Toxicokinetics of 2-ethylhexyl salicylate (EHS) and its seven metabolites in humans after controlled single dermal exposure to EHS.

The chemical UV filter 2-ethylhexyl salicylate (EHS) is used in various personal-care products. The dermal and oral metabolism of EHS have already been targeted by different studies. However, toxicokinetic data after a single dermal exposure to EHS was missing. In our study, three volunteers were dermally exposed to a commercial EHS-containing sunscreen for 9 h with an application dose of 2 mg sunscreen per cm2 body surface area. The exposure was performed indoors, and sunscreen was applied on about 75% of the total skin area. Complete urine voids were collected over 72 h and eight blood samples were drawn from each subject. Urine samples were analyzed for EHS and seven known metabolites (5OH-EHS, 4OH-EHS, 2OH-EHS, 6OH-EHS, 4oxo-EHS, 5oxo-EHS, and 5cx-EPS) by online-SPE UPLC MS/MS. The peaks of urinary elimination occurred 10-11 h after application. The elimination half-lives (Phase 1) were between 6.6 and 9.7 h. The dominant urinary biomarkers were EHS itself, followed by 5OH-EHS, 5cx-EPS, 5oxo-EHS, and 4OH-EHS. 2OH-EHS, 6OH-EHS, and 4oxo-EHS were detected only in minor amounts. An enhanced analysis of conjugation species revealed marginal amounts of unconjugated metabolites and up to 40% share of sulfate conjugates for 5OH-EHS, 5oxo-EHS, and 5cx-EPS. The results demonstrated a delayed systemic resorption of EHS via the dermal route. Despite an extensive metabolism, the parent compound occurred as main urinary parameter. The delayed dermal resorption as well as the slow elimination of EHS indicate an accumulation up to toxicological relevant doses during daily repeated dermal application to large skin areas.

Implementation of an electronic patient portal in routine mental health care of hospitals in Germany - evaluation of attitudes of healthcare providers.

BACKGROUND: The use of digital tools such as electronic patient portals in different health care disciplines and settings has been increasing, but the rate of implementation in clinical practice still lags behind expectations. While studies have linked the use of electronic patient portals to positive health outcomes for patients, studies addressing the viewpoints of healthcare providers are rare. METHODS: We performed an online survey of attitudes of healthcare providers towards an electronic patient portal for mental health hospitals. The portal was developed by five communal providers of mental health care in different regions in Germany. The survey was carried out during the early phase of implementation of the portal. RESULTS: Twenty project leaders and 37 clinicians from five different mental health hospitals answered the questionnaire (response rate: 45% and 28%). Overall, acceptance of online applications among respondents was high. The healthcare providers mentioned perceived benefits (e.g. accessibility of new patient groups, use of therapy-free periods) as well as a number of technical, structural, organizational and staffing barriers for successful implementation in hospital settings (e.g. workload of healthcare providers and lack of staff, limited digital competences, unstable WLAN). CONCLUSION: The perceived barriers and facilitators of the implementation of online applications and electronic patient portals in mental health hospitals identified by healthcare providers may be taken into account. Improving commitment of the healthcare providers to implementation and use of digital interventions may help foster digitalisation in mental health hospitals.

Assessment of the plasticizer exposure of hospital workers regularly handling medical devices: A pilot study.

Plastic medical devices, e.g. infusion sets, blood bags or tubing material, that are used manifold in the medical treatment of hospital patients, usually contain considerable amounts of plasticizers. Whereas several studies showed highly elevated inner plasticizer levels of patients treated with plasticized medical devices, little is known about the exposure situation of hospital staff. The present pilot study aimed to evaluate the urinary plasticizer metabolite levels of selected hospital workers of the blood bank (medical technical assistants, MTA) and of perfusionists that are regularly handling plasticized medical devices in order to estimate the work-related amount of the inner individual plasticizer exposure. The study subjects were asked to collect pre- and post-shift spot urine samples over the course of a working week, that were subsequently analyzed for selected urinary metabolites of the plasticizers DEHP, DINCH, DEHTP and TEHTM. Although the observed differences were rather low, a differentiated approach revealed a perceptible impact of the respective workplace environment on the individual urinary plasticizer metabolite levels. Thus, the group of blood bank MTA showed significantly elevated increment levels of urinary DEHP and DINCH metabolites, while the group of perfusionists, showed a considerable higher detection frequency of the main urinary TEHTM metabolite. All in all, however, it can be cautiously concluded by the results of the presented pilot study that a regular handling of plasticized medical devices by hospital employees (via inhalation or dermal contact) contributes demonstrably but yet only marginally to the individual internal plasticizer exposure.

Glycoproteomics Identifies Plexin-B3 as a Targetable Cell Surface Protein Required for the Growth and Invasion of Triple-Negative Breast Cancer Cells.

Driven by the lack of targeted therapies, triple-negative breast cancers (TNBCs) have the worst overall survival of all breast cancer subtypes. Considering that cell surface proteins are favorable drug targets and are predominantly glycosylated, glycoproteome profiling has significant potential to facilitate the identification of much-needed drug targets for TNBCs. Here, we performed N-glycoproteomics on six TNBCs and five normal control (NC) cell lines using hydrazide-based enrichment. Quantitative proteomics and integrative data mining led to the discovery of Plexin-B3 (PLXNB3), a previously undescribed TNBC-enriched cell surface protein. Furthermore, siRNA knockdown and CRISPR-Cas9 editing of in vitro and in vivo models show that PLXNB3 is required for TNBC cell line growth, invasion, and migration. Altogether, we provide insights into N-glycoproteome remodeling associated with TNBCs and functional evaluation of an extracted target, which indicate the surface protein PLXNB3 as a potential therapeutic target for TNBCs.

Metabolites of 4-methylbenzylidene camphor (4-MBC), butylated hydroxytoluene (BHT), and tris(2-ethylhexyl) trimellitate (TOTM) in urine of children and adolescents in Germany - human biomonitoring results of the German Environmental Survey GerES V (2014-2017).

The UV filter 4-methylbenzylidene camphor (4-MBC), used in cosmetics, the antioxidant butylated hydroxytoluene (BHT), used inter alia as a food additive and in cosmetics, and the plasticizer tris(2-ethylhexyl) trimellitate (TOTM), used mainly in medical devices as substitute for di-(2-ethylhexyl) phthalate (DEHP), are suspected to have endocrine disrupting effects. Human biomonitoring methods that allow for assessing the internal exposure of the general population to these substances were recently developed in a German cooperation to enhance the use of human biomonitoring. First-morning void urine samples from 3- to 17-year-old children and adolescents living in Germany were analysed for metabolites of 4-MBC (N = 447), BHT (N = 2091), and TOTM (N = 431) in the population-representative German Environmental Survey on Children and Adolescents 2014-2017 (GerES V). 4-MBC metabolites were found in quantifiable amounts only in single cases and exposure levels remained well below health-based guidance values. In contrast, ubiquitous exposure to BHT became evident with a geometric mean (GM) urinary concentration of the metabolite BHT acid of 2.346 µg/L (1.989 µg/gcreatinine) and a maximum concentration of 248 µg/L (269 µg/gcrea). The highest GM concentration was found in young children aged 3-5 years, yet no specific sources of exposure could be identified. Also, TOTM metabolites were found in quantifiable amounts only in very few samples. None of these findings could be related to previous hospital treatment or exposure via house dust. The presented results will be the basis to derive reference values for exposure of children and adolescents in Germany to BHT and will facilitate to identify changing exposure levels in the general population.

Cell-surface proteomics for the identification of novel therapeutic targets in cancer.

INTRODUCTION: Cancer is the second most common cause of death worldwide and its heterogeneity complicates therapy. Standard cytotoxic regiments disrupt rapidly dividing cells, regardless of their neoplastic status. The introduction of less toxic targeted therapies has partially contributed to the observed decrease in cancer-related mortality. Cell-surface proteins represent attractive targets for therapy, due to their easily-accessible localization and their involvement in essential signaling pathways, often dysregulated in cancer. Despite their clinical appeal, cell-surface proteins are often underrepresented in standard proteomic data sets, due to their poor solubility and lower expression levels compared to intracellular proteins. Areas covered: This review will summarize some of the available techniques for enriching the cell-surface proteome, and discuss their advantages, limitations and applicability to clinical sample-testing. Moreover, we discuss currently available strategies for the development of novel targeted therapies in cancer. Expert commentary: The interest in elucidating the cancer-associated surfaceome is growing and will likely benefit from recent advancements in instrument sensitivity, method development, and a growing body of high-quality proteomics databases. Multiomics studies, in combination with functional validations (e.g. dropout screens), and evaluation of the healthy surfaceome, will likely aid in the selection of relevant targets for future therapy development.

New metabolites of 2-ethylhexyl salicylate in human urine after simulated real-life dermal sunscreen application.

2-Ethylhexyl salicylate (EHS) is an organic UV filter which is used in sunscreen and other personal care products. The dermal uptake of EHS was studied in several dermal-exposure experiments. This paper aims to coherently assess urine samples after dermal exposure for the biomarkers EHS, 5OH-EHS, 5oxo-EHS, and 5cx-EPS as well as further biomarkers of interest, specifically 4OH-EHS, 4oxo-EHS, 2OH-EHS, and 6OH-EHS, for the first time. Samples from 18 participants of a pre-existing dermal exposure study under real-life conditions were reassessed using a comprehensive LC-MS/MS method. EHS accounts for 34 % of the cumulative excretion of all analytes within 24 h after exposure, followed by 5OH-EHS (19 %), 5cx-EPS (18 %), 4OH-EHS (15 %) and 5oxo-EHS (11 %). Further metabolites were only quantified in minor amounts. EHS as the most prominent excretion parameter in this study demonstrates the missing first-pass effect after dermal absorption. Furthermore, the applied novel comprehensive analytical procedure revealed oxidation at the ω (5cx-EPS, 6OH-EHS), ω-1 (5OH-EHS, 5oxo-EHS), and ω-2 positions (4OH-EHS, 4oxo-EHS) in the main chain of the ethylhexyl group as well as oxidation in the side chain (2OH-EHS). The presented data are of high relevance for a reliable toxicological risk assessment of dermal exposure to EHS.

Comprehensive assessment of the UV-filter 2-ethylhexyl salicylate and its phase I/II metabolites in urine by extended enzymatic hydrolysis and on-line SPE LC-MS/MS.

2-ethylhexyl salicylate (EHS) is used as a UV filter in personal-care products, such as sunscreen, to prevent skin damage through UV radiation. The application of EHS-containing products leads to systemic EHS absorption, metabolization and excretion. To measure EHS and its corresponding metabolite levels in urine, a comprehensive analytical procedure based on an extended enzymatic hydrolysis, on-line-SPE, and UPLC-MS/MS was developed. The method covers a large profile of seven metabolites (including isomeric structures) as well as EHS itself in a run time only of 18 min. Easy sample preparation, consisting of a 2-h hydrolysis step, followed by on-line enrichment and purification, add to the efficiency of the method. An update, compared to a previous method for the determination of EHS and metabolites in urine, is that, during hydrolysis, both glucuronide and sulfate conjugates are considered. The method was furthermore applied to urine samples after a real-life exposure scenario to EHS-containing sunscreen. The method is highly sensitive with limits of detection ranging from 6 to 65 ng/L. Moreover, it is characterized by good precision data, accuracy, and robustness to matrix influences. Application of the method to urine samples following dermal exposure to an EHS-containing sunscreen revealed EHS as the main biomarker after dermal exposure, followed by the major biomarkers 5OH-EHS, 5cx-EPS, 4OH-EHS and 5oxo-EHS. The expansion and optimization of this method decisively contributes to the research on the dermal metabolism of EHS and can be applied in exposure studies and for human biomonitoring.

Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood-brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50-100% of mice. Our study identifies a promising multi-targetable PTP4A-ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

Dynamic profiling of medulloblastoma surfaceome.

Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. The current standard of care (SOC) involves maximal safe resection and chemoradiotherapy in individuals older than 3 years, often leading to devastating neurocognitive and developmental deficits. Out of the four distinct molecular subgroups, Group 3 and 4 have the poorest patient outcomes due to the aggressive nature of the tumor and propensity to metastasize and recur post therapy. The toxicity of the SOC and lack of response in specific subtypes to the SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. To identify differentially enriched surface proteins that could be evaluated for potential future immunotherapeutic interventions, we leveraged N-glycocapture surfaceome profiling on Group 3 MB cells from primary tumor, through therapy, to recurrence using our established therapy-adapted patient derived xenograft model. Integrin 𝛼5 (ITGA5) was one of the most differentially enriched targets found at recurrence when compared to engraftment and untreated timepoints. In addition to being enriched at recurrence, shRNA-mediated knockdown and small molecule inhibition of ITGA5 have resulted in marked decrease in proliferation and self-renewal in vitro and demonstrated a survival advantage in vivo. Together, our data highlights the value of dynamic profiling of cells as they evolve through therapy and the identification of ITGA5 as a promising therapeutic target for recurrent Group 3 MB.

Randomized Phase II Trial of Sunitinib or Cediranib in Alveolar Soft Part Sarcoma.

PURPOSE: Alveolar soft part sarcoma (ASPS) is a rare, highly vascular tumor with few treatment options. We designed a phase II randomized trial to determine the activity and tolerability of single-agent cediranib or sunitinib in patients with advanced metastatic ASPS. PATIENTS AND METHODS: Patients 16 years of age and older were randomized to receive cediranib (30 mg) or sunitinib (37.5 mg) in 28-day cycles. Patients could cross over to the other treatment arm at disease progression. The primary endpoint was to measure the objective response rate (ORR) for each agent. Median progression-free survival (mPFS) for the two arms was also determined. RESULTS: Twenty-nine of 34 enrolled patients were evaluable for response. One patient on each of the initial two treatment arms had a partial response (ORR: 6.7% and 7.1% for cediranib and sunitinib, respectively). Twenty-four patients had a best response of stable disease (86.7% and 78.6% for cediranib and sunitinib, respectively). There were no significant differences in mPFS for the two treatment arms. Clinical benefit (i.e., objective response or stable disease for a minimum of four or six cycles of therapy) on the first-line tyrosine kinase inhibitor (TKI) therapy did not predict benefit on the second-line TKI. Both drugs were well tolerated. As of August 2021, 1 patient (unevaluable for ORR) remains on study. CONCLUSIONS: The study did not meet its endpoints for ORR. Although both TKIs provided clinical benefit, the outcomes may have been attenuated in patients who had progressed ≤6 months before enrollment, potentially accounting for the low response rates. See related commentary by Wilky and Maleddu, p. 1163.

Simultaneous and sensitive determination of the main metabolites of the plasticizer DEHP and its substitutes DEHTP, DINCH and TEHTM in human urine by coupling of on-line SPE, UHPLC and tandem mass spectrometry.

With the prominent but toxicologically critical plasticizer di-(2-ethylhexyl) phthalate (DEHP) declining, alternative plasticizers are increasingly used leading to a continuously more diverse exposure situation of humans with multiple plasticizers. Therefore, an on-line SPE-LC-MS/MS method for the simultaneous determination of the most relevant urinary biomarkers of exposure to DEHP and the alternative plasticizers 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH), di-(2-ethylhexyl) terephthalate (DEHTP) and tri-(2-ethylhexyl) trimellitate (TEHTM) was developed. The method is characterized by a high sensitivity with limits of detection ranging from 0.006 to 0.047 µg L-1 combined with an easy and straightforward sample preparation procedure. The wide linear working range of the method enables a reliable determination of analyte background levels in the general population as well as its potential use for monitoring studies investigating elevated plasticizer exposure settings. The method was successfully applied to urine samples from ten volunteers without occupational exposure to plasticizers revealing ubiquitous background exposure levels of the common plasticizers DEHP, DEHTP and DINCH.

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment.

PURPOSE: Glioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs. EXPERIMENTAL DESIGN: In the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70's role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples. RESULTS: CD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells. CONCLUSION: CD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

Effects of Speaking Rate on Breathing and Voice Behavior.

OBJECTIVES: The objective of this study was to investigate the effects of speaking rate (habitual and fast) and speech task (reading and spontaneous speech) on seven dependent variables: Breath group size (in syllables), Breath group duration (in seconds), Lung volume at breath group initiation, Lung volume at breath group termination, Lung volume excursion for each breath group (in % vital capacity), Lung volume excursion per syllable (in % vital capacity) and mean speaking Fundamental frequency (fO). METHODS: Ten women and seven men were included as subjects. Lung volume and breathing behaviors were measured by respiratory inductance plethysmography and fO was measured from audio recordings by the Praat software. Statistical significance was tested by analysis of variance. RESULTS: For both reading and spontaneous speech, the group increased mean breath group size and breath group duration significantly in the fast speaking rate condition. The group significantly decreased lung volume excursion per syllable in fast speech. Females also showed a significant increase of fO in fast speech. The lung volume levels for initiation and termination of breath groups, as well as lung volume excursions in % vital capacity, showed great individual variations and no significant effects of rate. Significant effects of speech task were found for breath group size and lung volume excursion per syllable, where reading induced more syllables produced per breath group and less % VC spend per syllable as compared to spontaneous speech. Interaction effects showed that the increases in breath group size and breath group duration associated with fast rate were significantly larger in reading than in spontaneous speech. CONCLUSION: Our data from 17 vocally untrained, healthy subjects showed great individual variations but still significant group effects regarding increased speaking rate, where the subjects seemed to spend less air per syllable and inhaled less often as a consequence of greater breath group sizes in fast speech. Subjects showed greater changes in breath group patterns as a consequence of fast speech in reading than in spontaneous speech, indicating that effects of speaking rate are dependent on the speech task.

Sensitive monitoring of the main metabolites of tri-(2-ethylhexyl) trimellitate (TOTM) in urine by coupling of on-line SPE, UHPLC and tandem mass spectrometry.

Tri-(2-ethylhexyl) trimellitate (TOTM or TEHTM) is a substitute for the plasticizer di-(2-ethylhexyl) phthalate (DEHP). Here, a fast and sensitive UHPLC-MS/MS method is presented enabling the simultaneous quantification of the six main TOTM metabolites in urine. These include the primary metabolites 1-MEHTM and 2-MEHTM (1-/2-mono-(2-ethylhexyl) trimellitate) and two oxidized metabolites of each to ensure a precise determination and comparison of the regioselective pathways. The method is based on online enrichment of the analytes after enzymatic hydrolysis with subsequent UHPLC separation and tandem mass spectrometry using isotopically labeled internal standards. The method is distinguished by its high sensitivity with detection limits ranging from 0.01 to 0.04 µg/l and a proficient precision with relative standard deviations well below 10% for each analyte. The application of UHPLC-MS/MS analysis proved to significantly enhance the sensitivity of the method due to the efficient separation of the regioisomeric structures of the TOTM metabolites considered. Additionally, a proficient repeatability and recovery was achieved by the use of structurally identical isotopically labeled internal standard substances. The method was successfully applied to urine samples of infant patients indicating urinary levels of the TOTM metabolites examined in a very low concentration range.

Unintended imitation of fundamental frequency in female speakers.

Objective: Former studies have shown that humans unintentionally adopt speech characteristics from their conversation partners, a phenomenon known as speech convergence. This study investigated imitation of fundamental frequency (Fo) in nine healthy females in a repetition task.Method: The design included two conditions; loud reading (baseline) and repetition after a model talker. The model talker was a female with a high-pitched voice and stimuli included both words and nonwords. All productions were analyzed with regard to mean and SD of Fo and compared between conditions and word types.Results: The participants showed different patterns regarding the effect on Fo, thus indicating that some individuals are more likely to change their speaking Fo in a repetition task than others. Group results showed somewhat but not significantly higher mean Fo in the repetition task as compared to baseline (reading). Moreover, nonwords were produced with a significantly higher mean Fo than real words, in both reading and repetition conditions. No interaction effect between condition and word type was found.

Identification and Validation of Novel Subtype-Specific Protein Biomarkers in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development. METHODS: A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers. Cell surface protein identification was achieved by in vitro biotinylation, followed by mass spectrometric analysis of purified biotin-tagged proteins. Proteins secreted into a chemically defined serum-free cell culture medium were analyzed by shotgun proteomics. RESULTS: Of 3288 identified proteins, 2 pan-PDAC (protocadherin-1 and lipocalin-2) and 2 exocrine-like-specific (cadherin-17 and galectin-4) biomarker candidates have been validated. Proximity ligation assay analysis of the 2 exocrine-like biomarkers revealed their co-localization on the surface of exocrine-like cells. CONCLUSIONS: The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.