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Up to one-third of all described marine species inhabit coral reefs, but the future of these hyperdiverse ecosystems is insecure due to local and global threats, such as overfishing, eutrophication, ocean warming, and acidification. Although these impacts are expected to have a net detrimental effect on reefs, it has been shown that some organisms like octocorals may remain unaffected, or benefit from, anthropogenically induced environmental change, and may replace stony corals in future reefs. Despite their potential importance in future shallow-water coastal environments, the molecular mechanisms leading to the resilience to anthropogenic-induced stress observed in octocorals remain unknown. Here, we use manipulative experiments, proteomics, and transcriptomics to show that the molecular toolkit used by Pinnigorgia flava, a common Indo-Pacific gorgonian octocoral, to deposit its calcium-carbonate skeleton is resilient to heat and seawater acidification stress. Sublethal heat stress triggered a stress response in P. flava but did not affect the expression of 27 transcripts encoding Skeletal Organic Matrix (SOM) proteins. Exposure to seawater acidification did not cause a stress response but triggered the downregulation of many transcripts, including an osteonidogen homolog present in the SOM. The observed transcriptional decoupling of the skeletogenic and stress-response toolkits provides insights into the mechanisms of resilience to anthropogenically-driven environmental change observed in octocorals.

Copper (Cu) and iron (Fe) are essential for plant growth and are often in short supply under natural conditions. Molecular responses to simultaneous lack of both metals (-Cu-Fe) differ from those seen in the absence of either alone. Metabolome profiling of plant leaves previously revealed that fumarate levels fall under -Cu-Fe conditions. We employed lines lacking cytosolic FUMARASE2 (FUM2) activity to study the impact of constitutive suppression of cytosolic fumarate synthesis on plant growth under Cu and/or Fe deficiency. In fum2 mutants, photosynthesis and growth were less impaired under -Cu-Fe conditions than in wild-type (WT) seedlings. In particular, levels of photosynthetic proteins, chloroplast ultrastructure, amino acid profiles and redox state were less perturbed by simultaneous Cu-Fe deficiency in lines that cannot produce fumarate in the cytosol. Although cytosolic fumarate has been reported to promote acclimation of photosynthesis to low temperatures when metal supplies are adequate, the photosynthetic efficiency of fum2 lines grown under Cu-Fe deficiency in the cold was higher than in WT. Uptake and contents of Cu and Fe are similar in WT and fum2 plants under control and -Cu-Fe conditions, and lack of FUM2 does not alter the ability to sense metal deficiency, as indicated by marker gene expression. Collectively, we propose that reduced levels of cytosolic fumarate synthesis ultimately increase the availability of Fe for incorporation into metalloproteins.

This paper presents the results of extensive investigations on the lamination strength grading, the production and the mechanical properties of European beech (Fagus sylvatica L.) glued laminated timber (GLT). Based on the analysis of potential influencing parameters on strength and stiffness as well as subsequent tension tests parallel to the grain on single boards, a combined visual/machine approach for grading the raw material into tensile strength classes T50, T42, T33 and T22 was developed. Boards strength graded with the developed procedure were then finger-jointed by a Swiss GLT producer and the strength of the finger joints was investigated by means of tension and bending tests. The strength and durability of the bonding was investigated and verified by means of tensile-shear and delamination tests. It could be shown that the required finger-joint and bondline strengths for GLT of strength classes GL40 and GL48 can be achieved, but that the process parameters for finger jointing (in particular the geometrical properties of the finger joint profile) have to be optimized in order to be able to produce GLT of strength class GL55. Finally, an extensive experimental testing campaign was performed to investigate the mechanical properties of European beech GLT produced based on the strength grading rules and production techniques developed before. Bending, tensile and compressive parallel to the grain, as well as shear tests were carried out on GLT specimens of strength classes GL40, GL48 and GL55 in different sizes in terms of cross-section and length. Based on these investigations and complementing numerical simulations, characteristic strength and stiffness values and formulae for consideration of size effects in bending, tension and shear were determined.

Singlet oxygen ( 1 O 2 ), the major reactive oxygen species (ROS) produced in chloroplasts, has been demonstrated recently to be a highly versatile signal that induces various stress responses. In the fluorescent ( flu ) mutant, its release causes seedling lethality and inhibits mature plant growth. However, these drastic phenotypes are suppressed when EXECUTER1 (EX1) is absent in the flu ex1 double mutant. We identified SAFEGUARD1 (SAFE1) in a screen of ethyl methanesulfonate (EMS) mutagenized flu ex1 plants for suppressor mutants with a flu -like phenotype. In flu ex1 safe1 , all 1 O 2 -induced responses, including transcriptional rewiring of nuclear gene expression, return to levels, such as, or even higher than, those in flu . Without SAFE1, grana margins (GMs) of chloroplast thylakoids (Thys) are specifically damaged upon 1 O 2 generation and associate with plastoglobules (PGs). SAFE1 is localized in the chloroplast stroma, and release of 1 O 2 induces SAFE1 degradation via chloroplast-originated vesicles. Our paper demonstrates that flu -produced 1 O 2 triggers an EX1-independent signaling pathway and proves that SAFE1 suppresses this signaling pathway by protecting GMs.

The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissue. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615 and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows non-invasive imaging, highlighting differentially-accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using widefield microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insight into GLP1R biology.

Jasmonates are vital plant hormones that not only act in the stress response to biotic and abiotic influences, such as wounding, pathogen attack, and cold acclimation, but also drive developmental processes in cooperation with other plant hormones. The biogenesis of jasmonates starts in the chloroplast, where several enzymatic steps produce the jasmonate precursor 12-oxophytodienoic acid (OPDA) from alpha-linolenic acid. OPDA in turn is exported into the cytosol for further conversion into active jasmonates, which subsequently induces the expression of multiple genes in the nucleus. Despite its obvious importance, the export of OPDA across the chloroplast membranes has remained elusive. In this study, we characterized a protein residing in the chloroplast outer membrane, JASSY, which has proven indispensable for the export of OPDA from the chloroplast. We provide evidence that JASSY has channel-like properties and propose that it thereby facilitates OPDA transport. Consequently, a lack of JASSY in Arabidopsis leads to a deficiency in accumulation of jasmonic acids, which results in impaired expression of jasmonate target genes on exposure to various stresses. This results in plants that are more susceptible to pathogen attack and also exhibit defects in cold acclimation.

The plastoglobular protein PG18 plays an important role in thylakoid formation and its loss has a significant impact on photosynthesis. This emphasizes the general importance of plastoglobules in chloroplast biogenesis., Plastoglobules are lipoprotein particles that are found in different types of plastids. They contain a very specific and specialized set of lipids and proteins. Plastoglobules are highly dynamic in size and shape, and are therefore thought to participate in adaptation processes during either abiotic or biotic stresses or transitions between developmental stages. They are suggested to function in thylakoid biogenesis, isoprenoid metabolism, and chlorophyll degradation. While several plastoglobular proteins contain identifiable domains, others provide no structural clues to their function. In this study, we investigate the role of plastoglobular protein 18 (PG18), which is conserved from cyanobacteria to higher plants. Analysis of a PG18 loss-of-function mutant in Arabidopsis thaliana demonstrated that PG18 plays an important role in thylakoid formation; the loss of PG18 results in impaired accumulation, assembly, and function of thylakoid membrane complexes. Interestingly, the mutant accumulated less chlorophyll and carotenoids, whereas xanthophyll cycle pigments were increased. Accumulation of photosynthetic complexes is similarly affected in both a Synechocystis and an Arabidopsis PG18 mutant. However, the ultrastructure of cyanobacterial thylakoids is not compromised by the lack of PG18, probably due to its less complex architecture.

Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants. Iterating mutagenesis and exposure to increasing light dramatically enhanced the light tolerance of a Synechocystis cyanobacterium strain. This involved over 100 mutations grouped around five haplotypes, as well as putative epistatic interactions.

Arabidopsis contains eight different h-type thioredoxins (Trx) being distributed in different cell organelles. Although Trx h2 is deemed to be confined to mitochondria, its subcellular localization and function are discussed controversially. Here, cell fractionation studies were used to clarify this question, showing Trx h2 protein to be exclusively localized in microsomes rather than mitochondria. Furthermore, Arabidopsis trxo1, trxh2 and trxo1h2 mutants were analyzed to compare the role of Trx h2 with mitochondrial Trx o1. Under medium light, trxo1 and trxo1h2 showed impaired growth, while trxh2 was similar to wild type. In line with this, trxo1 and trxo1h2 clustered differently from wild type with respect to nocturnal metabolite profiles, revealing a decrease in ascorbate and glutathione redox states. Under fluctuating light, these genotypic differences were attenuated. Instead, the trxo1h2 double mutant showed an improved NADPH redox balance, compared to wild type, accompanied by increased photosynthetic efficiency, specifically in the high-light phases. Conclusively, Trx h2 and Trx o1 are differentially localized in microsomes and mitochondria, respectively, which is associated with different redox-active functions and effects on plant growth in constant light, while there is a joint role of both Trxs in regulating NADPH redox balance and photosynthetic performance in fluctuating light.

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA‐targeting fluorescent fusion proteins in a cell‐type‐ and subcellular‐specific manner, it relies on the introduction of foreign genes. On the other hand, DNA‐binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short‐term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild‐type organisms., Precision without genetic engineering: pcHoechst allows light‐triggered DNA staining by photochemical uncaging. As such, it can be used to visualize and track nuclei with cellular resolution, which is demonstrated in live cells and in vivo. This chemical tool does not rely on genetic engineering and can ultimately be used to target and track DNA in subnuclear regions with high resolution.

Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. 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 a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

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.

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.

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

Sucrose (Suc) is one of the most important types of sugars in plants, serving inter alia as a long-distance transport molecule, a carbon and energy storage compound, an osmotically active solute, and fuel for many anabolic reactions. Suc biosynthesis and degradation pathways are well known; however, the regulation of Suc intracellular distribution is poorly understood. In particular, the cellular function of chloroplast Suc reserves and the transporters involved in accumulating these substantial Suc levels remain uncharacterized. Here, we characterize the plastidic sugar transporter (pSuT) in Arabidopsis (Arabidopsis thaliana), which belongs to a subfamily of the monosaccharide transporter-like family. Transport analyses with yeast cells expressing a truncated, vacuole-targeted version of pSuT indicate that both glucose and Suc act as substrates, and nonaqueous fractionation supports a role for pSuT in Suc export from the chloroplast. The latter process is required for a correct transition from vegetative to reproductive growth and influences inflorescence architecture. Moreover, pSuT activity affects freezing-induced electrolyte release. These data further underline the central function of the chloroplast for plant development and the modulation of stress tolerance.

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.

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.

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.

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.

The lifetime and stability of nanoscale endocytic structures are shown to be coupled to their growth by an endocytic protein., Clathrin-mediated endocytosis, an essential process for plasma membrane homeostasis and cell signaling, is characterized by stunning heterogeneity in the size and lifetime of clathrin-coated endocytic pits (CCPs). If and how CCP growth and lifetime are coupled and how this relates to their physiological function are unknown. We combine computational modeling, automated tracking of CCP dynamics, electron microscopy, and functional rescue experiments to demonstrate that CCP growth and lifetime are closely correlated and mechanistically linked by the early-acting endocytic F-BAR protein FCHo2. FCHo2 assembles at the rim of CCPs to control CCP growth and lifetime by coupling the invagination of early endocytic intermediates to clathrin lattice assembly. Our data suggest a mechanism for the nanoscale control of CCP growth and stability that may similarly apply to other metastable structures in cells.

NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPHthioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality

Protein scaffolds at presynaptic active zone membranes control information transfer at synapses. For scaffold biogenesis and maintenance, scaffold components must be safely transported along axons. A spectrum of kinases was suggested to control transport of scaffold components, but direct kinase/substrate relationships and operational principles steering phosphorylation-dependent active zone protein transport are presently unknown. Here we show that extensive phosphorylation of a 150-residue unstructured region at the N-terminus of the highly elongated BRP/ELKS active zone proteins is crucial for ordered active zone precursor transport. Point mutations that block SRPK79D-kinase-mediated phosphorylation of the BRP/ELKS N-terminus interfered with axonal transport, leading to BRP/ELKS-positive axonal aggregates that also contain additional active zone scaffold proteins. Axonal aggregates formed only in the presence of non-phosphorylatable BRP/ELKS isoforms containing the SRPK79D-targeted N-terminal stretch. We assume that specific active zone proteins are pre-assembled in transport packages and thus co-transported as functional scaffold building blocks. Our results suggest that transient post-translational modification of a discrete unstructured domain of the master scaffold component Bruchpilot blocks oligomerization of such building blocks during their long-range transport.

Cunningham and Littleton preview work from Goel et al. that describes a mechanism by which neurons regulate synaptic output after alterations in synapse size or active zone number., How neurons stabilize their overall synaptic strength following conditions that alter synaptic morphology or function is a key question in neuronal homeostasis. In this issue, Goel et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807165) find that neurons stabilize synaptic output despite disruptions in synapse size, active zone number, or postsynaptic function by controlling the delivery of active zone material and active zone size.

SummaryNeurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor AP180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in AP180−/−/Syb2+/− mice results in perinatal lethality, whereas Syb2+/− mice partially phenocopy loss of AP180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation.

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable "steady state" after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions.

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

Clathrin-mediated endocytosis (CME) involves membrane-associated scaffolds of the bin-amphiphysin-rvs (BAR) domain protein family as well as the GTPase dynamin, and is accompanied and perhaps triggered by changes in local lipid composition. How protein recruitment, scaffold assembly and membrane deformation is spatiotemporally controlled and coupled to fission is poorly understood. We show by computational modelling and super-resolution imaging that phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] synthesis within the clathrin-coated area of endocytic intermediates triggers selective recruitment of the PX-BAR domain protein SNX9, as a result of complex interactions of endocytic proteins competing for phospholipids. The specific architecture induces positioning of SNX9 at the invagination neck where its self-assembly regulates membrane constriction, thereby providing a template for dynamin fission. These data explain how lipid conversion at endocytic pits couples local membrane constriction to fission. Our work demonstrates how computational modelling and super-resolution imaging can be combined to unravel function and mechanisms of complex cellular processes., The spatiotemporal regulation of membrane scaffolds recruitment and coupling between membrane deformation and fission in endocytosis are unclear. Here the authors show that lipid conversion at endocytic pits recruits SNX9, which couples local membrane constriction to fission in endocytosis.

Background/Aims: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. Methods: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF 1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. Results: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and I f currents as well as functional hormonal regulation and sensitivity to I f channel blockers. Conclusion: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed I f current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes.

Subgroup-VII-ethylene-response-factor (ERF-VII) transcription factors are involved in the regulation of hypoxic gene expression and regulated by proteasome-mediated proteolysis via the oxygen-dependent branch of the N-end-rule pathway. While research into ERF-VII mainly focused on their role to regulate anoxic gene expression, little is known on the impact of this oxygen-sensing system in regulating plant metabolism and growth. By comparing Arabidopsis (Arabidopsis thaliana) plants overexpressing N-end-rule-sensitive and insensitive forms of the ERF-VII-factor RAP2.12, we provide evidence that oxygen-dependent RAP2.12 stability regulates central metabolic processes to sustain growth, development, and anoxic resistance of plants. (1) Under normoxia, overexpression of N-end-rule-insensitive Δ13RAP2.12 led to increased activities of fermentative enzymes and increased accumulation of fermentation products, which were accompanied by decreased adenylate energy states and starch levels, and impaired plant growth and development, indicating a role of oxygen-regulated RAP2.12 degradation to prevent aerobic fermentation. (2) In Δ13RAP2.12-overexpressing plants, decreased carbohydrate reserves also led to a decrease in anoxic resistance, which was prevented by external Suc supply. (3) Overexpression of Δ13RAP2.12 led to decreased respiration rates, changes in the levels of tricarboxylic acid cycle intermediates, and accumulation of a large number of amino acids, including Ala and γ-amino butyric acid, indicating a role of oxygen-regulated RAP2.12 abundance in controlling the flux-modus of the tricarboxylic acid cycle. (4) The increase in amino acids was accompanied by increased levels of immune-regulatory metabolites. These results show that oxygen-sensing, mediating RAP2.12 degradation is indispensable to optimize metabolic performance, plant growth, and development under both normoxic and hypoxic conditions.

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell–specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture.

HIV-1 cell-to-cell transmission confers a strong advantage as it increases efficiency of transfer up to 100-fold compared with a cell-free route. Mechanisms of HIV-1 cell-to-cell transmission are still unclear and can in part be explained by the presence of actin-containing cellular protrusions. Such protrusions have been shown to facilitate cell-to-cell viral dissemination. Using fluorescence microscopy, electron tomography, and ion abrasion scanning electron microscopy we show that HIV-1 induces membrane extensions in immature dendritic cells through activation of Cdc42. We demonstrate that these extensions are induced after engagement of DC-SIGN by HIV-1env via a cascade that involves Src kinases, Cdc42, Pak1, and Wasp. Silencing of Cdc42 or treatment with a specific Cdc42 inhibitor, Secramine A, dramatically reduced the number of membrane protrusions visualized on the cell surface and decreased HIV-1 transfer via infectious synapses. Ion abrasion scanning electron microscopy of cell-cell contact regions showed that cellular extensions from immature dendritic cells that have the appearance of thin filopodia in thin section images are indeed extended membranous sheets with a narrow cross section. Our results demonstrate that HIV-1 binding on immature dendritic cells enhances the formation of membrane extensions that facilitate HIV-1 transfer to CD4+ T lymphocytes.

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3'-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P2-rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis.

International audience; Dendritic cells (DCs) in mucosal surfaces are early targets for human immunodeficiency virus-1 (HIV-1). DCs mount rapid and robust immune responses upon pathogen encounter. However, immune response in the early events of HIV-1 transmission appears limited, suggesting that HIV-1 evade early immune control by DCs. We report that HIV-1 induces a rapid shutdown of autophagy and immunoamphisomes in DCs. HIV-1 envelope activated the mammalian target of rapamycin pathway in DCs, leading to autophagy exhaustion. HIV-1-induced inhibition of autophagy in DC increased cell-associated HIV-1 and transfer of HIV-1 infection to CD4(+) T cells. HIV-1-mediated downregulation of autophagy in DCs impaired innate and adaptive immune responses. Immunoamphisomes in DCs engulf incoming pathogens and appear to amplify pathogen degradation as well as Toll-like receptor responses and antigen presentation. The findings that HIV-1 downregulates autophagy and impedes immune functions of DCs represent a pathogenesis mechanism that can be pharmacologically countered with therapeutic and prophylactic implications.

Riboflavin (III, Fig. 1) was discovered 50 years ago by Kuhn and coworkers (1) as an essential vitamin; it is today produced in large quantities industrially, both by chemical synthesis and by fermentation, and is widely used as a food additive and as part of most vitamin supplements. Considering these facts it is surprising that the biosynthetic origin of almost half of the carbon atoms in the molecule is very poorly understood. Open image in new window Figure 1 Conversion of guanosine triphosphate into riboflavin

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production.

Strawberries are a very popular fruit among berries, for both their commercial and economic importance, but especially for their beneficial effects for human health. However, their bioactive compound content is strictly related to the nutritional status of the plant and might be affected if nutritional disorders (e.g. Fe or P shortage) occur. To overcome nutrient shortages, plants evolved different mechanisms, which often involve the release of root exudates. The biochemical and molecular mechanisms underlying root exudation and its regulation are as yet still poorly known, in particular in woody crop species. The aim of this work was therefore to characterize the pattern of root exudation of strawberry plants grown in either P or Fe deficiency, by investigating metabolomic changes of root tissues and the expression of genes putatively involved in exudate extrusion. Although P and Fe deficiencies differentially affected the total metabolism, some metabolites (e.g. raffinose and galactose) accumulated in roots similarly under both conditions. Moreover, P deficiency specifically affected the content of galactaric acid, malic acid, lysine, proline, and sorbitol-6-phosphate, whereas Fe deficiency specifically affected the content of sucrose, dehydroascorbic acid, galactonate, and ferulic acid. At the same time, the citrate content did not change in roots under both nutrient deficiencies with respect to the control. However, a strong release of citrate was observed, and it increased significantly with time, being +250% and +300% higher in Fe- and P-deficient plants, respectively, compared with the control. Moreover, concomitantly, a significant acidification of the growth medium was observed in both treatments. Gene expression analyses highlighted for the first time that at least two members of the multidrug and toxic compound extrusion (MATE) transporter family and one member of the plasma membrane H(+)-ATPase family are involved in the response to both P and Fe starvation in strawberry plants.

Summary For biochemical modification of the root–soil interface, the engineered secretion of stable enzymes from trichoblasts (= root hair bearing rhizodermal cells) is proposed. As a reporter activity, we chose to express a synthetic gene encoding a secretory phytase (PHY) directed by a trichoblast-specific promoter in root hair cells of the crop plant potato. Transgenic plants produced and secreted phytase in sufficient amounts to release phosphate from phytate in liquid medium. When grown in an unsterile substrate containing phytate, transgenic plants accumulated 40% more P in leaves than wild-type plants. The improved P nutrition driven by trichoblast-targeted expression and subsequent secretion of PHY illustrates the potential of using trichoblast-targeted expression of suitable enzymes for future applications in plant nutrition, phytoremediation and molecular farming.

Thirty patients had mild cognitive impairment and increased homocysteine levels in serum. On average, they were supplemented orally with a high dose of a vitamin B12-B6-folate combination for 270 days. All patients had normal serum B12 and folate levels at baseline. Cerebrospinal fluid levels of the tau protein (CSF-tau) and the albumin ratio were measured before and after treatment. The serum homocysteine levels were normalised after treatment. The albumin ratio significantly correlated with vascular risk factors. At baseline, the ratio was higher in the patients in comparison with age-matched controls. After treatment, the ratio was significantly reduced, which may indicate a tightening of the blood-brain barrier. The CSF-tau levels did not change significantly although there was a numeric decline. None of the patients progressed into dementia during the treatment period. When treated with a vitamin B12-B6-folate combination, patients with mild cognitive impairment and hyperhomocysteinaemia appear to improve their blood-brain barrier function. They may also stabilise their cognitive status. Further investigations are warranted on the role of blood-brain barrier dysfunction in the pathogenesis of dementia.

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway.

In recent years, depleted resources and environmental concerns have stimulated research in renewable and recyclable materials for particleboard production. This paper presents the research work on the production of particleboards using maize (Zea mays) cobs, rice (Oryza glaberrima) husks, and groundnut (Arachis hypogaea) shells, which are abundantly available as agricultural residues. The goal of this project was to study the feasibility of medium-density particleboard panels made of agricultural residues for use as internal partition wall cladding in residential buildings. The panels of densities between 600 and 900 kg/m3 were produced using a natural-based adhesive from acacia mimosa tannin extract and hexamine. Some other parameters like the moisture content (after adhesive application) and the press temperature were varied during the production so as to investigate their effect on some mechanical and physical properties like internal bond strength, bending modulus of rupture, swelling, and har...

With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.

This paper presents a system for computer-assisted quantification of axe-somatic boutons at motoneuron cell-surface membranes. Different immunohistochemical stains can be used to prepare tissue of the spinal cord. Based on micrographs displaying single neurons, a finite element balloon model has been applied to determine the exact location of the cell membrane. A synaptic profile is extracted next to the cell membrane and normalized with reference to the intracellular brightness. Furthermore, a manually selected reference cell is used to normalize settings of the microscope as well as variations in histochemical processing for each stain. Thereafter, staining, homogeneity, and allocation of boutons are determined automatically from the synaptic profiles. The system is evaluated by applying the coefficient of variation (C/sub v/) to repeated measurements of a quantity. Based on 1856 motoneuronal images acquired from four animals with three stains, 93% of the images are analyzed correctly. The others were rejected, based on process protocols. Using only rabbit anti-synaptophysin as primary antibody, the correctness increases above 96%, C/sub v/ values are below 3%, 5%, and 6% for all measures with respect to stochastic optimization, cell positioning, and a large range of microscope settings, respectively. A sample size of about 100 is required to validate a significant reduction of staining in motoneurons below a hemi-section (Wilcoxon rank-sum test, /spl alpha/=0.05, /spl beta/=0.9). The authors' system yields statistically robust results from light micrographs. In future, it is hoped that this system will substitute for the expensive and time-consuming analysis of spinal cord injury at the ultra-structural level, such as by manual interpretation of nonoverlapping electron micrographs.

With the ultimate goal to develop preparations of phytase (myo-inositol hexakisphosphate phosphohydrolase) with improved thermal resistance for inclusion in animal feed, several thermostabilization approaches were investigated with a set of fungal (Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger phytase) and consensus phytases. Screening of different low-Mr additives revealed that polyethylene glycols increase the thermostability of all phytases in a molecular weight-dependent fashion. The polyols ribitol, xylitol (C5 sugars) and sorbitol (C6 sugar) also improved their thermostability, whereas polyols containing more or less carbon atoms, such as glycerol, erythritol and mannoheptulose, showed only minor effects. The stabilizing effects of PEGs and polyols were concentration dependent. In a second series of experiments, crosslinking of the carbohydrate chains of A. fumigatus and consensus phytase using sodium periodate and adipic acid dihydrazide resulted in the for...