A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the ‘Phenylalanine, tyrosine and tryptophan biosynthesis’ pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU

Minimal Viral Potassium Channels for Studying Protein/Lipid Interaction

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Histogram Analysis of Diffusion Weighted Imaging in Low-Grade Gliomas: in vivo Characterization of Tumor Architecture and Corresponding Neuropathology

Background: Low-grade gliomas (LGG) in adults are usually slow growing and frequently asymptomatic brain tumors, originating from glial cells of the central nervous system (CNS). Although regarded formally as “benign” neoplasms, they harbor the potential of malignant transformation associated with high morbidity and mortality. Their complex and unpredictable tumor biology requires a reliable and conclusive presurgical magnetic resonance imaging (MRI). A promising and emerging MRI approach in this context is histogram based apparent diffusion coefficient (ADC) profiling, which recently proofed to be capable of providing prognostic relevant information in different tumor entities. Therefore, our study investigated whether histogram profiling of ADC distinguishes grade I from grade II glioma, reflects the proliferation index Ki-67, as well as the IDH (isocitrate dehydrogenase) mutation and MGMT (methylguanine-DNA methyl-transferase) promotor methylation status. Material and Methods: Pre-treatment ADC volumes of 26 LGG patients were used for histogram-profiling. WHO-grade, Ki-67 expression, IDH mutation, and MGMT promotor methylation status were evaluated. Comparative and correlative statistics investigating the association between histogram-profiling and neuropathology were performed. Results: Almost the entire ADC profile (p25, p75, p90, mean, median) was significantly lower in grade II vs. grade I gliomas. Entropy, as second order histogram parameter of ADC volumes, was significantly higher in grade II gliomas compared with grade I gliomas. Mean, maximum value (ADCmax) and the percentiles p10, p75, and p90 of ADC histogram were significantly correlated with Ki-67 expression. Furthermore, minimum ADC value (ADCmin) was significantly associated with MGMT promotor methylation status as well as ADC entropy with IDH-1 mutation status. Conclusions: ADC histogram-profiling is a valuable radiomic approach, which helps differentiating tumor grade, estimating growth kinetics and probably prognostic relevant genetic as well as epigenetic alterations in LGG

Interactions of Mycorrhiza and Protists in the Rhizosphere Systemically Alter Microbial Community Composition, Plant Shoot-to-Root Ratio and Within-Root System Nitrogen Allocation

Arbuscular mycorrhizal fungi (AMF) are important symbionts for plant nutrient uptake, but their exact role in plant nitrogen (N) nutrition is unclear. Protists on the other hand play an acknowledged role in plant N acquisition, and there is increasing evidence for a close interaction with AMF. In a split root set up, we investigated the distinct roles of mycorrhiza (Rhizophagus irregularis), protists (Acanthamoeba castellanii), and their interaction on plant N uptake, within-root system allocation patterns, and shoot-to-root ratio of winter wheat. In addition, we applied a quantitative metabolomics approach to characterize associated changes in soil microbial communities by microbial phospholipid fatty acid (PLFA) analysis from rhizosphere soil. AMF markedly altered plant shoot-to-root allometry by reducing root biomass of wheat, and mycorrhiza partly took over root system functioning. Protists promoted shoot and root growth, and improved plant N uptake by the release of N from consumed bacterial biomass, a mechanism known as microbial loop. The shoot system however responded little to these alterations of the root system and of the rhizosphere community composition, indicating that the plants optimized shoot growth despite varying investment into roots. Mycorrhiza reduced root biomass and plant N, especially in the combined treatments with protists by changing within root system allocation of N and root biomass. These systemic effects on root allocation pattern suggest that mycorrhiza also gained control over N provided by protist grazers. Protists and mycorrhiza altered rhizosphere bacterial communities in contrasting but consistent ways as shown by quantitative shifts in microbial PLFA profiles. Remarkably, the changes in bacterial community composition were systemically conveyed within the root system to the split-root chamber where the symbionts were lacking. Accordingly the synergistic effects of protists and mycorrhiza indicated systemic effects on nutrient- and on root-allocation within root systems as an emergent property that could not be predicted from single treatments with mycorrhiza or protists alone. The tight plant and microbial feed backs uncovered in this study have far reaching implications for understanding the assembly of plant microbiomes, and testify central roles of both protists and mycorrhizas in the assembly process

Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K+ Channels

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels. Includes Supporting Informatio

Identification of Intrahelical Bifurcated H‑Bonds as a New Type of Gate in K+ Channels

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels. Includes Supporting Informatio

Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called “snorkeling” of a cationic amino acid, which is conserved in the outer TMD of small viral K+ channels. Experimentally, snorkeling activity is not mandatory for KcvPBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, KcvATCV-1 and KcvMT325, lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of KcvPBCV-1 and N-terminally truncated mutants; the truncated mutants mimic KcvATCV-1 and KcvMT325. Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K+ channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains

Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called “snorkeling” of a cationic amino acid, which is conserved in the outer TMD of small viral K+ channels. Experimentally, snorkeling activity is not mandatory for KcvPBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, KcvATCV-1 and KcvMT325, lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of KcvPBCV-1 and N-terminally truncated mutants; the truncated mutants mimic KcvATCV-1 and KcvMT325. Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K+ channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains

Flow Diversion for Reconstruction of Intradural Vertebral Artery Dissecting Aneurysms Causing Subarachnoid Hemorrhage—A Retrospective Study From Four Neurovascular Centers

Objective: Dissecting aneurysms (DAs) of the vertebrobasilar territory manifesting with subarachnoid hemorrhage (SAH) are associated with significant morbi-mortality, especially in the case of re-hemorrhage. Sufficient reconstruction of the affected vessel is paramount, in particular, if a dominant vertebral artery (VA) is impacted. Reconstructive options include stent-assisted coiling and flow diversion (FD). The latter is technically less challenging and does not require catheterization of the fragile aneurysm. Our study aims to report a multicentric experience with FD for reconstruction of DA in acute SAH. Materials and Methods: This retrospective study investigated 31 patients (age: 30–78 years, mean 55.5 years) who had suffered from SAH due to a DA of the dominant VA. The patients were treated between 2010 and 2020 in one of the following German neurovascular centers: University Hospital Leipzig, Katharinenhospital Stuttgart, BG Hospital Bergmannstrost Halle/Saale, and Heinrich-Braun-Klinikum Zwickau. Clinical history, imaging, implanted devices, and outcomes were reviewed for the study. Results: Reconstruction with flow-diverting stents was performed in all cases. The p64 was implanted in 14 patients; one of them required an additional balloon expandable stent to reconstruct severe stenosis in the target segment. One case demanded additional liquid embolization after procedural rupture, and in one case, p64 was combined with a PED. Further 13 patients were treated exclusively with the PED. The p48MW-HPC was used in two patients, one in combination with two additional Silk Vista Baby (SVB). Moreover, one patient was treated with a single SVB, one with a SILK+. Six patients died [Glasgow Outcome Scale (GOS) 1]. Causes of death were periprocedural re-hemorrhage, thrombotic occlusion of the main pulmonary artery, and delayed parenchymal hemorrhage. The remaining three patients died in the acute–subacute phase related to the severity of the initial hemorrhage and associated comorbidities. One patient became apallic (GOS 2), whereas two patients had severe disability (GOS 3) and four had moderate disability (GOS 4). Eighteen patients showed a complete recovery (GOS 5). Conclusion: Reconstruction of VA-DA in acute SAH with flow-diverting stents is a promising approach. However, the severity of the condition is reflected by high overall morbi-mortality, even despite technically successful endovascular treatment