The delivery of genetically encoded fluorescent sensors via adeno-associated viral vectors (AAVs) enables the quantification of biological analytes with high spatiotemporal resolution in living animals. In this study, we expose an unreported problem of the approach, in which the presence of repeated subsequences in the sensor's DNA sequence triggers recombination during AAV production. In the case of Förster Resonance Energy Transfer (FRET) sensors, recombination leads to a mixture of fluorescent products, severely compromising in vivo functionality. To counter this phenomenon, we introduce Abundance-Biased Codon Diversification (ABCD), a modification of a previously reported codon diversification method that prevents recombination without sacrificing codon optimization for a target organism. We demonstrate that ABCD greatly facilitates in vivo studies by restoring the functionality of FRET sensors and advanced inducible expression systems delivered via AAV vectors. Our approach offers a robust solution to a previously overlooked challenge, significantly expanding the range of future applications in quantitative imaging and genetic manipulation in living animals using AAV-mediated strategies.

Microsomal epoxide hydrolase (mEH), first identified as detoxifying enzyme, can hydrolyze epoxyeicosatrienoic acids (EETs) to less active diols (DHETs). EETs are potent vasodilatory and pro-angiogenic lipids, also implicated in neurovascular coupling. In mouse brain, mEH is strongly expressed in vascular and perivascular cells in contrast to the related soluble epoxide hydrolase (sEH), predominantly found in astrocytes. While sEH inhibition in stroke has demonstrated neuroprotective effects and increases cerebral blood flow (CBF), data regarding the role of mEH in brain are scarce. Here, we explored the function of mEH in cerebral vasculature by comparing mEH-KO, sEH-KO and WT mice. Basal cerebral volume (CBV0) was significantly higher in various mEH-KO brain areas compared to WT and sEH-KO. In line, quantification of cerebral vasculature in cortex and thalamus revealed a higher capillary density in mEH-KO, but not in sEH-KO brain. Whisker-stimulated CBF changes were by factor two higher in both mEH-KO and sEH-KO. In acutely isolated cerebral endothelial cells the loss of mEH, but not of sEH, augmented total EET levels and decreased the DHET:EET ratio. Collectively, these data suggest an important function of mEH in the regulation of cerebral vasculature and activity-modulated CBF, presumably by controlling local levels of endothelial-derived EETs.

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

Drug-induced cholestasis (DIC) is recognized as a major safety concern in drug development, as it represents one of the three types of drug-induced liver injury (DILI). Cholestasis is characterized by the disruption of bile flow, leading to intrahepatic accumulation of toxic bile acids. Bile acid regulation is a multifarious process, orchestrated by several hepatic mechanisms, namely sinusoidal uptake and efflux, canalicular secretion and intracellular metabolism. In the present study, we developed a prediction model of DIC using in vitro inhibition data for 47 marketed drugs on nine transporters and five enzymes known to regulate bile acid homeostasis. The resulting model was able to distinguish between drugs with or without DILI concern (p-value = 0.039) and demonstrated a satisfactory predictive performance, with the area under the precision–recall curve (PR AUC) measured at 0.91. Furthermore, we simplified the model considering only two processes, namely reversible inhibition of OATP1B1 and time-dependent inhibition of CYP3A4, which provided an enhanced performance (PR AUC = 0.95). Our study supports literature findings suggesting a contribution not only from a single process inhibition, but a rather synergistic effect of the key bile acid clearance processes in the development of cholestasis. The use of a quantitative model in the preclinical investigations of DIC is expected to reduce attrition rate in advanced development programs and guide the discovery and development of safe medicines.

Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed.

Bile acid (BA) homeostasis is a complex and precisely regulated process to prevent impaired BA flow and the development of cholestasis. Several reactions, namely hydroxylation, glucuronidation and sulfation are involved in BA detoxification. In the present study, we employed a comprehensive approach to identify the key enzymes involved in BA metabolism using human recombinant enzymes, human liver microsomes (HLM) and human liver cytosol (HLC). We showed that CYP3A4 was a crucial step for the metabolism of several BAs and their taurine and glycine conjugated forms and quantitatively described their metabolites. Glucuronidation and sulfation were also identified as important drivers of the BA detoxification process in humans. Moreover, lithocholic acid (LCA), the most hydrophobic BA with the highest toxicity potential, was a substrate for all investigated processes, demonstrating the importance of hepatic metabolism for its clearance. Collectively, this study identified CYP3A4, UGT1A3, UGT2B7 and SULT2A1 as the major contributing (metabolic) processes in the BA detoxification network. Inhibition of these enzymes by drug candidates is therefore considered as a critical mechanism in the manifestation of drug-induced cholestasis in humans and should be addressed during the pre-clinical development.

The "totality" of the human exposure is conceived to encompass life-associated endogenous and exogenous aggregate exposures. Process-related contaminants (PRCs) are not only formed in foods by heat processing, but also occur endogenously in the organism as physiological components of energy metabolism, potentially also generated by the human microbiome. To arrive at a comprehensive risk assessment, it is necessary to understand the contribution of in vivo background occurrence as compared to the ingestion from exogenous sources. Hence, this review provides an overview of the knowledge on the contribution of endogenous exposure to the overall exposure to putative genotoxic food contaminants, namely ethanol, acetaldehyde, formaldehyde, acrylamide, acrolein, α,β-unsaturated alkenals, glycation compounds, N-nitroso compounds, ethylene oxide, furans, 2- and 3-MCPD, and glycidyl esters. The evidence discussed herein allows to conclude that endogenous formation of some contaminants appears to contribute substantially to the exposome. This is of critical importance for risk assessment in the cases where endogenous exposure is suspected to outweigh the exogenous one (e.g. formaldehyde and acrolein).

Cell stress perturbations in the liver are frequently the result of inhibitory insults on hepatic enzyme- and/or transporter-mediated processes involved in the disposition of bile acids and/or bilirubin. Hence, in recent years, more or less complex drug–drug interaction (DDI) prediction techniques (so-called R-value models) were derived to anticipate the overall drug-induced liver injury (DILI) risk of new chemical entities. This article aims to provide an integrated overview of our latest in-house efforts to forecast quantitatively, cell stress-based DILI predominantly by integrating principles of the extended clearance model (ECM).

Presently, adenovirus-based vaccines are dosed by viral particle or viral genome number. Given that the ratio of these to the actually infectious units, the active principle of the vaccines, can vary by several orders of magnitudes in a given preparation this is criticized by the author as inappropriate. Based on existing data from both, animal as well as human vaccination trials, the commentary details this problem and proposes solutions to the problem.

Understanding the physiological impact of transcranial ultrasound in rodent brains may offer an important preclinical model for human scale magnetic resonance–guided focused ultrasound methods. However, precision tools for high-resolution transcranial ultrasound targeting and real-time in vivo tracking of its effects at the mouse brain scale are currently lacking. We report a versatile bidirectional hybrid fluorescence-ultrasound (FLUS) system incorporating a 0.35-mm precision spherical-phased array ultrasound emission with a fiberscope-based wide-field fluorescence imaging. We show how the marriage between cortex-wide functional imaging and targeted ultrasound delivery can be used to transcranially map previously undocumented localized fluorescence events caused by reversible thermal processes and perform high-speed large-scale recording of neural activity induced by focused ultrasound. FLUS thus naturally harnesses the extensive toolbox of fluorescent tags and ultrasound’s localized bioeffects toward visualizing and causally perturbing a plethora of normal and pathophysiological processes in the living murine brain.

The working group “Analyses in Biological Materials” of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area developed and verified the presented biomonitoring method. This method enables the selective detection of the vulcanisation accelerator 2‐mercaptobenzothiazole (MBT) in urine. After adding a labelled internal standard (MBT‐d4), the samples are enzymatically hydrolysed to release free MBT from the conjugated MBT compounds. After online purification and enrichment, the analytes are separated from accompanying components by liquid chromatography and analysed using tandem mass spectrometry. Calibration standards are prepared in pooled urine and processed in the same way as the samples to be analysed. The MAK Collection for Occupational Health and Safety 2020, Vol 5, No 3 1 Biomonitoring-Methoden – 2-Mercaptobenzothiazol in Urin 1 Kenndaten der Methode Matrix Urin Analytisches Messprinzip LC-MS/MS Parameter und entsprechende Arbeitsstoffe Arbeitsstoff CAS-Nr. Parameter CAS-Nr. 2‐Mercaptobenzothiazol (MBT) 149-30‐4 Zinksalz des 2‐Mercaptobenzothiazols 155-04‐4 MBT 149-30‐4 Zuverlässigkeitskriterien der Methode 2-Mercaptobenzothiazol (MBT) Standardabweichung (rel.) sw = 2,3 %, 1,9 % bzw. 1,6 % Streubereich u = 5,1 %, 4,2 % bzw. 3,6 % Präzision in der Serie: bei einer dotierten Konzentration von 10 μg, 100 μg bzw. 1000 μg MBT pro Liter Urin und n = 10 Bestimmungen Standardabweichung (rel.) sw = 4,3 %, 5,8 % bzw. 3,4 % Streubereich u = 9,6 %, 13,0 % bzw. 7,6 % Präzision von Tag zu Tag: bei einer dotierten Konzentration von 10 μg, 100 μg bzw. 1000 μg MBT pro Liter Urin und n = 10 Bestimmungen Wiederfindungsrate (rel.) r = 86 %, 96 % bzw. 96 % Richtigkeit: bei einer Sollkonzentration von 10 μg, 100 μg bzw. 1000 μg MBT pro Liter Urin und n = 10 Bestimmungen Nachweisgrenze: 0,4 μg MBT pro Liter Urin Bestimmungsgrenze: 1,2 μg MBT pro Liter Urin 2 Allgemeine Informationen zu 2-Mercaptobenzothiazol 2‐Mercaptobenzothiazol (MBT) ist eine organische Verbindung aus der Gruppe der Heteroaromaten. Die Substanz liegt unter Normalbedingungen in Form von leicht gelblichen Kristallen vor, die in Wasser schwer löslich sind. Die industrielle Herstellung erfolgt durch Umsetzung von Anilin, Schwefelkohlenstoff und Schwefel unter Hochdruck bei hohen Temperaturen von etwa 230 °C (Greim 1999; IARC 2018). MBT wird hauptsächlich als Vulkanisationsbeschleuniger in der Reifenproduktion und der Herstellung von technischen Gummiartikeln eingesetzt. Zusätzlich kommen das Zinksalz des Mercaptobenzothiazols sowie die Sulfenamide, eine vom MBT ausgehende Verbindungsklasse, als mögliche Quelle für MBT in Frage. So ist MBT potentiell in vielen täglichen Gebrauchsgegenständen zu finden, wie z. B. in Reifen, Kabeln, Gummihandschuhen, GummiThe MAK Collection for Occupational Health and Safety 2020, Vol 5, No 3 2 Biomonitoring-Methoden – 2-Mercaptobenzothiazol in Urin bändern und -dichtungen, sowie Bohrund Schneidölen. Weiterhin wird es als Reagenz bei der quantitativen nasschemischen Metallanalytik eingesetzt (IARC 2018). 2‐Mercaptobenzothiazol liegt in zwei tautomeren Formen vor, bei denen das Gleichgewicht auf die Seite des 2‐(3H)Benzothiazolthions (NH-Form) verschoben ist (vgl. Abbildung 1).

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has re-evaluated α‑aluminium oxide [1344-28‑1]. α‑Aluminium oxide is a biopersistent granular dust. There are no inhalation studies from which a NOAEC could be derived. Therefore, the respirable fraction of α‑aluminium oxide dust is classified in Carcinogen Category 4 and a maximum concentration at the workplace (MAK value) of 0.3 mg/m 3 × material density is established for the respirable fraction

The working group “Analyses in Biological Materials” of the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area developed the presented biomonitoring method. The method enables the determination of ochratoxin A in small volumes of serum. The samples are purified by liquid-liquid extraction and the analyte is extracted from the acidified sample with dichloromethane. After the extraction agent has been removed, the residue is dissolved in water. The extracts are injected into the capillary, then the individual components are separated by capillary electrophoresis and detected by laser-induced fluorescence. Coumarin-3-carboxylic acid is used as an internal standard. Calibration standards are prepared in serum and processed in the same way as the samples to be analysed. The MAK Collection for Occupational Health and Safety 2020, Vol 5, No 4 1 Biomonitoring-Methoden – Ochratoxin A in Serum 1 Kenndaten der Methode Matrix Serum Analytisches Messprinzip Kapillarelektrophorese mit laserinduzierter Fluoreszenzdetektion (CE-LIF) Parameter und entsprechender Arbeitsstoff Arbeitsstoff CAS-Nr. Parameter CAS-Nr. Ochratoxin A 303-47‐9 Ochratoxin A 303-47‐9 Zuverlässigkeitskriterien

The German Commission for the Investigation of Health Hazards of Chemical Com-pounds in the Work Area has re-evaluated methylamine [74-89-5]. The critical effect is irritation of the nasal airways as observed in a 2-week study in rats with a NOAEC of 75 ml/m 3 . The RD 50 data for methylamine show that its irritation potency