Cobalamins function as allosteric activators of an Angelman syndrome-associated UBE3A/E6AP variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution.

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Brain-derived Tau for Monitoring Brain Injury in Acute Ischemic Stroke.

The evolution of infarcts varies widely among patients with acute ischemic stroke (IS) and influences treatment decisions. Neuroimaging is not applicable for frequent monitoring and there is no blood-based biomarker to track ongoing brain injury in acute IS. Here, we examined the utility of plasma brain-derived tau (BD-tau) as a biomarker for brain injury in acute IS. We conducted the prospective, observational Precision Medicine in Stroke [PROMISE] study with serial blood sampling upon hospital admission and at days 2, 3, and 7 in patients with acute ischemic stroke (IS) and for comparison, in patients with stroke mimics (SM). We determined the temporal course of plasma BD-tau, its relation to infarct size and admission imaging-based metrics of brain injury, and its value to predict functional outcome. Upon admission (median time-from-onset, 4.4h), BD-tau levels in IS patients correlated with ASPECTS (ρ=-0.21, P<.0001) and were predictive of final infarct volume (ρ=0.26, P<.0001). In contrast to SM patients, BD-tau levels in IS patients increased from admission (median, 2.9 pg/ml [IQR, 1.8-4.8]) to day 2 (median time-from-onset, 22.7h; median BD-tau, 5.0 pg/ml [IQR, 2.6-10.3]; P<.0001). The rate of change of BD-tau from admission to day 2 was significantly associated with collateral supply (R2=0.10, P<.0001) and infarct progression (ρ=0.58, P<.0001). At day 2, BD-tau was predictive of final infarct volume (ρ=0.59, P<.0001) and showed superior value for predicting the 90-day mRS score compared with final infarct volume. In conclusion, in 502 patients with acute IS, plasma BD-tau was associated with imaging-based metrics of brain injury upon admission, increased within the first 24 hours in correlation with infarct progression, and at 24 hours was superior to final infarct volume in predicting 90-day functional outcome. Further research is needed to determine whether BD-tau assessments can inform decision-making in stroke care.

A miniTurbo-based proximity labeling protocol to identify conditional protein interactomes in vivo in Myxococcus xanthus.

Protein-protein interactions are foundational for many cellular processes. Such interactions are especially challenging to identify if they are transient or depend on environmental conditions. This protocol details steps to identify stable and transient protein interactomes in the bacterium Myxococcus xanthus using biotin ligase miniTurbo-based proximity labeling. We include instructions for optimizing the expression of control proteins, in vivo biotin labeling of bacteria grown on a surface or in suspension culture, enrichment of biotinylated proteins, and sample processing for proteomic analysis. For complete details on the use and execution of this protocol, please refer to Branon et al. (2018).1.

Enrichment and structural assignment of geometric isomers of unsaturated furan fatty acids.

Furan fatty acids (FuFAs) are valuable minor fatty acids, which are known for their excellent radical scavenging properties. Typically, the furan moiety is embedded in an otherwise saturated carboxyalkyl chain. Occasionally, these classic FuFAs are accompanied by low amounts of unsaturated furan fatty acids (uFuFAs), which additionally feature one double bond in conjugation with the furan moiety. A recent study produced evidence for the occurrence of two pairs of E-/Z-uFuFA isomers structurally related to saturated uFuFAs. Here, we present a strategy that allowed such trace compounds to be enriched to a level suited for structure determination by NMR. Given the low amounts and the varied abundance ratio of the four uFuFA isomers, the isolation of individual compounds was not pursued. Instead, the entire isomer mixture was enriched to an amount and purity suitable for structure investigation with contemporary NMR methods. Specifically, lipid extracted from 150 g latex, the richest known source of FuFAs, was subsequently fractionated by countercurrent chromatography (CCC), silver ion, and silica gel column chromatography. Analysis of the resulting mixture of four uFuFAs isomers (2.4 mg in an abundance ratio of 56:23:11:9) by different NMR techniques including PSYCHE verified that the structures of the two most abundant isomers were E-9-(3-methyl-5-pentylfuran-2-yl)non-8-enoic acid and E-9-(3-methyl-5-pent-1-enylfuran-2-yl)nonanoic acid. Additionally, we introduced a computer-based method to generate an averaged chromatogram from freely selectable GC/MS runs of CCC fractions without the necessity of pooling aliquots. This method was found to be suitable to simplify subsequent enrichment steps.

Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry.

Regulated protein degradation controls protein levels of all short-lived proteins to ensure cellular homeostasis and also protects cells from misfolded or other abnormal proteins. The most important players in the degradation system are E3 ubiquitin ligases which recognize exposed sequence motifs, so-called degrons, of target proteins and mark them through the attachment of ubiquitin for degradation. N-terminal (Nt) sequences are extensively used as degrons (N-degrons) and all 20 amino acids are able to feed proteins in 1 of the 5 known N-degron pathways. Studies have mainly focused on characterizing systematically the role of the starting amino acid on protein stability and less on the identification of the E3 ligases involved. Recent data from our lab and literature suggest that there is an extensive interplay of N-recognins and Nt-modifying enzymes like Nt-acetyltransferases (NATs) or N-myristoyltransferases which only starts to be elucidated. It suggests that improperly modified or unexpectedly unmodified proteins become rapidly removed after synthesis ensuring protein maturation and quality control of specific subsets of proteins. Here, we describe a peptide pull-down and down-stream bioinformatics workflow conducted in the MaxQuant and Perseus computational environment to identify N-recognin candidates in an unbiased way using quantitative mass spectrometry (MS)-based proteomics. Our workflow allows the identification of N-recognin candidates for specific N-degrons, to determine their sequence specificity and it can be applied as well more general to identify binding partners of N-terminal modifications. This method paves the way to identify pathways involved in protein quality control and stability acting at the N-terminus.

Biomolecular condensate drives polymerization and bundling of the bacterial tubulin FtsZ to regulate cell division.

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we study the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization and bundle FtsZ filaments, suggesting a cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. This mechanism shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism's ancient origin.

Amisulpride as a potential disease-modifying drug in the treatment of tauopathies.

INTRODUCTION: Hyperphosphorylation and aggregation of the microtubule-associated protein tau cause the development of tauopathies, such as Alzheimer's disease and frontotemporal dementia (FTD). We recently uncovered a causal link between constitutive serotonin receptor 7 (5-HT7R) activity and pathological tau aggregation. Here, we evaluated 5-HT7R inverse agonists as novel drugs in the treatment of tauopathies. METHODS: Based on structural homology, we screened multiple approved drugs for their inverse agonism toward 5-HT7R. Therapeutic potential was validated using biochemical, pharmacological, microscopic, and behavioral approaches in different cellular models including tau aggregation cell line HEK293 tau bimolecular fluorescence complementation, primary mouse neurons, and human induced pluripotent stem cell-derived neurons carrying an FTD-associated tau mutation as well as in two mouse models of tauopathy. RESULTS: Antipsychotic drug amisulpride is a potent 5-HT7R inverse agonist. Amisulpride ameliorated tau hyperphosphorylation and aggregation in vitro. It further reduced tau pathology and abrogated memory impairment in mice. DISCUSSION: Amisulpride may be a disease-modifying drug for tauopathies.

Biochemical characterisation of Mer3 helicase interactions and the protection of meiotic recombination intermediates.

Crossing over between homologs is critical for the stable segregation of chromosomes during the first meiotic division. Saccharomyces cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of the majority of crossovers during meiosis. Here, we describe the structural organisation of Mer3 and using AlphaFold modelling and XL-MS we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1 helicase, but only in the presence of Dmc1. Thus we provide a mechanism whereby Mer3 interacts with a network of proteins to protect Dmc1 derived D-loops from dissolution.

Epigenomic Profiling of Dorsal Root Ganglia upon Regenerative and Non-regenerative Axonal Injury.

RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and assay for transposase-accessible chromatin sequencing (ATAC-seq) are genome-wide techniques that provide information relative to gene expression, chromatin binding sites, and chromatin accessibility, respectively. Here we describe RNA-seq, H3K9ac, H3K27ac and H3K27me3 ChIP-seq, and ATAC-seq in dorsal root ganglia (DRG) after sciatic nerve or dorsal column axotomy, to characterize the transcriptional and epigenetic signatures of DRG upon regenerative vs non-regenerative axonal lesion.

Induced Remodelling of Astrocytes In Vitro and In Vivo by Manipulation of Astrocytic RhoA Activity.

Structural changes of astrocytes and their perisynaptic processes occur in response to various physiological and pathophysiological stimuli. They are thought to profoundly affect synaptic signalling and neuron-astrocyte communication. Understanding the causal relationship between astrocyte morphology changes and their functional consequences requires experimental tools to selectively manipulate astrocyte morphology. Previous studies indicate that RhoA-related signalling can play a major role in controlling astrocyte morphology, but the direct effect of increased RhoA activity has not been documented in vitro and in vivo. Therefore, we established a viral approach to manipulate astrocytic RhoA activity. We tested if and how overexpression of wild-type RhoA, of a constitutively active RhoA mutant (RhoA-CA), and of a dominant-negative RhoA variant changes the morphology of cultured astrocytes. We found that astrocytic expression of RhoA-CA induced robust cytoskeletal changes and a withdrawal of processes in cultured astrocytes. In contrast, overexpression of other RhoA variants led to more variable changes of astrocyte morphology. These induced morphology changes were reproduced in astrocytes of the hippocampus in vivo. Importantly, astrocytic overexpression of RhoA-CA did not alter the branching pattern of larger GFAP-positive processes of astrocytes. This indicates that a prolonged increase of astrocytic RhoA activity leads to a distinct morphological phenotype in vitro and in vivo, which is characterized by an isolated reduction of fine peripheral astrocyte processes in vivo. At the same time, we identified a promising experimental approach for investigating the functional consequences of astrocyte morphology changes.

[Support needs of care dependent individuals : Survey of general and specific needs for information, assistance and support of care dependent individuals and their relatives]. / Unterstützungsbedarfe pflegeempfangender Menschen : Erhebung allgemeiner und spezieller Informations­, Hilfe- und Unterstützungsbedarfe pflegeempfangender Menschen und ihrer Angehörigen.

BACKGROUND: In 2009, statutory regulations on information and counselling regarding nursing care needs, performed by so-called care advisors have been implemented for persons in need of long-term care and their relatives. In order to adequately prepare these care advisors, contemporary needs and requirements must be determined. The aim of the study was to determine the different needs of persons in need of long-term care and their relatives. METHOD: Care advisors were interviewed via an online survey tool using a standardized questionnaire. A 5-point Likert scale was used to determine the needs regarding information and advice on 16 specific topics. In general, overall needs regarding information and advice of care recipients and relatives were recorded using a 10-point scale (1 low and 10 high). Using classification and regression trees (CRT) and random forest, the correlation between the individual main topics and the general need for advice was analyzed. RESULTS: The participating care advisors (n = 276) rated the general demand for information of people in need of care and their relatives with a mean of 7.8 and 9.2, respectively. For those in need of care, the strongest association of general information needs was the topic of housing advice For the relatives, the topic social law aspects and benefits was the most relevant association. CONCLUSION: The general demand for information was rated very high. Since differences became obvious between those in need of care and their relatives, it is necessary to adjust care advice for these two groups.

Geometrical and positional isomers of unsaturated furan fatty acids in food.

Furan fatty acids (FuFA) are important antioxidants found in low concentrations in many types of food. In addition to conventional FuFA which normally feature saturated carboxyalkyl and alkyl chains, a few previous studies indicated the FuFA co-occurrence of low shares of unsaturated furan fatty acids (uFuFA). For their detailed analysis, the potential uFuFA were enriched by centrifugal partition chromatography (CPC) or countercurrent chromatography (CCC) followed by silver ion chromatography from a 4,7,10,13,16,19-docosahexaenoic acid ethyl ester oil, a 5,8,11,14,17-eicosapentaenoic acid ethyl ester oil and a latex glove extract. Subsequent gas chromatography with mass spectrometry (GC/MS) analysis enabled the detection of 16 individual uFuFA isomers with a double bond in conjugation with the central furan moiety. In either case, four instead of two uFuFA isomers previously reported in food, respectively, were detected by GC/MS. These isomers showed characteristic elution and abundance patterns in GC/MS chromatograms which indicated the presence of two pairs of cis/trans-isomers (geometrical isomers).

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots.

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes.

Furan Fatty Acids in Some 20 Fungi Species: Unique Profiles and Quantities.

Furan fatty acids (FuFAs) are a group of excellent antioxidants in food. Since data in fungi were scarce, 37 commercial or collected edible and meadow fungi were analyzed on FuFA patterns and contents. FuFA amounts in fresh fungi ranged from not detectable (n = 2) to 40 mg/100 g fungi dry weight. Fresh samples of the popular edible fungi genera Agaricus and Pleurotus showed comparable FuFA contents of 9.0-33 mg/100 g fungi dry weight. The unique FuFA profile of the fungi was dominated by 9-(3,4-dimethyl-5-pentylfuran-2-yl)-nonanoic acid (9D5). In addition, the uncommon 9-(3,4-dimethyl-5-butylfuran-2-yl)-nonanoic acid (9D4) was present in 30% of the samples with contents of up to 0.2 mg/100 g fungi dry weight. Countercurrent separation techniques were used to isolate the main FuFA 9D5, to verify the presence of 9D4, and to determine ultra-traces of 11-(3,4-dimethyl-5-pentylfuran-2-yl)-undecanoic acid (11D5), which may have been assimilated by the fungi from the substrate.

CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin.

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

Serotonin Receptor 5-HT2A Regulates TrkB Receptor Function in Heteroreceptor Complexes.

Serotonin receptor 5-HT2A and tropomyosin receptor kinase B (TrkB) strongly contribute to neuroplasticity regulation and are implicated in numerous neuronal disorders. Here, we demonstrate a physical interaction between 5-HT2A and TrkB in vitro and in vivo using co-immunoprecipitation and biophysical and biochemical approaches. Heterodimerization decreased TrkB autophosphorylation, preventing its activation with agonist 7,8-DHF, even with low 5-HT2A receptor expression. A blockade of 5-HT2A receptor with the preferential antagonist ketanserin prevented the receptor-mediated downregulation of TrkB phosphorylation without restoring the TrkB response to its agonist 7,8-DHF in vitro. In adult mice, intraperitoneal ketanserin injection increased basal TrkB phosphorylation in the frontal cortex and hippocampus, which is in accordance with our findings demonstrating the prevalence of 5-HT2A-TrkB heteroreceptor complexes in these brain regions. An expression analysis revealed strong developmental regulation of 5-HT2A and TrkB expressions in the cortex, hippocampus, and especially the striatum, demonstrating that the balance between TrkB and 5-HT2A may shift in certain brain regions during postnatal development. Our data reveal the functional role of 5-HT2A-TrkB receptor heterodimerization and suggest that the regulated expression of 5-HT2A and TrkB is a molecular mechanism for the brain-region-specific modulation of TrkB functions during development and under pathophysiological conditions.

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts.

Easy and reliable identification of pathogenic species such as yeasts, emerging as problematic microbes originating from the genus Candida, is a task in the management and treatment of infections, especially in hospitals and other healthcare environments. Aptamers are seizing an already indispensable role in different sensing applications as binding entities with almost arbitrarily tunable specificities and optimizable affinities. Here, we describe a polyclonal SELEX library that not only can specifically recognize and fluorescently label Candida cells, but is also capable to differentiate C. albicans, C. auris and C. parapsilosis cells in flow-cytometry, fluorometric microtiter plate assays and fluorescence microscopy from human cells, exemplified here by human dermal fibroblasts. This offers the opportunity to develop diagnostic tools based on this library. Moreover, these specific and robust affinity molecules could also serve in the future as potent binding entities on biomaterials and as constituents of technical devices and will thus open avenues for the development of cost-effective and easily accessible next generations of electronic biosensors in clinical diagnostics and novel materials for the specific removal of pathogenic cells from human bio-samples.

The gut metabolite indole-3 propionate promotes nerve regeneration and repair.

The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate1. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms2. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration3. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and the release of neurotrophins, can be activated by intermittent fasting (IF)4,5. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in mice through an unexpected mechanism that relies on the gram-positive gut microbiome and an increase in the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating the recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype, which was confirmed by inhibition of neutrophil chemotaxis. Our results demonstrate the ability of a microbiome-derived metabolite, such as IPA, to facilitate regeneration and functional recovery of sensory axons through an immune-mediated mechanism.