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Zeugenschaft ist heute wichtiger denn je – und das nicht trotz, sondern vielmehr gerade wegen der gewachsenen Möglichkeiten, jederzeit und überall das Geschehen technisch aufzunehmen und einer breiten Öffentlichkeit zugänglich zu machen. Zeugenschaft ist jedoch nicht nur eine epistemische Praxis, sondern auch eine Form sozialen Engagements, wie die sozialen Protestbewegungen der letzten Jahre gezeigt haben. In den aktuellen Debatten um Zeugenschaft ist allerdings eine gewisse Verengung hin auf die epistemischen Aspekte des Zeugnisgebens zu beobachten. Dagegen versucht der Artikel, Praktiken der Zeugenschaft auf ihre ethischen und politischen Implikationen hin zu befragen. Dabei gilt es, kritisch auf die Normen zu reflektieren, die Zeugenschaft ermöglichen oder verhindern. Zugleich impliziert jedes Zeugnis ein politisches Moment, insofern sich das Wahrsprechen des Zeugen bzw. der Zeugin selbst als politischer Akt interpretieren lässt. Zeugnisgeben erweist sich damit, so die These, nicht nur als eine epistemische, sondern auch als eine ethische und politische Praxis, ohne dass diese Elemente aufeinander reduzierbar wären.

Abstract Background Beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on the lipid levels of dyslipidemic subjects are widely described in the literature. However, the underlying molecular mechanisms are largely unknown. The aim of this study was to investigate the effects of n-3 PUFAs on the expression of lipid metabolism-related genes in normo- and dyslipidemic men to unveil potential genes and pathways affecting lipid metabolism. Methods Ten normo- and ten dyslipidemic men were supplemented for twelve weeks with six fish oil capsules per day, providing 1.14 g docosahexaenoic acid and 1.56 g eicosapentaenoic acid. The gene expression levels were determined by whole genome microarray analysis and quantitative real-time polymerase chain reaction. Results Several transcription factors (peroxisome proliferator-activated receptor α (PPARα), retinoid X receptor (RXR) α, RXRγ, hepatic nuclear factor (HNF) 6, and HNF1ß) as well as other genes related to triacylglycerol (TG) synthesis or high-density lipoprotein (HDL-C) and cholesterol metabolism (phospholipids transfer protein, ATP-binding cassette sub-family G member 5, 2-acylglycerol O-acyltransferase (MOGAT) 3, MOGAT2, diacylglycerol O-acyltransferase 1, sterol O-acyltransferase 1, apolipoprotein CII, and low-density lipoprotein receptor) were regulated after n-3 PUFA supplementation, especially in dyslipidemic men. Conclusion Gene expression analyses revealed several possible molecular pathways by which n-3 PUFAs lower the TG level and increase the HDL-C and low-density lipoprotein level, whereupon the regulation of PPARα appear to play a central role. Trial registration ClinicalTrials.gov (ID: NCT01089231)

Abstract Background Epidemiological studies have suggested the benefits of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on cardiovascular health, but only limited data are available describing n-3 PUFA regulated pathways in humans. The aim of this study was to investigate the effects of n-3 PUFA administration on whole genome expression profiles in the blood of normo- and dyslipidemic subjects. Methods Differentially expressed genes were detected after four hours, one week and twelve weeks of supplementation with either fish oil (FO) or corn oil in normo- and dyslipidemic men using whole genome microarrays. Results Independent of the oil, a significantly higher number of genes was regulated in dyslipidemic subjects compared to normolipidemic subjects. Pathway analyses discovered metabolisms dominantly affected by FO after twelve weeks of supplementation, including the lipid metabolism, immune system and cardiovascular diseases. Several pro-inflammatory genes, in particular, were down-regulated in dyslipidemic subjects, indicating the immune-modulatory and anti-inflammatory capability of FO and its bioactive FAs, eicosapentaenoic acid and docosahexaenoic acid. Conclusions This is the first study showing significant differences in gene expression profiles between normo- and dyslipidemic men after FO supplementation. Further studies need to clarify the exact role of n-3 PUFAs in pathways and metabolisms which were identified as being regulated after FO supplementation in this study. Trial registration ClinicalTrials.gov (ID: NCT01089231)

Abstract Background The beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs), especially in dyslipidemic subjects with a high risk of cardiovascular disease, are widely described in the literature. A lot of effects of n-3 PUFAs and their oxidized metabolites are triggered by regulating the expression of genes. Currently, it is uncertain if the administration of n-3 PUFAs results in different expression changes of genes related to antioxidative mechanisms in normo- and dyslipidemic subjects, which may partly explain their cardioprotective effects. The aim of this study was to investigate the effects of n-3 PUFA supplementation on expression changes of genes involved in oxidative processes. Methods Ten normo- and ten dyslipidemic men were supplemented for twelve weeks with fish oil capsules, providing 1.14 g docosahexaenoic acid and 1.56 g eicosapentaenoic acid. Gene expression levels were determined by whole genome microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Results Using microarrays, we discovered an increased expression of antioxidative enzymes and a decreased expression of pro-oxidative and tissue enzymes, such as cytochrome P450 enzymes and matrix metalloproteinases, in both normo- and dyslipidemic men. An up-regulation of catalase and heme oxigenase 2 in both normo- and dyslipidemic subjects and an up-regulation of cytochrome P450 enzyme 1A2 only in dyslipidemic subjects could be observed by qRT-PCR analysis. Conclusions Supplementation of normo- and dyslipidemic subjects with n-3 PUFAs changed the expression of genes related to oxidative processes, which may suggest antioxidative and potential cardioprotective effects of n-3 PUFAs. Further studies combining genetic and metabolic endpoints are needed to verify the regulative effects of n-3 PUFAs in antioxidative gene expression to better understand their beneficial effects in health and disease prevention. Trial registration ClinicalTrials.gov (ID: NCT01089231)

Abstract Background Transcript profiling of closely related species provides a means for identifying genes potentially important in species diversification. However, the predictive value of transcript profiling for inferring downstream-physiological processes has been unclear. In the present study we use shotgun proteomics to validate inferences from microarray studies regarding physiological differences in three Pachycladon species. We compare transcript and protein profiling and evaluate their predictive value for inferring glucosinolate chemotypes characteristic of these species. Results Evidence from heterologous microarrays and shotgun proteomics revealed differential expression of genes involved in glucosinolate hydrolysis (myrosinase-associated proteins) and biosynthesis (methylthioalkylmalate isomerase and dehydrogenase), the interconversion of carbon dioxide and bicarbonate (carbonic anhydrases), water use efficiency (ascorbate peroxidase, 2 cys peroxiredoxin, 20 kDa chloroplastic chaperonin, mitochondrial succinyl CoA ligase) and others (glutathione-S-transferase, serine racemase, vegetative storage proteins, genes related to translation and photosynthesis). Differences in glucosinolate hydrolysis products were directly confirmed. Overall, prediction of protein abundances from transcript profiles was stronger than prediction of transcript abundance from protein profiles. Protein profiles also proved to be more accurate predictors of glucosinolate profiles than transcript profiles. The similarity of species profiles for both transcripts and proteins reflected previously inferred phylogenetic relationships while glucosinolate chemotypes did not. Conclusions We have used transcript and protein profiling to predict physiological processes that evolved differently during diversification of three Pachycladon species. This approach has also identified candidate genes potentially important in adaptation, which are now the focus of ongoing study. Our results indicate that protein profiling provides a valuable tool for validating transcript profiles in studies of adaptive divergence.

Abstract Background Parental rearing behavior is a significant etiological factor for the vulnerability of psychopathology and has been an issue of clinical research for a long time. For this scope instruments are important who asses economically recalled parental rearing behavior in a clinical practice. Therefore, a short German instrument for the assessment of the recalled parental rearing behavior Fragebogen zum erinnerten elterlichen Erziehungsverhalten (FEE) was psychometrically evaluated [Recalled Parental Rearing Behavior]. Methods This questionnaire was evaluated in a representative population sample (N = 2.948) in Germany which included 44.2% male and 55.8% female persons with a mean age of M = 47.35 (SD = 17.10, range = 18–92). For the content evaluation of the FEE the Life Satisfaction Questionnaire (FLZ) and the Inventory of Interpersonal Problems (IIP) was filled out by the participants. Results The FEE scales yielded a good to satisfactory internal consistency and split-half reliability. Its three factors (rejection/punishment, emotional warmth, control/overprotection) correlated positively with most of the areas of life satisfaction. Furthermore, positive associations between interpersonal problems and parental rejection and control could be identified. Conclusion The FEE is a short, reliable and valid instrument that can be applied in the clinical practice. In addition, the data proved an association between recalled parental rearing behavior, life satisfaction and interpersonal problems conform to the literature. Finally, specific problems with the retrospective assessment of parental rearing behavior were addressed as well.

Abstract Background Age-related macular degeneration (AMD) is a complex disorder that is responsible for the majority of central vision loss in older adults living in developed countries. Phenotypic and genetic heterogeneity complicate the analysis of genome-wide scans for AMD susceptibility loci. The ordered subset analysis (OSA) method is an approach for reducing heterogeneity, increasing statistical power for detecting linkage, and helping to define the most informative data set for follow-up analysis. OSA assesses the linkage evidence in subsets of potentially more homogeneous families by rank-ordering family-specific lod scores with respect to trait-associated covariates or phenotypic features. Here, we present results of incorporating five continuous covariates into our genome-wide linkage analysis of 389 microsatellite markers in 62 multiplex families: Body mass index (BMI), systolic (SBP) and diastolic (DBP) blood pressure, intraocular pressure (IOP), and pack-years of cigarette smoking. Chromosome-wide significance of increases in nonparametric multipoint lod scores in covariate-defined subsets relative to the overall sample was assessed by permutation. Results Using a correction for testing multiple covariates, statistically significant lod score increases were observed for two chromosomal regions: 14q13 with a lod score of 3.2 in 28 families with average IOP ≤ 15.5 (p = 0.002), and 6q14 with a lod score of 1.6 in eight families with average BMI ≥ 30.1 (p = 0.0004). On chromosome 16p12, nominally significant lod score increases (p ≤ 0.05), up to a lod score of 2.9 in 32 families, were observed with several covariate orderings. While less significant, this was the only region where linkage evidence was associated with multiple clinically meaningful covariates and the only nominally significant finding when analysis was restricted to advanced forms of AMD. Families with linkage to 16p12 had higher averages of SBP, IOP and BMI and were primarily affected with neovascular AMD. For all three regions, linkage signals at or very near the peak marker have previously been reported. Conclusion Our results suggest that a susceptibility gene on chromosome 16p12 may predispose to AMD, particularly to the neovascular form, and that further research into the previously suggested association of neovascular AMD and systemic hypertension is warranted.

Rising atmospheric temperature and CO 2 impact all freshwater systems. In groundwater, one such impact is CO 2 - and temperature-induced weathering, which leads to more intense weathering of silicate rocks. Here, we tested whether the increased CO 2 levels, the weathering, or rather the increasing temperature, impacted on fauna and prokaryotes in the groundwater ecosystem. We also conducted the analyses separately for two groups of wells, one of which contained wells that were secluded from the surface (and often rather deep), and wells tapping the quaternary aquifers (often rather shallow) which exchange with the surface more intensely. Organism abundances and relative composition did not correlate with temperature or CO 2 levels. While many organisms rely on silica, in contrast, we found negative correlations between silica concentrations and fauna. The increases in silica concentrations over time, i.e. temporal trends, also partly correlated negatively with organisms. We hypothesize that the unexpected negative correlations are not direct effects, but indirectly indicate that groundwater communities do not adapt rapidly enough to changes in silica concentrations, but also more generally to changes for which silica might only be a proxy. Groundwater fauna take part in the ecosystem service of water self-cleaning and are thus considered beneficial for sustainable raw water for drinking water production. The propensity of groundwater fauna to decrease with increases in silica, jeopardizes future drinking water production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

N-acetylgalactosaminyl-transferases (GalNAc-Ts) initiate mucin-type O-glycosylation, an abundant and complex posttranslational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a lectin domain consisting of three homologous repeats (α, β, and γ), where α and β can potentially interact with O-GalNAc on substrates to enhance activity toward a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both α and β in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The α repeat directs O-glycosylation to acceptor sites carboxyl-terminal to an existing GalNAc, while the β repeat directs O-glycosylation to amino-terminal sites. In addition, GalNAc-T1 incorporates α and β into various substrate binding modes to cooperatively increase the specificity toward an acceptor site located between two existing O-glycans. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and provide crucial information for identifying the biological substrates of GalNAc-T1.

Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change., (© 2023 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models., Competing Interests: Declaration of interests The authors’ current additional affiliations, unrelated to this work, are as follows: D.L.E.V., Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands; L.N.K., Nature Reviews Neurology, London, UK; U.C., School of Medicine, Cardiff University, Cardiff, UK; F.S.B., School of Biological Sciences, University of Edinburgh, Edinburgh, UK; J.B., Clinical Neurosciences, University of Cambridge, Cambridge, UK; J.P.C., Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, UK; and T.M.C., Mend the Gap, University of British Columbia, Vancouver, BC, Canada., (Copyright © 2023. Published by Elsevier Inc.)

Subterranean ecosystems are among the most widespread environments on Earth, yet we still have poor knowledge of their biodiversity. To raise awareness of subterranean ecosystems, the essential services they provide, and their unique conservation challenges, 2021 and 2022 were designated International Years of Caves and Karst. As these ecosystems have traditionally been overlooked in global conservation agendas and multilateral agreements, a quantitative assessment of solution-based approaches to safeguard subterranean biota and associated habitats is timely. This assessment allows researchers and practitioners to understand the progress made and research needs in subterranean ecology and management. We conducted a systematic review of peer-reviewed and grey literature focused on subterranean ecosystems globally (terrestrial, freshwater, and saltwater systems), to quantify the available evidence-base for the effectiveness of conservation interventions. We selected 708 publications from the years 1964 to 2021 that discussed, recommended, or implemented 1,954 conservation interventions in subterranean ecosystems. We noted a steep increase in the number of studies from the 2000s while, surprisingly, the proportion of studies quantifying the impact of conservation interventions has steadily and significantly decreased in recent years. The effectiveness of 31% of conservation interventions has been tested statistically. We further highlight that 64% of the reported research occurred in the Palearctic and Nearctic biogeographic regions. Assessments of the effectiveness of conservation interventions were heavily biased towards indirect measures (monitoring and risk assessment), a limited sample of organisms (mostly arthropods and bats), and more accessible systems (terrestrial caves). Our results indicate that most conservation science in the field of subterranean biology does not apply a rigorous quantitative approach, resulting in sparse evidence for the effectiveness of interventions. This raises the important question of how to make conservation efforts more feasible to implement, cost-effective, and long-lasting. Although there is no single remedy, we propose a suite of potential solutions to focus our efforts better towards increasing statistical testing and stress the importance of standardising study reporting to facilitate meta-analytical exercises. We also provide a database summarising the available literature, which will help to build quantitative knowledge about interventions likely to yield the greatest impacts depending upon the subterranean species and habitats of interest. We view this as a starting point to shift away from the widespread tendency of recommending conservation interventions based on anecdotal and expert-based information rather than scientific evidence, without quantitatively testing their effectiveness., (© 2022 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Forest damage by insect infestation directly affects the trees themselves, but also indirectly affects water quality via soil processes. The changes in water composition may undergo different pathways depending on site-specific characteristics and forest components, especially the proportion of coniferous and deciduous trees. Here, we test whether changes in forest components and the intensity of disturbance can predict the chemical properties of water outflow from affected lake catchments. Information about forest regeneration (a phase dominated by deciduous trees) and the proportions of damaged and healthy coniferous trees and treeless areas were obtained from satellite data. The four study catchments of Prášilské, Laka, Plešné, and Čertovo lakes are geographically close and located in the same mountain range (Šumava Mts., Czech Republic) at similar altitude, but they differ in extents of forest disturbances and recoveries. The water quality measured at the lake catchment outflows differed, and better reflected the development of forest components and health than did meteorological (temperature and precipitation) or hydrological (discharge) variables. Several of the outflow properties (concentrations of inorganic aluminium, protons, potassium, calcium, magnesium, alkalinity, dissolved organic carbon (DOC), nitrate, and total phosphorus), responded catchment-specifically and with different delays to forest disturbance. The most pronounced differences occurred in DOC concentrations, which started to increase in the most disturbed Plešné and Laka catchments 7 and 6 years, respectively, after the peak in tree dieback, but did not increase significantly in the Prášilské catchment, which was disturbed several times during the last 3-4 decades. This study demonstrates an importance of extents of forest disturbances, the following changes in forest composition, and catchment-specific characteristics on water composition., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ras homolog gene family member A (RhoA) is a small GTPase of the Rho family involved in regulating multiple signal transduction pathways that influence a diverse range of cellular functions. RhoA and many of its downstream effector proteins are highly expressed in the nervous system, implying an important role for RhoA signaling in neurons and glial cells. Indeed, emerging evidence points toward a role of aberrant RhoA signaling in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. In this review, we summarize the current knowledge of RhoA regulation and downstream cellular functions with an emphasis on the role of RhoA signaling in neurodegenerative diseases and the therapeutic potential of RhoA inhibition in neurodegeneration.

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology., Competing Interests: The authors declare that no competing interests exist.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

PARK2 (parkin) mutations cause early-onset Parkinson's disease (PD). Parkin is an ubiquitin E3 ligase that participates in several cellular functions, including mitochondrial homeostasis. However, the specific metabolomic changes caused by parkin depletion remain unknown. Here, we used isogenic human induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) to investigate the effect of parkin loss of function by comparative metabolomics supplemented with ultrastructural and functional analyses. PARK2 KO neurons displayed increased tricarboxylic acid (TCA) cycle activity, perturbed mitochondrial ultrastructure, ATP depletion, and dysregulation of glycolysis and carnitine metabolism. These perturbations were combined with increased oxidative stress and a decreased anti-oxidative response. Key findings for PARK2 KO cells were confirmed using patient-specific iPSC-derived neurons. Overall, our data describe a unique metabolomic profile associated with parkin dysfunction and show that combining metabolomics with an iPSC-derived dopaminergic neuronal model of PD is a valuable approach to obtain novel insight into the disease pathogenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Caves and other subterranean habitats fulfill the requirements of experimental model systems to address general questions in ecology and evolution. Yet, the harsh working conditions of these environments and the uniqueness of the subterranean organisms have challenged most attempts to pursuit standardized research.Two main obstacles have synergistically hampered previous attempts. First, there is a habitat impediment related to the objective difficulties of exploring subterranean habitats and our inability to access the network of fissures that represents the elective habitat for the so-called "cave species." Second, there is a biological impediment illustrated by the rarity of most subterranean species and their low physiological tolerance, often limiting sample size and complicating laboratory experiments.We explore the advantages and disadvantages of four general experimental setups ( in situ , quasi in situ , ex situ , and in silico ) in the light of habitat and biological impediments. We also discuss the potential of indirect approaches to research. Furthermore, using bibliometric data, we provide a quantitative overview of the model organisms that scientists have exploited in the study of subterranean life.Our over-arching goal is to promote caves as model systems where one can perform standardized scientific research. This is important not only to achieve an in-depth understanding of the functioning of subterranean ecosystems but also to fully exploit their long-discussed potential in addressing general scientific questions with implications beyond the boundaries of this discipline., Competing Interests: None declared., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Background: Parkinson's disease is characterized by motor dysfunctions including bradykinesia. In a recent study, eight weeks of daily transcranial stimulation with bipolar pulsed electromagnetic fields improved functional rate of force development and decreased inter-hand tremor coherence in patients with mild Parkinson's disease., Objective: To investigate the effect of long-term treatment with transcranial bipolar pulsed electromagnetic fields on motor performance in terms of movement speed and on neurotrophic and angiogenic factors., Methods: Patients diagnosed with idiopathic Parkinson's disease had either daily 30-min treatment with bipolar (±50 V) transcranial pulsed electromagnetic stimulation (squared pulses, 3ms duration) for three eight-week periods separated by one-week pauses (T-PEMF group) (n = 16) or were included in a PD-control group (n = 8). Movement speed was assessed in a six-cycle sit-to-stand task performed on a force plate. Cerebrospinal fluid and venous blood were collected and analyzed for erythropoietin and vascular endothelial growth factor., Results: Major significant improvement of movement speed compared to the natural development of the disease was found (p = 0.001). Thus, task completion time decreased gradually during the treatment period from 10.10s to 8.23s (p<0.001). The untreated PD-control group did not change (p = 0.458). The treated group did not differ statistically from that of a healthy age matched reference group at completion of treatment. Erythropoietin concentration in the cerebrospinal fluid also increased significantly in the treated group (p = 0.012)., Conclusion: Long-term treatment with transcranial bipolar pulsed electromagnetic fields increased movement speed markedly and elevated erythropoietin levels. We hypothesize that treatment with transcranial bipolar pulsed electromagnetic fields improved functional performance by increasing dopamine levels in the brain, possibly through erythropoietin induced neural repair and/or protection of dopaminergic neurons., Competing Interests: The authors have declared that no competing interests exist.

Microglia have recently been established as key regulators of brain development. However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1β, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate in vitro derivation of dopaminergic neurons., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Parkin and α -synuclein are two key proteins involved in the pathophysiology of Parkinson's disease (PD). Neurotoxic alterations of α -synuclein that lead to the formation of toxic oligomers and fibrils contribute to PD through synaptic dysfunction, mitochondrial impairment, defective endoplasmic reticulum and Golgi function, and nuclear dysfunction. In half of the cases, the recessively inherited early-onset PD is caused by loss of function mutations in the PARK2 gene that encodes the E3-ubiquitin ligase, parkin. Parkin is involved in the clearance of misfolded and aggregated proteins by the ubiquitin-proteasome system and regulates mitophagy and mitochondrial biogenesis. PARK2 -related PD is generally thought not to be associated with Lewy body formation although it is a neuropathological hallmark of PD. In this review article, we provide an overview of post-mortem neuropathological examinations of PARK2 patients and present the current knowledge of a functional interaction between parkin and α -synuclein in the regulation of protein aggregates including Lewy bodies. Furthermore, we describe prevailing hypotheses about the formation of intracellular micro-aggregates (synuclein inclusions) that might be more likely than Lewy bodies to occur in PARK2 -related PD. This information may inform future studies aiming to unveil primary signaling processes involved in PD and related neurodegenerative disorders.

Mutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. We used such cells to investigate the impact of PARK2 KO on the lysosomal compartment and found a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology with enlarged electron-lucent lysosomes and an increased lysosomal content, which was exacerbated by mitochondrial stress and could be ameliorated by antioxidant treatment. We also found decreased lysosomal enzyme activity and autophagic perturbations, suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, β-glucocerebrosidase, was increased, suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons and reveal an important new connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

Background: The research focused on the evaluation of the impact of cover cropping on trace metals (Fe, Mn, Cu, Zn, Pb, Co and Cd) and nutrients in vineyard soils and Vitis vinifera L. For this purpose, two types of cover crops (Lolium perenne L. and Medicago sativa L.) and their mixture were planted between vine rows of Muscat white in the vineyard in South Crimea. Trace elements, nutrients and other parameters were analyzed in the soil layers, leaves and grapevines of control and cover cropped plots., Results: The effect of cover cropping was dependent on applied plant species. Ryegrass (L. perenne L.) seems to compete with V. vinifera L. for nutrients - these were lower in the soil and vines of the treated plot. In parallel, lead (Pb) bioconcentration in grapevines was reduced. In contrast, under lucerne (M. sativa L.), nitrogen in the soil and vines, and trace metal bioconcentration (Fe, Pb and Co) were higher., Conclusions: Our results indicate that cover cropping can influence the chemical composition of soil and vines. This should be considered when selecting cover crops. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Mutations in parkin, encoded by the PARK2 gene, causes early-onset familial Parkinson's disease (PD), but dysfunctional parkin has also been implicated in sporadic PD. By combining human isogenic induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) and a novel large-scale mass spectrometry based proteomics and post-translational modification (PTM)-omics approach, we have mapped changes in protein profiles and PTMs caused by parkin deficiency in neurons. Our study identifies changes to several proteins previously shown to be dysregulated in brains of sporadic PD patients. Pathway analysis and subsequent in vitro assays reveal perturbations in migration and neurite outgrowth in the PARK2 KO neurons. We confirm the neurite defects using long-term engraftment of neurons in the striatum of immunosuppressed hemiparkinsonian adult rats. The GTP-binding protein RhoA was identified as a key upstream regulator, and RhoA activity was significantly increased in PARK2 KO neurons. By inhibiting RhoA signalling the migration and neurite outgrowth phenotypes could be rescued. Our study provides new insight into the pathogenesis of PD and demonstrates the broadly applicable potential of proteomics and PTMomics for elucidating the role of disease-causing mutations., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, sources of recharge and contamination in urban groundwater and in groundwater underneath a forest in the same aquifer were determined and compared. Data on hydro-chemical parameters and stable isotopes of water were collected in urban and forest springs in the Kharkiv region, Ukraine, over a period of 12 months. Groundwater transit time and precipitation contribution were calculated using hydrogeological data and stable isotopes of water to delineate groundwater recharge conditions. Hydro-chemical data, stable isotopes and emerging contaminants were used to trace anthropogenic groundwater recharge and approximate sewage and tap water contributions to the aquifer. The results indicated that each spring had unique isotopic signatures that could be explained by recharge conditions, groundwater residence time, and specific mixing patterns with sewage and water leaks. Elevated nitrate content, stable isotopes of nitrate, and the presence of emerging pollutants (mainly illicit drugs) in most of the urban springs confirmed mixing of urban groundwater with sewage leaks. These leaks amounted to up to 25% of total recharge and exhibited seasonal variations in some springs. Overall, the results show that urban groundwater receives variable seasonal contributions of anthropogenic components that increase the risk to the environment and human health, and reduce its usability for drinking water production. The multi-tracing approach presented can be useful for other cities worldwide that have similar problems of poor water management and inadequate sewage and water supply infrastructure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a consequence of the growing global dependence on groundwater resources, environmental risk assessments (ERA) for groundwater are increasingly required and, with that, ecotoxicological studies with groundwater fauna (stygofauna). However, the literature on the ecotoxicological studies with stygobiotic species (i.e. species that complete their life cycle exclusively in groundwater) has not expanded significantly since the first paper published in this field. The limitations regarding the use of stygobiotic species for ecotoxicological testing are clear and consistent across the globe; stygobiotic species are often 1) naturally present in low numbers, 2) difficult to collect, and 3) difficult to culture under laboratory conditions. This paper reviews the methods used in ecotoxicological studies performed with stygobiotic species, and provides ten recommendations for Good Laboratory Practice (GLP) for such tests. The recommendations focused on the following 10 points: 1) the taxonomic identification, the life stage/size and gender of the test organisms; 2) collection methodology of the organisms, including collection location, conditions and methods; 3) holding and acclimation conditions in the laboratory; 4) exposure conditions such as test set up and exposure time, number of replicates and densities of organisms in tests and in test vessels; 5) range-finding test set up and schedule; 6) final test design, including details of controls and treatments, and replication options; 7) incubation conditions, specifying temperature, pH and oxygenation levels throughout the test; 8) test duration; 9) observations and endpoints; 10) test validity criteria and compliance. The recommendations were developed for the purpose of supporting future short-term ecotoxicological testing with stygofauna through providing consistency in the protocols. A discussion of the potential implications for groundwater managers and decision-makers committed to ERA for groundwater is included., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The protein parkin, encoded by the PARK2 gene, is vital for mitochondrial homeostasis, and although it has been implicated in Parkinson's disease (PD), the disease mechanisms remain unclear. We have applied mass spectrometry-based proteomics to investigate the effects of parkin dysfunction on the mitochondrial proteome in human isogenic induced pluripotent stem cell-derived neurons with and without PARK2 knockout (KO). The proteomic analysis quantified nearly 60% of all mitochondrial proteins, 119 of which were dysregulated in neurons with PARK2 KO. The protein changes indicated disturbances in oxidative stress defense, mitochondrial respiration and morphology, cell cycle control, and cell viability. Structural and functional analyses revealed an increase in mitochondrial area and the presence of elongated mitochondria as well as impaired glycolysis and lactate-supported respiration, leading to an impaired cell survival in PARK2 KO neurons. This adds valuable insight into the effect of parkin dysfunction in human neurons and provides knowledge of disease-related pathways that can potentially be targeted for therapeutic intervention.

Microbial communities are the driving force behind the degradation of contaminants like aromatic hydrocarbons in groundwater ecosystems. However, little is known about the response of native microbial communities to contamination in pristine environments as well as their potential to recover from a contamination event. Here, we used an indoor aquifer mesocosm filled with sandy quaternary calciferous sediment that was continuously fed with pristine groundwater to study the response, resistance and resilience of microbial communities to toluene contamination over a period of almost two years, comprising 132days of toluene exposure followed by nearly 600days of recovery. We observed an unexpectedly high intrinsic potential for toluene degradation, starting within the first two weeks after the first exposure. The contamination led to a shift from oxic to anoxic, primarily nitrate-reducing conditions as well as marked cell growth inside the contaminant plume. Depth-resolved community fingerprinting revealed a low resistance of the native microbial community to the perturbation induced by the exposure to toluene. Distinct populations that were dominated by a small number of operational taxonomic units (OTUs) rapidly emerged inside the plume and at the plume fringes, partially replacing the original community. During the recovery period physico-chemical conditions were restored to the pristine state within about 35days, whereas the recovery of the biological parameters was much slower and the community composition inside the former plume area had not recovered to the original state by the end of the experiment. These results demonstrate the low resilience of sediment-associated groundwater microbial communities to organic pollution and underline that recovery of groundwater ecosystems cannot be assessed solely by physico-chemical parameters., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Micro scale processes are expected to have a fundamental role in shaping groundwater ecosystems and yet they remain poorly understood and under-researched. In part, this is due to the fact that sampling is rarely carried out at the scale at which microorganisms, and their grazers and predators, function and thus we lack essential information. While set within a larger scale framework in terms of geochemical features, supply with energy and nutrients, and exchange intensity and dynamics, the micro scale adds variability, by providing heterogeneous zones at the micro scale which enable a wider range of redox reactions. Here we outline how understanding micro scale processes better may lead to improved appreciation of the range of ecosystems functions taking place at all scales. Such processes are relied upon in bioremediation and we demonstrate that ecosystem modelling as well as engineering measures have to take into account, and use, understanding at the micro scale. We discuss the importance of integrating faunal processes and computational appraisals in research, in order to continue to secure sustainable water resources from groundwater., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The acidification of the oceans could potentially alter marine plankton communities with consequences for ecosystem functioning. While several studies have investigated effects of ocean acidification on communities using traditional methods, few have used genetic analyses. Here, we use community barcoding to assess the impact of ocean acidification on the composition of a coastal plankton community in a large scale, in situ, long-term mesocosm experiment. High-throughput sequencing resulted in the identification of a wide range of planktonic taxa (Alveolata, Cryptophyta, Haptophyceae, Fungi, Metazoa, Hydrozoa, Rhizaria, Straminipila, Chlorophyta). Analyses based on predicted operational taxonomical units as well as taxonomical compositions revealed no differences between communities in high CO2 mesocosms (~ 760 μatm) and those exposed to present-day CO2 conditions. Observed shifts in the planktonic community composition were mainly related to seasonal changes in temperature and nutrients. Furthermore, based on our investigations, the elevated CO2 did not affect the intraspecific diversity of the most common mesozooplankter, the calanoid copepod Pseudocalanus acuspes. Nevertheless, accompanying studies found temporary effects attributed to a raise in CO2. Differences in taxa composition between the CO2 treatments could, however, only be observed in a specific period of the experiment. Based on our genetic investigations, no compositional long-term shifts of the plankton communities exposed to elevated CO2 conditions were observed. Thus, we conclude that the compositions of planktonic communities, especially those in coastal areas, remain rather unaffected by increased CO2.

Spontaneous cytosolic calcium transients and oscillations have been reported in various tissues of nonhuman and human origin but not in human midbrain-derived stem cells. Using confocal microfluorimetry, we studied spontaneous calcium transients and calcium-regulating mechanisms in a human ventral mesencephalic stem cell line undergoing proliferation and neuronal differentiation. Spontaneous calcium transients were detected in a large fraction of both proliferating (>50%) and differentiating (>55%) cells. We provide evidence for the existence of intracellular calcium stores that respond to muscarinic activation of the cells, having sensitivity for ryanodine and thapsigargin possibly reflecting IP 3 receptor activity and the presence of ryanodine receptors and calcium ATPase pumps. The observed calcium transient activity potentially supports the existence of a sodium-calcium antiporter and the existence of calcium influx induced by depletion of calcium stores. We conclude that the cells have developed the most important mechanisms governing cytosolic calcium homeostasis. This is the first comparative report of spontaneous calcium transients in proliferating and differentiating human midbrain-derived stem cells that provides evidence for the mechanisms that are likely to be involved. We propose that the observed spontaneous calcium transients may contribute to mechanisms involved in cell proliferation, phenotypic differentiation, and general cell maturation.

Research into the causes of neurodegenerative diseases like Parkinson's- and Alzheimer's disease has long been hampered by the lack of access to live disease-afflicted neurons for in vitro studies. The introduction of induced pluripotent stem (iPS) cells has made such studies possible. iPS cells can be reprogrammed from somatic patient-derived cells (e.g. skin cells) and differentiated into any cell type of the body. This allows for the production of neurons, which have the genetic background of the patients and show disease-relevant phenotypes.

Biodegradation is one of the most favored and sustainable means of removing organic pollutants from contaminated aquifers but the major steering factors are still surprisingly poorly understood. Growing evidence questions some of the established concepts for control of biodegradation. Here, we critically discuss classical concepts such as the thermodynamic redox zonation, or the use of steady state transport scenarios for assessing biodegradation rates. Furthermore, we discuss if the absence of specific degrader populations can explain poor biodegradation. We propose updated perspectives on the controls of biodegradation in contaminant plumes. These include the plume fringe concept, transport limitations, and transient conditions as currently underestimated processes affecting biodegradation.

The lac promoter is one of the most commonly used promoters for expression control of recombinant genes in E. coli. In the absence of galactosides, the lac promoter is repressed by its repressor protein LacI. Since the lac promoter is regulated by a repressor, overexpression of LacI is necessary for regulation when the promoter is introduced on a high-copy plasmid. For that purpose, a modified variant of LacI, a LVA-tagged LacI, was submitted to the Registry of Standard Biological Parts and has been used for more than 500 constructs since then. We have found, however, that natural LacI is superior to the LVA-tagged LacI as controller of expression.

Temperature stress in invertebrates is known to be reflected by changes in catecholamine levels. However, the mechanisms of stress response are not fully understood. Groundwater and surface water amphipods are expected to be differently adapted to temperature elevations due to the different temperature regimes in their habitats and consequently, show a different stress response. No data have been published so far regarding the effects of stress on catecholamine patterns in groundwater invertebrates and accordingly, comparisons with surface water fauna are also missing. In this study, we compared the average catecholamine levels in two taxonomically related amphipod species: Niphargus inopinatus, living in groundwater with constant water temperatures throughout the year, and Gammarus pulex, a surface water stream amphipod frequently exposed to diurnal and seasonal temperature fluctuations. Furthermore, we tracked the immediate changes in whole-animal catecholamine levels in response to heat stress in both species. Pronounced differences in the catecholamine levels of the two species became apparent, with the average dopamine (DA) level of N. inopinatus being almost 1000 times higher than that in G. pulex. The noradrenaline (NA) concentrations in N. inopinatus were on average two orders of magnitude higher than in G. pulex, and for adrenaline (A), the difference constituted one order of magnitude. When exposed to short-term heat stress, both species showed a response in terms of catecholamine levels, but the observed patterns were different. In N. inopinatus, temperature stress was reflected by the appearance of adrenaline, while in G. pulex a significant increase in noradrenaline levels occurred in the treatment with the highest temperature elevation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A protocol was developed for testing the ecotoxicological effects of volatile organic compounds (VOCs) on groundwater invertebrates. Test substance volatility was addressed in a "closed from start to analysis"-design. Since manifestation of toxic effects may be delayed in 'slower metabolizing' organisms such as groundwater fauna, a time-independent (TI-) approach was adopted. Toluene was used as a model substance and its toxicity to the groundwater amphipod Niphargus inopinatus was assessed as an example. The method evaluation process considered various methodological issues such as partitioning of the toxicant between the water and the gas phase (Henry equilibrium), the possible depletion of oxygen in closed test vials, as well as microbial biodegradation of the test substance. For N. inopinatus, an LC50,14 days of 46.6mgL(-1) toluene was obtained. The ultimate LC50 value was estimated at 23.3mgL(-1) toluene. No oxygen depletion occurred in the test vials and Henry equilibrium was found to be established after 6h. The new test system proposed now awaits broad practical application., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The use of ecological criteria for the assessment of aquatic ecosystem status is routine for surface waters. So far no ecological parameters are considered for the assessment and monitoring of groundwater quality. It has been well known for decades that aquifers are ecosystems harbouring a vast diversity of invertebrates and microorganisms. The growing knowledge on groundwater microbial and faunal communities as well as the molecular and statistical tools available form a solid ground for the development of first ecologically sound assessment schemes. The sensitivity of groundwater communities towards impacts from land use and surface waters is exemplarily demonstrated by a data set of two geologically similar but hydrologically partially separated aquifer systems. Subgroups of the fauna in groundwater (stygobites vs. stygophiles and stygoxenes) successfully indicated elevated nitrate impacts linked to land use activities. Within the microbial communities, impacts from land use are mirrored by high bacterial biodiversity values atypical for pristine groundwater of comparable systems. The data show that there is legitimate hope for the application of ecological criteria for groundwater quality assessment in the future.

The use of groundwater as a carrier of thermal energy is an important source of sustainable heating and cooling. However, the effects of thermal use on geochemical and biological aquifer characteristics are poorly understood. Here, we have assessed the impacts of heat discharge on an uncontaminated, shallow aquifer by monitoring the hydrogeochemical, bacterial and faunal parameters at an active thermal discharge facility. The observed variability between wells was considerable. Yet, no significant temperature impacts on bacterial or faunal abundance and on bacterial productivity were observed. Also, we did not observe an improved survival or growth of coliforms with temperature. In contrast, the diversity of bacterial terminal restriction fragment (T-RF) length polymorphism fingerprints and faunal populations was either positively or negatively affected by temperature, respectively, and the abundance of selected T-RFs was clearly temperature dependent. Canonical correspondence analysis indicated that both the impact of temperature and of surface water from a nearby river, were important drivers of aquifer biotic variability. These results demonstrate that aquifer thermal energy discharge can affect aquifer bacteria and fauna, while at the same time controlling only a minor part of the total seasonal and spatial variability and therefore posing no likely threat to ecosystem functioning and drinking water protection in uncontaminated, shallow aquifers.

The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) was found to be unstable in air when adsorbed on a thin-layer silica gel chromatography plate, a result that is in good agreement with the relatively high reactivity of this compound. Three novel main products were separated from the reaction mixture and identified by mass spectrometry and nuclear magnetic resonance data as: (i) 3-acetamidomethyl-6-methoxycinnolinone (AMMC), (ii) 3-nitro-AMK (AMNK, N1-acetyl-5-methoxy-3-nitrokynuramine), and (iii) N-[2-(6-methoxyquinazolin-4-yl)-ethyl]-acetamide (MQA). AMMC and AMNK are shown to be nonenzymatically formed also in solution, by nitric oxide (NO) in the first case, and by a mixture of peroxynitrite and hydrogen carbonate, in the second one. The use of three different NO donors, PAPA-NONOate, S-nitroso-N-acetylpenicillamine and sodium nitroprussiate led to essentially the same results, with regard to a highly preferential formation of AMMC; AMNK was not detected in these reaction systems. Competition experiments with the NO scavenger N-acetylcysteine indicate a somewhat lower reactivity compared with the competitor. Peroxynitrite led to AMNK formation in the presence of physiological concentrations of hydrogen carbonate at pH 7.4, but not in its absence, indicating that nitration involves a mixture of carbonate radicals and NO2, formed from the peroxynitrite-CO2 adduct. No AMMC was detected after AMK exposure to peroxynitrite. Both AMNK and AMMC exhibited a much lower reactivity toward 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radicals than did AMK. In a competition assay for hydroxyl radicals, AMMC showed prooxidant properties, whereas AMNK was a moderate antioxidant. AMMC and AMNK should represent relatively stable physiological products, although their rates of synthesis are still unknown and may be low. Formation of these compounds may contribute to the disappearance of AMK from tissues and body fluids.

The 28-gauge catheter supplied by Kendall Healthcare Products Company (Mansfield, MA) for continuous spinal anesthesia has two important characteristics. The ink used for the centimeter calibration marks and to identify the tip of the catheter "washes off" while it is inserted in the patient. In addition, the catheter has a tendency to stretch while it is in the patient or being removed. These two characteristics produced a situation in which it was impossible to determine whether a catheter fragment had been left in a patient. This predicament had a serious impact on the postoperative management of the patient. A computerized tomographic scan and spine films were negative, while other evidence suggested that a fragment was left behind. When the patient developed a postdural puncture headache, the authors felt that an epidural blood patch was contraindicated. The patient's headache did resolve with 48 hours of conservative therapy.

This report describes five patients with failed continuous spinal anesthetics with lidocaine. Lidocaine concentrations of 420 to 880 micrograms/ml were assayed in the spinal fluid despite lack of anesthesia. There was free flow of cerebrospinal fluid (CSF) through the catheter in all patients before and after the injection of lidocaine. Inactive lidocaine was not responsible for the failures, since lidocaine from the same manufacturing batch produced anesthesia in other patients. Incorrect catheter placement could not explain the failures, since bupivacaine injected through the same intraspinal catheter produced anesthesia in four of the five patients. The authors, therefore, suggest that some patients have a physiologic resistance to lidocaine.