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SUMMARYPathogenic gene variants in humans affecting the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown genetic modifiers. To identify such modifiers, we established novel congenic mouse models. LRP2 deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on FVB/N background indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identifiedPttg1andUlk4as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are new positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized PTTG1 as a novel primary cilia component in the neuroepithelium. The identification of genes, that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart, is likely relevant to understand variability in human congenital disorders.

Rhodamine molecules are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling of the four methyl groups, which improves photophysical (i.e. brightness, lifetimes) and chemical (i.e. bleaching) properties. We explore this finding for SNAP- and Halo-tag labelling, and highlight enhanced properties in several applications, such as Förster resonance energy transfer, fluorescence activated cell sorting, fluorescence lifetime microscopy and stimulated emission depletion nanoscopy. We envision deuteration as a generalizable concept to improve existing and develop new Chemical Biology Probes.

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Summary Plant metabolism is broadly reprogrammed during acclimation to abiotic changes. Most previous studies have focused on transitions from standard to single stressful conditions. Here, we systematically analyze acclimation processes to levels of light, heat, and cold stress that subtly alter physiological parameters and assess their reversibility during de-acclimation. Metabolome and transcriptome changes were monitored at 11 different time points. Unlike transcriptome changes, most alterations in metabolite levels did not readily return to baseline values, except in the case of cold acclimation. Similar regulatory networks operate during (de-)acclimation to high light and cold, whereas heat and high-light responses exhibit similar dynamics, as determined by surprisal and conditional network analyses. In all acclimation models tested here, super-hubs in conditional transcriptome networks are enriched for components involved in translation, particularly ribosomes. Hence, we suggest that the ribosome serves as a common central hub for the control of three different (de-)acclimation responses., Graphical Abstract, Highlights • Time series of acclimation and de-acclimation without pleiotropic stresses • Kinetics of transcriptomes and metabolomes under three environmental conditions • Transcriptomes return to a baseline, but HL and heat metabolomes do not • All acclimation responses involve the translational machinery and photosynthesis, Biological Sciences; Metabolomics; Plant Biology; Plants; Transcriptomics

Presynaptic precursors deliver synaptic proteins to nascent and growing synapses. The authors provide evidence that during an early precursor formation step, the small GTPase Rab2 regulates immature precursors formation at the trans-Golgi prior to subsequent Arl8-dependent maturation steps., Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi., Graphical Abstract

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC50s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.

SummaryReliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals is prerequisite for faithful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. Here we show that in mutants of the small GTPase RAB2 active zone and synaptic vesicle proteins accumulated in the neuronal somata at the trans-Golgi network and were consequently depleted at synaptic terminals, provoking neurotransmission deficits. The ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40×60 nm and segregated in subfractions either positive for active zone proteins or co-positive for synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, rab2 behaved epistatic over arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we here identified a Golgi-associated assembly sequence in presynaptic precursor vesicle biogenesis controlled by RAB2 dependent membrane remodelling and protein sorting at the trans-Golgi.

An untreated hearing impairment could have a severe influence on the morbidity. The Mini-Audio-Test (MAT) has been developed for early detecting of a relevant hearing loss. This follow-up investigation should determine the sensitivity, specificity, and the predictive values for a minimum-level of detected hearing loss of 25, 30, 35, and 40 dB, both, in one octave-frequency between 0.5 and 4 kHz and the average of hearing loss for these four frequencies.This survey uses data which were collected in 2016 and 2017. 943 patients parted into two groups (aged 50 to 59 years and aged 60 years and more), were investigated using the MAT. Statistical analysis on the sensitivity, specificity, and predictive values in respect were done as proportions together with the 95 %-confidence interval by a logistic regression.The sensitivity of the MAT was increasing in both groups of age by increasing the minimum of to be detected hearing loss. The specificity was decreasing as well, but less. The positive predictive values were higher for the older age-group and are decreasing by increasing of the minimum of to be detected hearing loss. In general, the values using the averaged hearing loss are noticeably lower than for the single frequency detecting. The values for the negative predictive values were contrary.The results for persons aged 60 years and more are worse than for the younger. Therefore, a first screening on hearing loss starting at the age of 50 years seems to be recommended urgently. Only by this, a sufficient rehabilitation of the hearing loss could be done in time so that the negative consequences of an untreated hearing impairment could be influenced positively.Eine nichtbehandelte Schwerhörigkeit hat einen erheblichen Einfluss auf die Morbidität. Zur Früherkennung wurde der Mini-Audio-Test (MAT) entwickelt. Im Rahmen einer Nachuntersuchung soll untersucht werden, welche Werte sich für die Sensitivität, Spezifität und prädiktiven Werte für einen minimal zu detektierenden Hörverlust von 25, 30, 35 und 40 dB sowohl in mindestens 1 der 4 Oktavfrequenzen zwischen 0,5 und 4 kHz als auch in den über diesen Frequenzen gemittelten Hörverlust ergeben.Nachuntersucht wurden Daten, die 2016 und 2017 erhoben wurden. Bestimmt wurden die Sensitivität, Spezifität und die negativ und positiv prädiktiven Werte mittels einer logistischen Regression.Die Sensitivität des MAT stieg in beiden Altersgruppen mit zunehmendem zu detektierendem Hörverlust an, die Spezifität sank analog. Die positiv prädiktiven Werte waren für die Älteren höher als für die Jüngeren und sanken mit steigendem, minimal zu detektierendem Hörverlust. Dabei lagen die Werte für die gemittelten Frequenzen deutlich unter denen, die den Hörverlust nur in 1 Frequenz berücksichtigen. Die negativ prädiktiven Werte verhielten sich genau entgegengesetzt.Die Werte sind für Ältere ab dem 60. Lebensjahr deutlich schlechter als für Jüngere ab dem 50. Lebensjahr. Dies lässt es umso wichtiger erscheinen, dass bereits ein erstes Hörscreening ab dem 50. Lebensjahr durchgeführt wird, um einen individuellen Hörverlust realistisch einschätzen zu können. Nur durch eine rechtzeitige Hörrehabilitation können negative Folgen einer unbehandelten Schwerhörigkeit positiv beeinflusst werden.

The cellular analysis of mushroom body (MB)-dependent memory forming processes is far advanced, whereas, the molecular and physiological understanding of their synaptic basis lags behind. Recent analysis of the Drosophila olfactory system showed that Unc13A, a member of the M(Unc13) release factor family, promotes a phasic, high release probability component, while Unc13B supports a slower tonic release component, reflecting their different nanoscopic positioning within individual active zones. We here use STED super-resolution microscopy of MB lobe synapses to show that Unc13A clusters closer to the active zone centre than Unc13B. Unc13A specifically supported phasic transmission and short-term plasticity of Kenyon cell:output neuron synapses, measured by combining electrophysiological recordings of output neurons with optogenetic stimulation. Knockdown of unc13A within Kenyon cells provoked drastic deficits of olfactory aversive short-term and anaesthesia-sensitive middle-term memory. Knockdown of unc13B provoked milder memory deficits. Thus, a low frequency domain transmission component is probably crucial for the proper representation of memory-associated activity patterns, consistent with sparse Kenyon cell activation during memory acquisition and retrieval. Notably, Unc13A/B ratios appeared highly diversified across MB lobes, leaving room for an interplay of activity components in memory encoding and retrieval.

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease.

Urban areas are hubs for innovation, economic activity and growth that hugely influence the development of our society, making those areas important drivers in the global transition towards circular economy. Although global policies are necessary to set the general ambition, local interventions are crucial to realize it. This paper presents a methodological framework aimed at facilitating the understanding and application of circular economy strategies in urban systems, that being a single city, or urban regions. The framework is conceived as a flexible structure that contains a network of potential decisions, describing different convergence and divergence points, and that is meant as a supporting tool for future urban circular economy implementation initiatives. After a literature review and an analysis of specific case studies on urban circular economy implementation, a four-phased methodology is proposed where the territory is explored in order to identify and select initiatives in the areas with greatest potential for circular economy. Actions needed to implement the selected initiatives are finally summarized in a roadmap. Each phase contains recommendations of different tasks to complete and available tools for achieving the expected results. Different approaches to adopt in the application of this methodology are discussed as well, such as production-based vs. consumption-based, and top-down vs. bottom-up. Special emphasis is put on the importance of involving local agents, in order to obtain specific and validated proposals that are adapted to the reality of the territory and the concerns of the stakeholders. Through these comprehensive guidelines, the ultimate goal of this methodology is to help urban systems to foster circular economy principles, and therefore reaffirm their role in addressing and managing global sustainability issues.

Coastal settlements face many hazards from climate change. Consequently, there has been extensive focus on developing and implementing adaptation. However, these efforts have prodominantly centred on larger cities. Coastal towns and small cities (urban areas between 1000 and 100,000 people) have received little attention, despite experiencing a number of barriers to adaptation. The absence of information on the global scale of the adaptation challenge within coastal towns and small cities may have contributed to these settlements being overlooked. This paper develops a method that can be used to estimate the numbers, sizes, and locations of coastal towns and small cities worldwide from global population data (Global Human Settlement data). Denmark is used as a pilot for this method with settlements over 1000 people classified with relatively high accuracy. The method developed here represents a potentially fruitful approach to supporting coastal adaptation, as coastal towns and small cities are identifiable globally, they can be classified into types. This will support an assessment of their risk to coastal hazards, and could facilitate knowledge and practice sharing between similar coastal towns and small cities.

Petzoldt et al. investigate the functional protein architecture at the presynaptic active zone, an elaborate protein scaffold organizing synaptic vesicle (SV) release. They find that the conserved multidomain protein RIM-BP provides a relay to guide SVs during their recruitment into membrane close SV release sites., At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a “hinge” in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs’ conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites., Graphical Abstract

Background: Regional ventilation of the lung can be visualized by pulmonary electrical impedance tomography (EIT). The aim of this study was to examine the post‐operative redistribution of regional ventilation after lung surgery dependent on the side of surgery and its association with forced vital capacity. Methods: In this prospective, observational cohort study 13 patients undergoing right and 13 patients undergoing left‐sided open or video‐thoracoscopic procedures have been investigated. Pre‐operative measurements with EIT and spirometry were compared with data obtained 3 days post‐operation. The center of ventilation (COV) within a 32 × 32 pixel matrix was calculated from EIT data. The transverse axis coordinate of COV, COVx (left/right), was modified to COVx′ (ipsilateral/contralateral). Thus, COVx′ shows a negative change if ventilation shifts contralateral independent of the side of surgery. This enabled testing with two‐way ANOVA for repeated measurements (side, time). Results: The perioperative shift of COVx′ was dependent on the side of surgery (P = .007). Ventilation shifted away from the side of surgery after the right‐sided surgery (COVx′‐1.97 pixel matrix points, P

Corals are ecosystem engineers of the coral reefs, one of the most biodiverse but severely threatened marine ecosystems. The ability of corals to form the three dimensional structure of reefs depends on the precipitation of calcium carbonate under biologically control. However, the exact mechanisms underlying this biologically controlled biomineralization remain to be fully unelucidated, for example whether corals employ a different molecular machinery for the deposition of different calcium carbonate (CaCO3) polymorphs (i.e., aragonite or calcite). Here we used tandem mass spectrometry (MS/MS) to compare skeletogenic proteins, i.e., the proteins occluded in the skeleton of three octocoral and one scleractinian species: Tubipora musica and Sinularia cf. cruciata, both forming calcite sclerites, the blue coral Heliopora coerulea with an aragonitic rigid skeleton, and the scleractinian aragonitic Montipora digitata. We observed extremely low overlap between aragonitic and calcitic species, while a core set of proteins is shared between octocorals producing calcite sclerites. However, the same carbonic anhydrase (CruCA4) is employed for the formation of skeletons of both polymorphs. Similarities could also be observed between octocorals and scleractinians, including the presence of a galaxin-like protein. Additionally, as in scleractinians, some octocoral skeletogenic proteins, such as acidic proteins and scleritin, appear to have been secondarily co-opted for calcification and likely derive from proteins playing different extracellular functions. In H. coerulea, co-option was characterized by aspartic acid-enrichment of proteins. This work represents the first attempt to identify the molecular basis underlying coral skeleton polymorph diversity, providing several new research targets and enabling both future functional and evolutionary studies aimed at elucidating the origin and evolution of biomineralization in corals.

This paper investigates grammatical variation in the complementation of the verb provide. It describes the distribution of the four possible patterns with two internal arguments and the interaction between pattern choice and lexical choice. The study finds and documents significant differences in the preferred complementation patterns for American and British English as well as for spoken and written news genres. It also establishes the double object construction as a viable option for American English. Methodologically, this study is based on robust automatic syntactic annotation and computerized retrieval from a data-set comprising 2.5 billion words. It is this large amount of data that permits the observation of strong preferences in terms of pattern choice at the interface between grammar and lexis.

In contrast to coastal towns and small urban settlements, small coastal cities (population of between 50,000 and 100,000) may exhibit comparable knowledge and planning infrastructure as larger cities or be similarly connected to research institutions. However, at a global level, there is little statistical data about small cities, and their numbers and locations are thus so far unknown. In this paper, five cases of small coastal cities, two of which are located in the USA - Miami Beach and Milford - and three, in Australia (Mandurah), Bangladesh (Sundarbans), and South Africa (Knysna) respectively, are presented and discussed in view of adaptation to climate change. All cases reported vulnerability to sea level rise, with impacts compounded by increasing storminess (and even hurricanes of increasing intensity), tides, inland flooding and loss of natural habitat. Changes in precipitation resulted in both reduced river flow, leading to sediment deficits and coastal erosion (from the sea) or increased river flow with similar damage (from the land). Adaptation response was most clearly delineated along lines of development status with wealthier, more highly developed cities pursuing more technologically advanced solutions, whilst the two cities of the Global South focused more on governance and capacity building solutions. Also, it was clear from the Knysna case that if a city or municipality lacks the capacity to effectively respond with adaptation measures, local private wealth may dominate and result in haphazard adaptation responses to the detriment of regional adaptation planning efforts. While this paper provides a first step in establishing a richer understanding, it is clear that more case studies across global regions and economic categories are required to deepen our understanding of climate change adaptation planning in small cities.

Based on a dataset of 84 papers and industry studies, we analyse the impact of digital transformation on the insurance sector using Porter’s value chain (The Competitive Advantage: Creating and Sustaining Superior Performance, The Free Press, New York, 1985) and Berliner’s insurability criteria (Limits of Insurability of Risks, Prentice-Hall, Englewood Cliffs, NJ, 1982). We also present future research directions from the academic and practitioner points of view. The results illustrate four major tasks the industry is facing: enhancing the customer experience, improving its business processes, offering new products, and preparing for competition with other industries. Moreover, we identify three key areas of change with respect to insurability: the effect of new and more information on information asymmetry and risk pooling, the implications of new technologies on loss frequency and severity, and the increasing dependencies of systems through connectivity.

First impressions are formed by the external appearance and, in this respect, essentially by an examination of the face. In the literature, the teeth, especially the maxillary front, are among an eye-catching and sensitive area that plays a significant role in the overall evaluation of appearance. In this study, the first eye fixation of 60 subjects with different levels of dental training (layperson, trained layperson, dental student, and dentist) is recorded using an eye-tracking system, and their subsequent evaluation of the images is recorded. Ten unedited original photographs of different maxillary anterior teeth and ten subsequently edited photographs will be used to evaluate forensic aspects such as the effect of symmetry and color on the overall evaluation. The results will be used to determine which areas of the maxillary anterior are demonstrably viewed and whether knowledge of dental esthetics influences evaluation and viewing.

Introduction: The aim of this ex vivo study was to investigate the influence of different insertion torques on primary stability of a conical and a cylindrical implant system. Materials and Methods: Thirty-two dental implants (Astra Tech OsseoSpeed 5.0 S × 11 mm cylindrical [n = 16] and 5.0 × 11 mm conical [n = 16]) were inserted with 20, 30, 40, and 45 N·cm into fresh porcine bone of mixed trabecular-cortical quality. Before insertion, bone quality was assessed via cone beam tomography. After insertion, resonance frequency analysis was reported using the implant stability quotient (ISQ). Implant insertion depths were evaluated, and the implants were pushed out of the bone by force (measured in N). All experiments were done with n = 4 per group. Results: The highest ISQ (mean 78.25 ± 2.9) and pushout values (mean 675 N ± 5.8) were measured for the cylindrical implant after insertion using 30 N·cm. The conical implant showed the highest primary stability by means of ISQ (mean 76.25 ± 2.2) and pushout force (mean 502.5 N ± 9.6) after an insertion torque of 40 N·cm. If more insertion force was used, primary stability was reduced in all cases. Conclusion: The data indicate that different forms of an implant system need different insertion torques to obtain an optimal primary stability. These results have to be verified clinically.

Plant responses to coincident nutrient deficiencies cannot be predicted from the responses to individual deficiencies. Although copper (Cu) and iron (Fe) are essential micronutrients for plant growth that are often and concurrently limited in soils, the combinatorial response to Cu-Fe deficiency remains elusive. In the present study, we characterised the responses of Arabidopsis thaliana plants deprived of Cu, Fe or both (-Cu-Fe) at the level of plant development, mineral composition, and reconfiguration of transcriptomes, proteomes and metabolomes. Compared to single deficiencies, simultaneous -Cu-Fe leads to a distinct pattern in leaf physiology and microelement concentration characterised by lowered protein content and enhanced manganese and zinc levels. Conditional networking analysis of molecular changes indicates that biological processes also display different co-expression patterns among single and double deficiencies. Indeed, the interaction between Cu and Fe deficiencies causes distinct expression profiles for 15% of all biomolecules, leading to specific enhancement of general stress responses and protein homeostasis mechanisms, at the same time as severely arresting photosynthesis. Accordingly, central carbon metabolites, in particular photosynthates, decrease especially under -Cu-Fe conditions, whereas the pool of free amino acids increases. Further meta-analysis of transcriptomes and proteomes corroborated that protein biosynthesis and folding capacity were readjusted during the combinatorial response and unveiled important rearrangements in the metabolism of organic acids. Consequently, our results demonstrate that the response to -Cu-Fe imposes a distinct reconfiguration of large sets of molecules, not triggered by single deficiencies, resulting into a switch from autotrophy to heterotrophy and involving organic acids such as fumaric acid as central mediators of the response.

Sourdough fermentation influences several properties of leavened baked goods also because Lactic acid bacteria (LAB) and yeasts produce bioactive peptides with a positive effect on human health. In an early study, three Lactobacilli strains (L. farciminis H3 and A11 and L. sanfranciscensis I4) possessing different proteolytic activities were used to produce sourdoughs containing peptides equipped with anti-inflammatory and/or antioxidant properties. This work was aimed to assess whether these properties could be retained after cooking. The selected LABs were used to produce breads from which low molecular weight (LMW-) peptides were extracted. The results provide solid proofs of keeping both antioxidant and anti-inflammatory activities of peptides from cooked products. Sequences of LMW-peptides either from doughs and breads were determined by mass spectrometry: differences have been noticed in amino acidic composition and in sequences, however, all the strains produce peptides equipped with antioxidant and anti-inflammatory activities.

Co- and post-translational processing are crucial maturation steps to generate functional proteins. MetAP-2 plays an important role in this process, and inhibition of its proteolytic activity has been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitors of MetAP-2 may be promising options for the treatment of cancer. This work describes the discovery and structure-based hit optimization of a novel MetAP-2 inhibitory scaffold. Of critical importance, a cyclic tartronic diamide coordinates the MetAP-2 metal ion in the active site while additional side chains of the molecule were designed to occupy the lipophilic methionine side chain recognition pocket as well as the shallow cavity at the opening of the active site. The racemic screening hit from HTS campaign 11a was discovered with an enzymatic IC

The circumventricular organs (CVOs) in the central nervous system (CNS) lack a vascular blood-brain barrier (BBB), creating communication sites for sensory or secretory neurons, involved in body homeostasis. Wnt/β-catenin signaling is essential for BBB development and maintenance in endothelial cells (ECs) in most CNS vessels. Here we show that in mouse development, as well as in adult mouse and zebrafish, CVO ECs rendered Wnt-reporter negative, suggesting low level pathway activity. Characterization of the subfornical organ (SFO) vasculature revealed heterogenous claudin-5 (Cldn5) and Plvap/Meca32 expression indicative for tight and leaky vessels, respectively. Dominant, EC-specific β-catenin transcription in mice, converted phenotypically leaky into BBB-like vessels, by augmenting Cldn5+vessels, stabilizing junctions and by reducing Plvap/Meca32+ and fenestrated vessels, resulting in decreased tracer permeability. Endothelial tightening augmented neuronal activity in the SFO of water restricted mice. Hence, regulating the SFO vessel barrier may influence neuronal function in the context of water homeostasis., SCOPUS: ar.j, info:eu-repo/semantics/published

Epsl5-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice led to increased fat accumulation. This organismic phenotype was paralleled at the cellular level by increased lipid uptake via a caveolae-, dynamin- and CD36-dependent pathway, an elevated number of detached caveolae and higher caveolar mobility. Furthermore, EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent lipid uptake pathway and suggest that low EHD2 expression levels are linked to obesity.

Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) we show that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation., Synaptic plasticity ensures functionality during perturbations and enables memory formation. Here, the authors describe homeostatic functional and nano-modular active zone modifications for immediate and long-lasting enhancement of neurotransmitter release, and identify Unc13 as a presynaptic molecular target for homeostatic potentiation and learning.

Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. Here, we show that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. Our results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories., The role of macroautophagy in neuronal processes mediating brain maintenance remains enigmatic. Authors show here that impairing autophagy within the major learning related brain center of Drosophila, and not other regions, triggered a form of presynaptic metaplasticity that was invariably connected to the absence of the specific component of aversive olfactory memory, which normally only declines in the course of aging process

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi., Highlighted Article: SEPT1 associates with the Golgi complex through GM130, and is required for maintaining Golgi architecture and function by connecting cis-Golgi membranes to the microtubule-nucleating machinery.