmental health in the future.

Design of a routine in vitro inhalation approach to estimate biological effects of nebulized products.Application in a case study on a potential real product for purposeful room conditioning by use of a commercial nebulizer.Combining results from aerosol characterization and in vitro inhalation experiments allowed for comprehensive correlation of product composition, aerosol properties and biological effects.Assignment of sub-toxic biological effects to a specific product component enabled identification of a product composition with potentially even less biological effect.Combined in vivo exposure estimation and in vitro LOAEL determination enabled a QIVIVE approach.

Perampanel as precision therapy in rare genetic epilepsies.

OBJECTIVE: Perampanel, an antiseizure drug with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist properties, may have a targeted effect in genetic epilepsies with overwhelming glutamate receptor activation. Epilepsies with loss of γ-aminobutyric acid inhibition (e.g., SCN1A), overactive excitatory neurons (e.g., SCN2A, SCN8A), and variants in glutamate receptors (e.g., GRIN2A) hold special interest. We aimed to collect data from a large rare genetic epilepsy cohort treated with perampanel, to detect possible subgroups with high efficacy. METHODS: This multicenter project was based on the framework of NETRE (Network for Therapy in Rare Epilepsies), a web of pediatric neurologists treating rare epilepsies. Retrospective data from patients with genetic epilepsies treated with perampanel were collected. Outcome measures were responder rate (50% seizure reduction), and percentage of seizure reduction after 3 months of treatment. Subgroups of etiologies with high efficacy were identified. RESULTS: A total of 137 patients with 79 different etiologies, aged 2 months to 61 years (mean = 15.48 ± 9.9 years), were enrolled. The mean dosage was 6.45 ± 2.47 mg, and treatment period was 2.0 ± 1.78 years (1.5 months-8 years). Sixty-two patients (44.9%) were treated for >2 years. Ninety-eight patients (71%) were responders, and 93 (67.4%) chose to continue therapy. The mean reduction in seizure frequency was 56.61% ± 34.36%. Sixty patients (43.5%) sustained >75% reduction in seizure frequency, including 38 (27.5%) with >90% reduction in seizure frequency. The following genes showed high treatment efficacy: SCN1A, GNAO1, PIGA, PCDH19, SYNGAP1, POLG1, POLG2, and NEU1. Eleven of 17 (64.7%) patients with Dravet syndrome due to an SCN1A pathogenic variant were responders to perampanel treatment; 35.3% of them had >90% seizure reduction. Other etiologies remarkable for >90% reduction in seizures were GNAO1 and PIGA. Fourteen patients had a continuous spike and wave during sleep electroencephalographic pattern, and in six subjects perampanel reduced epileptiform activity. SIGNIFICANCE: Perampanel demonstrated high safety and efficacy in patients with rare genetic epilepsies, especially in SCN1A, GNAO1, PIGA, PCDH19, SYNGAP1, CDKL5, NEU1, and POLG, suggesting a targeted effect related to glutamate transmission.

Incorporating integrated testing strategy (ITSv1) defined approach into read-across (RAx) in predicting skin sensitization potency: ITSv1-based RAx.

Recently, due to regulatory and ethical demands, new approach methodologies (NAMs), defined approaches (DAs), and read-across (RAx) have been used in the risk assessment of skin sensitization. Integrated testing strategy (ITS)v1 DA, adopted in OECD Guideline No. 497, can be used for skin sensitization potency categorization. However, ITSv1 DA alone is not used for further refinement of the potency prediction based on EC3 (the estimated concentration that produces a stimulation index of 3 in murine local lymph node assay) values. Moreover, there is no explicit approach to incorporating NAM/DA data into RAx to fill the data gap of EC3 values with high confidence. This study developed a strategy incorporating ITSv1 DA into RAx to predict skin sensitization potency: ITSv1-based RAx. To examine the reliability of this novel strategy, a case study with lilial, a fragrance material, was performed. Based on ITSv1-based RAx, the skin sensitization potency of lilial was determined by extrapolating the EC3 value of 9.5% for the suitable analogue bourgeonal, which was close to the historical EC3 value of 8.6%. The result suggested that the strategy can refine the prediction of EC3 values with high confidence and be useful for the risk assessment of skin sensitization.

HIV Productively Infects Highly Differentiated and Exhausted CD4+ T Cells During AIDS.

Background: Throughout HIV infection, productively infected cells generate billions of viral particles and are thus responsible for body-wide HIV dissemination, but their phenotype during AIDS is unknown. As AIDS is associated with immunological changes, analyzing the phenotype of productively infected cells can help understand HIV production during this terminal stage. Methods: Blood samples from 15 untreated viremic participants (recent infection, n=5; long-term infection, n=5; active opportunistic AIDS-defining disease, n=5) and 5 participants virologically controlled on antiretroviral therapy (ART) enrolled in the Analysis of the Persistence, Reservoir and HIV Latency (APRIL) study (NCT05752318) were analyzed. Cells expressing the capsid protein p24 (p24+ cells) after 18 hours of resting or 24 hours of stimulation (HIV-Flow) revealed productively infected cells from viremic participants or translation-competent reservoir cells from treated participants, respectively. Results: The frequency of productively infected cells tended to be higher during AIDS in comparison with recent and long-term infections (median, 340, 72, and 32/million CD4+ T cells, respectively) and correlated with the plasma viral load at all stages of infection. Altogether, these cells were more frequently CD4low, HLA-ABClow, CD45RA-, Ki67+, PD-1+, with a non-negligible contribution from pTfh (CXCR5+PD-1+) cells, and were not significantly enriched in HIV coreceptors CCR5 nor CXCR4 expression. The comparison markers expression between stages showed that productively infected cells during AIDS were enriched in memory and exhausted cells. In contrast, the frequencies of infected pTfh were lower during AIDS compared to non-AIDS stages. A UMAP analysis revealed that total CD4+ T cells were grouped in 7 clusters and that productive p24+ cells were skewed to given clusters throughout the course of infection. Overall, the preferential targets of HIV during the latest stages seemed to be more frequently highly differentiated (memory, TTD-like) and exhausted cells and less frequently pTfh-like cells. In contrast, translation-competent reservoir cells were less frequent (5/million CD4+ T cells) and expressed more frequently HLA-ABC and less frequently PD-1. Conclusions: In long-term infection and AIDS, productively infected cells were differentiated and exhausted. This could indicate that cells with these given features are responsible for HIV production and dissemination in an immune dysfunction environment occurring during the last stages of infection.

AusTraits, a curated plant trait database for the Australian flora.

We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge.

Versailles Project on Advanced Materials and Standards interlaboratory study on intensity calibration for x-ray photoelectron spectroscopy instruments using low-density polyethylene.

We report the results of a Versailles Project on Advanced Materials and Standards interlaboratory study on the intensity scale calibration of x-ray photoelectron spectrometers using low-density polyethylene (LDPE) as an alternative material to gold, silver, and copper. An improved set of LDPE reference spectra, corrected for different instrument geometries using a quartz-monochromated Al Kα x-ray source, was developed using data provided by participants in this study. Using these new reference spectra, a transmission function was calculated for each dataset that participants provided. When compared to a similar calibration procedure using the NPL reference spectra for gold, the LDPE intensity calibration method achieves an absolute offset of ∼3.0% and a systematic deviation of ±6.5% on average across all participants. For spectra recorded at high pass energies (≥90 eV), values of absolute offset and systematic deviation are ∼5.8% and ±5.7%, respectively, whereas for spectra collected at lower pass energies (<90 eV), values of absolute offset and systematic deviation are ∼4.9% and ±8.8%, respectively; low pass energy spectra perform worse than the global average, in terms of systematic deviations, due to diminished count rates and signal-to-noise ratio. Differences in absolute offset are attributed to the surface roughness of the LDPE induced by sample preparation. We further assess the usability of LDPE as a secondary reference material and comment on its performance in the presence of issues such as variable dark noise, x-ray warm up times, inaccuracy at low count rates, and underlying spectrometer problems. In response to participant feedback and the results of the study, we provide an updated LDPE intensity calibration protocol to address the issues highlighted in the interlaboratory study. We also comment on the lack of implementation of a consistent and traceable intensity calibration method across the community of x-ray photoelectron spectroscopy (XPS) users and, therefore, propose a route to achieving this with the assistance of instrument manufacturers, metrology laboratories, and experts leading to an international standard for XPS intensity scale calibration.

Contact allergy to hair-colouring products: a cosmetovigilance follow-up study by four companies in Europe from 2014 to 2017.

BACKGROUND: A previous analysis of undesirable events (UEvs), reported to four major companies following the use of hair-colouring products in Europe, showed that the reporting rates were stable for both oxidative and direct hair-colouring products over the period 2003-2006. OBJECTIVES: In order to verify the impact of risk management measures implemented since 2006, as well as the impact of a new Commission Regulation (No 1223/2009), the same four companies analysed cosmetovigilance data collected over an additional four-year period (2014-2017). The objective was to determine whether there was any time effect, country effect, or product type effect, as well as identify risk factors. MATERIALS AND METHODS: Each company collected reports of alleged UEvs, undesirable effects (UEfs) and serious undesirable effects (SUEs) for their products in their key European markets, and calculated the respective reporting rates (number of events/million units sold). A detailed analysis was performed on allergic contact dermatitis-type events. RESULTS: The reporting rates for alleged UEvs and allergic-type UEfs associated with hair-colouring products remained stable over the four-year period, although a statistically significant decrease was observed for some companies. No time effect on SUEs was observed for three companies but a statistically significant decrease in SUEs was observed for one company. Black henna tattoos remained a major risk factor regarding SUEs due to hair dyes. CONCLUSION: The reporting rates of undesirable events, including contact allergy-type events, were stable over time. This was true for oxidative and direct hair dyes, for both home use and professional exposure scenarios.

Understanding the Lifetime of Battery Cells Based on Solid-State Li6PS5Cl Electrolyte Paired with Lithium Metal Electrode.

All-solid-state batteries with solid electrolytes having ionic conductivities in the range of those of liquid electrolytes have gained much interest as safety is still a major issue for applications. Meanwhile, lithium metal seems to be the anode material of choice to face the demand for higher capacities. Still, the main challenges that come with the use of a lithium metal anode, i.e., formation and growth of lithium dendrites, are still not understood very well. This work focuses on the reasons of the lifetime behavior of lithium symmetric cells with the solid electrolyte Li6PS5Cl and lithium electrode. In particular, the voltage increases during the application of a constant current density are investigated. The interface between the lithium metal electrode and the solid electrolyte is analyzed by X-ray photoelectron spectroscopy, and the resistance changes of each electrode during stripping and plating are investigated by impedance spectroscopy on a three-electrode cell. A main factor for the lifetime influenced by lithium dendrite formation and growth is the buildup of a lithium vacancy gradient, leading to voids which decrease the interface area and therefore increase the local current density. Additionally, those lithium vacancies in lithium metal represent a limitation for conductivity rather than migration in solid electrolyte. Further experiments indicate that the seedlike plating behavior of lithium also plays a key role in increased local current density and therefore decreased lifetime. Plating of only a small amount of lithium leads to small areas of well-connected interfaces, resulting in high local current density. A medium amount of plated lithium leads to larger areas of interface between lithium and electrolyte, balancing the current density distribution. In contrast, a high amount of repeatedly deposited lithium leads to lithium seed plating on top of already plated lithium. Those seed spots grown on top represent a better interface connection, which again leads to higher local current densities at those spots and therefore results in shorter lifetimes due to short circuits caused by lithium dendrites.

Threshold of Toxicological Concern (TTC) for Botanical Extracts (Botanical-TTC) derived from a meta-analysis of repeated-dose toxicity studies.

Threshold of Toxicological Concern (TTC) is a promising approach for evaluating the human health risk for systemic toxicity when there is a lack of toxicological information. The threshold for systemic toxicity is reportedly 1800, 540, and 90 µg/day for Cramer I-III chemical structures, according to Munro's structural decision tree, and 0.15 µg/day for genotoxic compounds. However, the concept of TTC has been developed for single substances; therefore, the applicability of TTC for mixtures remains unclear. To expand application of probability approach for mixtures, a validation study using the point of departures (PoDs) derived from mixtures is required. In the present study, we investigated novel TTC of botanical extracts (Botanical-TTC) for cosmetics from a meta-analysis based on the PoDs derived from repeated dose toxicity testing in botanical extracts. Accordingly, 213 PoDs were determined by repeated-dose toxicity studies and divided using a default uncertainty factor of 100 combined with the extrapolation factor of study duration to calculate the derived-no-effect-level (DNEL) and derived-minimal-effect-level (DMEL). The minimum DNEL/DMEL was 1.6-fold higher than the Cramer III TTC. In addition, because human health risk below the 1 st percentile value (663 µg/day) was considered as extremely limited, the exposure level can be proposed as Botanical-TTC.

A research agenda for seed-trait functional ecology.

Trait-based approaches have improved our understanding of plant evolution, community assembly and ecosystem functioning. A major challenge for the upcoming decades is to understand the functions and evolution of early life-history traits, across levels of organization and ecological strategies. Although a variety of seed traits are critical for dispersal, persistence, germination timing and seedling establishment, only seed mass has been considered systematically. Here we suggest broadening the range of morphological, physiological and biochemical seed traits to add new understanding on plant niches, population dynamics and community assembly. The diversity of seed traits and functions provides an important challenge that will require international collaboration in three areas of research. First, we present a conceptual framework for a seed ecological spectrum that builds upon current understanding of plant niches. We then lay the foundation for a seed-trait functional network, the establishment of which will underpin and facilitate trait-based inferences. Finally, we anticipate novel insights and challenges associated with incorporating diverse seed traits into predictive evolutionary ecology, community ecology and applied ecology. If the community invests in standardized seed-trait collection and the implementation of rigorous databases, major strides can be made at this exciting frontier of functional ecology.

Allergy Alert Test for p-Phenylenediamine-Allergic Hair Dye Users.

BACKGROUND: Contact dermatitis to hair dyes remains a health concern. Regulations in many countries require consumer self-testing for hair dyes, but no standardized procedure exists. OBJECTIVE: The aim of this study was to develop a self-test protocol for an allergy alert test (AAT) that can elicit a self-noticeable alert signal in p-phenylenediamine (PPD)-allergic consumers. METHODS: Simulating consumer use conditions (open application for 45 minutes after mixing with a developer), PPD-positive hair dye-allergic subjects and PPD-negative control subjects were tested on the forearm and behind the ear with experimental products containing 0.05%, 0.25%, 0.75%, and 2% PPD. Reactions were self-evaluated by subjects and independently assessed by dermatologists. CONCLUSIONS: The AAT caused a reaction self-noticeable on the forearm in 90.5% (38/42) and behind the ear in 93% (39/42) of the PPD-positive subjects. This was objectified by a dermatological evaluation. The strength of the AAT response and the number of responding subjects increased with increasing PPD concentrations. Allergy alert test responses were also dependent on the reaction strength of the diagnostic patch test to PPD before the study; in subjects with (+++) patch test reactions, 19 of 19 were positive. All 48 control subjects were negative to the AAT. Therefore, the AAT protocol provides a signal indicative of an allergic reaction in PPD-allergic hair dye consumers.

Skin sensitization quantitative risk assessment for occupational exposure of hairdressers to hair dye ingredients.

Occupational exposure of hairdressers to hair dyes has been associated with the development of allergic contact dermatitis (ACD) involving the hands. p-Phenylenediamine (PPD) and toluene-2,5-diamine (PTD) have been implicated as important occupational contact allergens. To conduct a quantitative risk assessment for the induction of contact sensitization to hair dyes in hairdressers, available data from hand rinsing studies following typical occupational exposure conditions to PPD, PTD and resorcinol were assessed. By accounting for wet work, uneven exposure and inter-individual variability for professionals, daily hand exposure concentrations were derived. Secondly, daily hand exposure was compared with the sensitization induction potency of the individual hair dye defined as the No Expected Sensitization Induction Levels (NESIL). For PPD and PTD hairdresser hand exposure levels were 2.7 and 5.9 fold below the individual NESIL. In contrast, hand exposure to resorcinol was 50 fold below the NESIL. Correspondingly, the risk assessment for PPD and PTD indicates that contact sensitization may occur, when skin protection and skin care are not rigorously applied. We conclude that awareness of health risks associated with occupational exposure to hair dyes, and of the importance of adequate protective measures, should be emphasized more fully during hairdresser education and training.

Distribution and reduction magnitude of HIV-DNA burden in CD4+ T cell subsets depend on art initiation timing.

OBJECTIVE: The aim of our study was to analyze the dynamics of HIV-DNA levels in CD4 T-cell subsets in individuals starting successful dolutegravir-based regimens. DESIGN: Twenty-seven individuals with acute infection (AI, n = 8) or chronic infection (CI, n = 5) and patients in virological success (VS, n = 10) or virological failure (VF, n = 4) on antiretroviral therapy (ART) who initiated a dolutegravir-based regimen were enrolled (NCT02557997). METHODS: CD4 T-cells from baseline and week 48 of successful treatment were sorted into effector memory (TEM), transitional memory (TTM), central memory (TCM) and naïve (TN) cell groups for total HIV-DNA measurements by qPCR. Bayesian methods were used to estimate the posterior probability of a HIV-DNA decrease more than 0.25 log copies/10 cells at week 48. RESULTS: All patients achieved HIV-RNA suppression at 48 weeks. At baseline and week 48, the highest contributions to the HIV-DNA-infected pool from CD4 T cells were observed in TTM cells in the AI group (62.4 and 60.2%, respectively), but in TCM cells for the CI, VS and VF groups (54.6 and 59.4%, 58.2 and 62.9%, 62.4 and 67.2%), respectively. HIV-DNA burden declined in all subsets after 48 weeks of treatment in the AI (probability (Pr) > 91%), CI (Pr > 52%) and VF (Pr > 52%) groups, but only in TEM cells in the VS group (Pr = 95%). CONCLUSION: Our study showed that dolutegravir-based treatment reduced the HIV-DNA cellular burden in individuals from the AI, CI and VF groups, though the reduction levels differed between the patient subgroups. Early treated patients had the highest probability of HIV-DNA reduction. Interestingly, in the aviremic VS group, HIV-DNA reduction was limited to TEM cells.

Necessity for retrospective evaluation of past-positive chemicals in in vitro chromosomal aberration tests using recommended cytotoxicity indices.

We have demonstrated that retrospective evaluation of existing data of in vitro chromosomal aberration test using the new cytotoxicity indices RICC (relative increase in cell count) or RPD (relative population doubling) reduces the false-positive rate. We have constructed an algorithm to predict the likelihood that past-positive results would differ when retested accordingly. Here, we emphasize the importance of reviewing existing in vitro chromosomal aberration test results. The present Letter not only supports the rediscovery of potentially useful chemicals excluded from further development as a result of misclassification due to in vitro false-positive results, but also contributes to the development of a precise Quantitative Structure-Activity Relationship (QSAR) model by providing an appropriate training data-set. Furthermore, re-evaluation is expected to provide novel insights into underlying mechanisms and/or key structures involved in the development of chromosomal aberrations.

Are pharmaceuticals with evolutionary conserved molecular drug targets more potent to cause toxic effects in non-target organisms?

The ubiquitous use of pharmaceuticals has resulted in a continuous discharge into wastewater and pharmaceuticals and their metabolites are found in the environment. Due to their design towards specific drug targets, pharmaceuticals may be therapeutically active already at low environmental concentrations. Several human drug targets are evolutionary conserved in aquatic organisms, raising concerns about effects of these pharmaceuticals in non-target organisms. In this study, we hypothesized that the toxicity of a pharmaceutical towards a non-target invertebrate depends on the presence of the human drug target orthologs in this species. This was tested by assessing toxicity of pharmaceuticals with (miconazole and promethazine) and without (levonorgestrel) identified drug target orthologs in the cladoceran Daphnia magna. The toxicity was evaluated using general toxicity endpoints at individual (immobility, reproduction and development), biochemical (RNA and DNA content) and molecular (gene expression) levels. The results provide evidence for higher toxicity of miconazole and promethazine, i.e. the drugs with identified drug target orthologs. At the individual level, miconazole had the lowest effect concentrations for immobility and reproduction (0.3 and 0.022 mg L-1, respectively) followed by promethazine (1.6 and 0.18 mg L-1, respectively). At the biochemical level, individual RNA content was affected by miconazole and promethazine already at 0.0023 and 0.059 mg L-1, respectively. At the molecular level, gene expression for cuticle protein was significantly suppressed by exposure to both miconazole and promethazine; moreover, daphnids exposed to miconazole had significantly lower vitellogenin expression. Levonorgestrel did not have any effects on any endpoints in the concentrations tested. These results highlight the importance of considering drug target conservation in environmental risk assessments of pharmaceuticals.

L-arginine/NO pathway is altered in children with haemolytic-uraemic syndrome (HUS).

The haemolytic uraemic syndrome (HUS) is the most frequent cause of acute renal failure in childhood. We investigated L-arginine/NO pathway in 12 children with typical HUS and 12 age-matched healthy control subjects. Nitrite and nitrate, the major NO metabolites in plasma and urine, asymmetric dimethylarginine (ADMA) in plasma and urine, and dimethylamine (DMA) in urine were determined by GC-MS and GC-MS/MS techniques. Urinary measurements were corrected for creatinine excretion. Plasma nitrate was significantly higher in HUS patients compared to healthy controls (P = 0.021), whereas urine nitrate was borderline lower in HUS patients compared to healthy controls (P = 0.24). ADMA plasma concentrations were insignificantly lower, but urine ADMA levels were significantly lower in the HUS patients (P = 0.019). Urinary DMA was not significantly elevated. In HUS patients, nitrate (R = 0.91) but not nitrite, L-arginine, or ADMA concentrations in plasma correlated with free haemoglobin concentration. Our results suggest that both NO production and ADMA synthesis are decreased in children with typical HUS. We hypothesize that in the circulation of children with HUS a vicious circle between the L-arginine/NO pathway and free haemoglobin-mediated oxidative stress exists. Disruption of this vicious circle by drugs that release NO and/or sulphydryl groups-containing drugs may offer new therapeutic options in HUS.