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Crocodilians are an order of ancient reptiles that thrive in pathogen-rich environments. The ability to inhabit these harsh environments is indicative of a resilient innate immune system. Defensins, a family of cysteine-rich cationic host defence peptides, are a major component of the innate immune systems of all plant and animal species, however crocodilian defensins are poorly characterised. We now show that the saltwater crocodile defensin CpoBD13 harbors potent antifungal activity that is mediated by a pH-dependent membrane-targeting action. CpoBD13 binds the phospholipid phosphatidic acid (PA) to form a large helical oligomeric complex, with specific histidine residues mediating PA binding. The utilisation of histidine residues for PA engagement allows CpoBD13 to exhibit differential activity at a range of environmental pH values, where CpoBD13 is optimally active in an acidic environment.

Das Projekt MakEd_digital zielt auf die Entwicklung digitalisierungsbezogener Kompetenzen von angehenden Lehrpersonen zur Nutzung, Entwicklung und Reflexion digitaler Medien und Werkzeuge in Lehr-/Lernkontexten ab. Dabei bilden unterschiedliche theoretische Modelle Basis und Bezugsperspektiven f��r die wissenschaftliche Konkretisierung und Validierung des hier angewandten Kompetenzbegriffes (nach Weinert, 2001). Diese Modellvielfalt ergibt sich zwingend aus den mehrfachen Bezugsdimensionen des Projekts, das wissenschaftliche Reflexion in der Hochschullehre, den Betrieb verschiedener p��dagogisch-didaktischer Makerspaces sowie den Aufbau einer Maker-orientierten Community ��ber Fachgrenzen hinweg umfasst. Der Artikel beschreibt diese Theorien und ihr Zusammenspiel im Sinne des Gedankens von (medien-)p��dagogischem Making.

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development.

Defensins are a class of host defence peptides (HDPs) that often harbour antimicrobial and anticancer activities, making them attractive candidates as novel therapeutics. In comparison with current antimicrobial and cancer treatments, defensins uniquely target specific membrane lipids via mechanisms distinct from other HDPs. Therefore, defensins could be potentially developed as therapeutics with increased selectivity and reduced susceptibility to the resistance mechanisms of tumour cells and infectious pathogens. In this review, we highlight recent advances in defensin research with a particular focus on membrane lipid-targeting in cancer and infection settings. In doing so, we discuss strategies to harness lipid-binding defensins for anticancer and anti-infective therapies.

Obwohl der Stadtteil H��chst ein Subzentrum f��r den Westen Frankfurts bildet, wird er oft nur mit einer Industriestadt assoziiert. H��chst ist aber gepr��gt durch eine dichte, lebendige Innenstadt, seiner Altstadt, vielf��ltige Kultur-& Kreativansiedelungen und eine vielschichtige Bev��lkerungsstruktur, bestehend aus vielen ethnischen Minderheiten, die mehr als 66% der lokalen Bewohner darstellen. Vor dem Hintergrund von Migration und des demografischen Wandels der urbanen Stadtgesellschaft in Minorty-Majority-St��dten waren st��dtebauliche Konzepten f��r die Innenstadt und seine Randbereiche gesucht. Welches Leitbild l��sst sich hier entwickeln, das einen divers zug��nglichen, breit nutzbaren, und inklusiven urbanen Stadtraum entfaltet und das die kulturellen Potentiale der Innenstadt, der nahen sowie der regionalen Umgebung st��rkt? Anhand verschiedener st��dtebaulicher Erfassungs-Methoden sind Alltagsr��ume verschiedener Bev��lkerungsgruppen erarbeitet und in Bezug gesetzt worden.

Parkinson’s Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo. To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensi...

Phosphorylated phosphatidylinositol lipids, or phosphoinositides, critically regulate diverse cellular processes, including signalling transduction, cytoskeletal reorganisation, membrane dynamics and cellular trafficking. However, phosphoinositides have been inadequately investigated in the context of cell death, where they are mainly regarded as signalling secondary messengers. However, recent studies have begun to highlight the importance of phosphoinositides in facilitating cell death execution. Here, we cover the latest phosphoinositide research with a particular focus on phosphoinositides in the mechanisms of cell death. This progress article also raises key questions regarding the poorly defined role of phosphoinositides, particularly during membrane-associated events in cell death such as apoptosis and secondary necrosis. The review then further discusses important future directions for the phosphoinositide field, including therapeutically targeting phosphoinositides to modulate cell death.

Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin–lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin–phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption., Defensins are cationic antimicrobial peptides that permeabilize membranes of pathogens presumably via the assembly of defensin–lipid oligomers. Here authors provide evidence for this by solving the crystal structure of a plant defensin in an oligomeric state with phospholipids.

The lattice expansion in ZnO, using in-situ X ray diffraction, has been investigated during flash sintering with varying current densities. While current flow through the specimen enhances the kinetics of sintering for ZnO, the temperature is not high enough to claim thermal runaway or localized melting. Unlike the case of yttria stabilized zirconia [1,2], experimental temperature approximations predict comparable specimen temperature to conventional sintering temperature of ZnO. Microstructural analysis supports the findings of the in-situ temperature approximations. In comparison with black body radiation, a gap between theoretical value and measured value was found due to flaws in the theoretical model. In addition, a new type of flash sintering was introduced, with current ramp, to avoid the power spike which has been the source of much debate about the transition from voltage to current control. The advantage of this method is in the controlled sintering kinetics thus avoiding the channeling found in dielectric materials [3].

Host defense peptides (HDPs) are well-characterized for their antimicrobial activities but also variously display potent immunomodulatory effects. Human β-defensin 3 (HBD-3) belongs to a well-known HDP family known as defensins and is able to induce leukocyte chemotactic recruitment, leukocyte activation/maturation, proinflammatory cytokine release, and co-stimulatory marker expression. HBD-3-stimulated cytokine induction is NF-κB-dependent and was initially suggested to act via G protein-coupled C-C chemokine receptor phospholipase C (PLC) and/or Toll-like receptor signaling. Subsequent pharmacological inhibition, however, revealed that NF-κB activation by HBD-3 is receptor-independent and instead involves the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) pathway, the mechanism of which remains undetermined. Recently, we have shown that HBD-3 can enter mammalian cells and bind to inner membrane phosphoinositide 4,5-bisphosphate [PI(4,5)P2], an important second lipid messenger of PLC and PI3K-Akt pathways. In this study, we report that the interaction of HBD-3 with PI(4,5)P2 is important for PI3K-Akt-NF-κΒ-mediated induction of tumor necrosis factor and interleukin-6. These data provide insights into the mechanism of immunomodulation by HBD-3, and more generally, highlight the complex multifaceted signaling roles of HDPs in innate defense. Furthermore, it is suggested that the proposed mode of action may be conserved in other HDPs.

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

Defensins are innate immune molecules that upon recognition of specific phospholipids can disrupt microbial membranes by forming oligomeric assemblies. Structures of two related plant defensins, NaD1 and NsD7, bound to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA), respectively, revealed striking differences in their oligomeric topologies. To understand how NsD7 binds different phospholipids and rationalize the different topologies, we determined the structure of an NsD7−PIP2 complex. This structure reveals fundamental differences in phospholipid binding compared to NsD7−PA, and an oligomeric topology nearly identical to the previously determined NaD1−PIP2 complex, establishing that the PIP2 fibril topology is conserved between NaD1 and NsD7. Our findings highlight the remarkable ability of defensins to bind different types of phospholipids to form oligomeric fibrils with diverse topologies. This article is protected by copyright. All rights reserved.

Poster presented atSMBE 2016Gordon AMPs 2017Defensins are small, charged, disulphide-richeukaryotic proteins with diverse sequences, structures, andfunctions. Their antimicrobial activities are of particular interestfor protecting crops and humans from pathogens.They have been traditionally treated as a single superfamily.However, we present evidence that there exist two independentevolutionary origins of defensins, based on their secondarystructure element order, disulphide topology, and tertiarystructures. These two superfamilies, the cis-defensins and transdefensins,exhibit some of the most extensive convergentevolution of protein sequence, structure and function.We have developed new methods of sequence alignment andanalysis to overcome the difficulties of investigating such shortand divergent sequences. Multivariate analysis of proteinsequence space allows grouping of defensins into naturallyoccurring clusters which describe the residue properties thatseparate phyla and functions. It can be further used to designsynthetic, cluster-central, archetypal defensin sequences.

Pathogenic microbes are developing resistance to established antibiotics, making the development of novel antimicrobial molecules paramount. One major resource for discovery of antimicrobials is the arsenal of innate immunity molecules that are part of the first line of pathogen defense in many organisms. Gene encoded cationic antimicrobial peptides are a major constituent of innate immune arsenals. Many of these peptides exhibit potent antimicrobial activity in vitro. However, a major hurdle that has impeded their development for use in the clinic is the loss of activity at physiological salt concentrations, attributed to weakening of the electrostatic interactions between the cationic peptide and anionic surfaces of the microbial cells in the presence of salt. Using plant defensins we have investigated the relationship between the charge of an antimicrobial peptide and its activity in media with elevated salt concentrations. Plant defensins are a large class of antifungal peptides that have remarkable stability at extremes of pH and temperature as well as resistance to protease digestion. A search of a database of over 1200 plant defensins identified ZmD32, a defensin from Zea mays, with a predicted charge of +10.1 at pH 7, the highest of any defensin in the database. Recombinant ZmD32 retained activity against a range of fungal species in media containing elevated concentrations of salt. In addition, ZmD32 was active against Candida albicans biofilms as well as both Gram negative and Gram-positive bacteria. This broad spectrum antimicrobial activity, combined with a low toxicity on human cells make ZmD32 an attractive lead for development of future antimicrobial molecules.

In combination with studies of post-mortem Parkinson's disease (PD) brains, pharmacological and genetic models of PD have suggested that two fundamental interacting cellular processes are impaired – proteostasis and mitochondrial respiration. We have re-examined the role of mitochondrial dysfunction in lymphoblasts isolated from individuals with idiopathic PD and an age-matched control group. As previously reported for various PD cell types, the production of reactive oxygen species (ROS) by PD lymphoblasts was significantly elevated. However, this was not due to an impairment of mitochondrial respiration, as is often assumed. Instead, basal mitochondrial respiration and ATP synthesis are dramatically elevated in PD lymphoblasts. The mitochondrial mass, genome copy number and membrane potential were unaltered, but the expression of indicative respiratory complex proteins was also elevated. This explains the increased oxygen consumption rates by each of the respiratory complexes in experimentally uncoupled mitochondria of iPD cells. However, it was not attributable to increased activity of the stress- and energy-sensing protein kinase AMPK, a regulator of mitochondrial biogenesis and activity. The respiratory differences between iPD and control cells were sufficiently dramatic as to provide a potentially sensitive and reliable biomarker of the disease state, unaffected by disease duration (time since diagnosis) or clinical severity. Lymphoblasts from control and PD individuals thus occupy two distinct, quasi-stable steady states; a ‘normal’ and a ‘hyperactive’ state characterized by two different metabolic rates. The apparent stability of the ‘hyperactive’ state in patient-derived lymphoblasts in the face of patient ageing, ongoing disease and mounting disease severity suggests an early, permanent switch to an alternative metabolic steady state. With its associated, elevated ROS production, the ‘hyperactive’ state might not cause pathology to cells that are rapidly turned over, but brain cells might accumulate long-term damage leading ultimately to neurodegeneration and the loss of mitochondrial function observed post-mortem. Whether the ‘hyperactive’ state in lymphoblasts is a biomarker specifically of PD or more generally of neurodegenerative disease remains to be determined., Editors' choice: Cultured Parkinson's disease lymphocytes are metabolically hyperactive, suggesting a new understanding of the underlying cytopathology and biomarkers for this and potentially other neurodegenerative diseases.

Defensins are cationic antimicrobial peptides that serve as important components of host innate immune defenses, often by targeting cell membranes of pathogens. Oligomerization of defensins has been linked to their antimicrobial activity; however, the molecular basis underpinning this process remains largely unclear. Here we show that the plant defensin NsD7 targets the phospholipid phosphatidic acid (PA) to form oligomeric complexes that permeabilize PA-containing membranes. The crystal structure of the NsD7-PA complex reveals a striking double helix of two right-handed coiled oligomeric defensin fibrils, the assembly of which is dependent upon the interaction with PA at the interface between NsD7 dimers. Using site-directed mutagenesis, we demonstrate that key residues in this PA-binding site are required for PA-mediated NsD7 oligomerization and coil formation, as well as permeabilization of PA-containing liposomes. These data suggest that multiple lipids can be targeted to induce oligomerization of defensins during membrane permeabilization and demonstrate the existence of a "phospholipid code" that identifies target membranes for defensin-mediated attack as part of a first line of defense across multiple species.

The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both divergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin

Die Stadt Wien besch��ftigt sich seit Jahren mit der Belebung von leerstehenden Gesch��ftsfl��chen in der Erdgescho��zone. Dabei geht es neben der Frage der Aktivierung auch um die Frage der Belebung von Gesch��ftsstra��en und der optischen Aufwertung des Stadtbildes. In den letzten Jahren wurden diverse Agenturen mit der gezielten Aufgabe zur Beratung von potentiellen MieterInnen und VermieterInnen von leerstehenden Objekten ins Leben gerufen. Weiters wurde die Frage der Aktivierung des Leerstandes in unterschiedliche Organisationen und deren T��tigkeitsfelder integriert. Auch die gezielte monet��re Unterst��tzung der Wirtschaftsagentur Wien f��r Gesch��ftsfl��chen, die seit mehr als einem Jahr leer stehen, zeigen die Bedeutung dieses Themas. MigrantInnen��konomien spielen in der Belebung eine zentrale Rolle, weil diverse Wiener Einkaufsstra��en ohne MigrantInnen��konomien nicht in ihrer Dynamik existieren w��rden. Festgestellt wird, dass 39% der Wiener UnternehmerInnen einen Migrationshintergrund haben und die Gr��ndungsintensit��t um einiges h��her ist, als die der autochthonen Bev��lkerung. Statistiken untermauern au��erdem, dass die Aktivit��ten von Personen mit Migrationshintergrund ��berwiegend in den Erdgescho��zonen angesiedelt sind. Dabei spielen Handel und Gastronomie eine zentrale Rolle. Weiters ist auch auf die Gr��ndungsdynamik der arabischen Community hinzuweisen, die sich binnen k��rzester Zeit in diversen Bezirken stark im Stadtbild etabliert hat. Bezogen auf unterschiedliche Themengebiete, kann das Potential von MigrantInnen��konomien beispielsweise gerade in der Belebung von leerstehenden Gesch��ftsfl��chen von Wiener Wohnen eingesetzt werden. Eigene ���Nahversorgerstruktur��� am Gemeindebau k��nnte die moderne Concierge- Serviceleistung ersetzen. Weiters m��ssten Rahmenbedingungen auch f��r VermieterInnen geschaffen werden, um das Potential von ���migrantischen��� Gesch��ftsideen abzusch��tzen und die Nachhaltigkeit diverser Gesch��ftsmodelle bewerten zu k��nnen. Grunds��tzlich ist davon auszugehen, dass infolge der st��rkeren Verdr��ngung des klassischen Handels durch Zunahme von online- Eink��ufen, die moderne Gastronomie zunehmen wird. Dies geht auch aus dem EHL-Gesch��ftsstra��enbericht 2018 hervor. Dieser Umstand unterstreicht mehr, dass MigrantInnen verst��rkt im ��ffentlichen Raum an Bedeutung zunehmen werden., Die Stadt Wien besch��ftigt sich seit Jahren mit der Belebung von leerstehenden Gesch��ftsfl��chen in der Erdgescho��zone. Dabei geht es neben der Frage der Aktivierung auch um die Frage der Belebung von Gesch��ftsstra��en und der optischen Aufwertung des Stadtbildes. In den letzten Jahren wurden diverse Agenturen mit der gezielten Aufgabe zur Beratung von potentiellen MieterInnen und VermieterInnen von leerstehenden Objekten ins Leben gerufen. Weiters wurde die Frage der Aktivierung des Leerstandes in unterschiedliche Organisationen und deren T��tigkeitsfelder integriert. Auch die gezielte monet��re Unterst��tzung der Wirtschaftsagentur Wien f��r Gesch��ftsfl��chen, die seit mehr als einem Jahr leer stehen, zeigen die Bedeutung dieses Themas. MigrantInnen��konomien spielen in der Belebung eine zentrale Rolle, weil diverse Wiener Einkaufsstra��en ohne MigrantInnen��konomien nicht in ihrer Dynamik existieren w��rden.

The fragile X premutation (PM) allele contains a CGG expansion of 55-200 repeats in the FMR1 gene's promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39-81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers - CGG repeat number and the levels of the FMR1 mRNA - were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85-90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings - suggestive of a role of this enzyme in the risk of FXTAS - need to be verified and expanded in future studies using larger samples and longitudinal assessment.

Human β-defensin 2 permeabilizes and kills fungal cells by specifically targeting the important plasma membrane lipid PIP2., Human defensins belong to a subfamily of the cationic antimicrobial peptides and act as a first line of defense against invading microbes. Their often broad-spectrum antimicrobial and antitumor activities make them attractive for therapeutic development; however, their precise molecular mechanism(s) of action remains to be defined. We show that human β-defensin 2 (HBD-2) permeabilizes Candida albicans cell membranes via a mechanism targeting the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the structure of HBD-2 bound to PIP2, which revealed two distinct PIP2-binding sites, and showed, using functional assays, that mutations in these sites ablate PIP2-mediated fungal growth inhibition by HBD-2. Our study provides the first insight into lipid-mediated human defensin membrane permeabilization at an atomic level and reveals a unique mode of lipid engagement to permeabilize cell membranes.

The fragile X premutation (PM) allele contains a CGG expansion of 55–200 repeats in the FMR1 gene’s promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39–81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers – CGG repeat number and the levels of the FMR1 mRNA – were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85–90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings – suggestive of a role of this enzyme in the risk of FXTAS – need to be verified and expanded in future studies using larger samples and longitudinal assessment.

Summary The human protein arginine methyltransferase NDUFAF7 controls the assembly of the ∼1-MDa mitochondrial complex I (CI; the NADH ubiquinone oxidoreductase) by methylating its subunit NDUFS2. We determined crystal structures of MidA, the Dictyostelium ortholog of NDUFAF7. The MidA catalytic core domain resembles other eukaryotic methyltransferases. However, three large core loops assemble into a regulatory domain that is likely to control ligand selection. Binding of MidA to NDUFS2 is weakened by methylation, suggesting a mechanism for methylation-controlled substrate release. Structural and bioinformatic analyses support that MidA and NDUFAF7 and their role in CI assembly are conserved from bacteria to humans, implying that protein methylation already existed in proteobacteria. In vivo studies confirmed the critical role of the MidA methyltransferase activity for CI assembly, growth, and phototaxis of Dictyostelium . Collectively, our data elucidate the origin of protein arginine methylation and its use by MidA/NDUFAF7 to regulate CI assembly.

// Thanh Kha Phan 1 , Fung T. Lay 1 , Ivan K.H. Poon 1 , Mark G. Hinds 2 , Marc Kvansakul 1 , Mark D. Hulett 1 1 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia 2 Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia Correspondence to: Mark D. Hulett, e-mail: m.hulett@latrobe.edu.au Keywords: HBD-3, defensin, necrosis, tumour cell lysis, PI(4,5)P 2 Received: November 05, 2015 Accepted: November 24, 2015 Published: December 09, 2015 ABSTRACT Cationic antimicrobial peptides (CAPs), including taxonomically diverse defensins, are innate defense molecules that display potent antimicrobial and immunomodulatory activities. Specific CAPs have also been shown to possess anticancer activities; however, their mechanisms of action are not well defined. Recently, the plant defensin NaD1 was shown to induce tumour cell lysis by directly binding to the plasma membrane phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). The NaD1–lipid interaction was structurally defined by X-ray crystallography, with the defensin forming a dimer that binds PI(4,5)P 2 via its cationic β2-β3 loops in a ‘cationic grip’ conformation. In this study, we show that human β-defensin 3 (HBD-3) contains a homologous β2-β3 loop that binds phosphoinositides. The binding of HBD-3 to PI(4,5)P 2 was shown to be critical for mediating cytolysis of tumour cells, suggesting a conserved mechanism of action for defensins across diverse species. These data not only identify an evolutionary conservation of CAP structure and function for lipid binding, but also suggest that PIP-binding CAPs could be exploited for novel multifunction therapeutics.

Defensins are innate immune molecules that upon recognition of specific phospholipids can disrupt microbial membranes by forming oligomeric assemblies. Structures of two related plant defensins, NaD1 and NsD7, bound to phosphatidylinositol 4,5-bisphosphate (PIP

There is an ongoing need for effective and targeted cancer treatments that can overcome the detrimental side effects presented by current treatment options. One class of novel anticancer molecules with therapeutic potential currently under investigation are cationic antimicrobial peptides (CAPs). CAPs are small innate immunity peptides found ubiquitously throughout nature that are typically membrane-active against a wide range of pathogenic microbes. A number of CAPs can also target mammalian cells and often display selective activity towards tumor cells, making them attractive candidates as novel anticancer agents warranting further investigation. This current and comprehensive review describes key examples of naturally occurring membrane-targeting CAPs and their modified derivatives that have demonstrated anticancer activity, across multiple species of origin and structural subfamilies. In addition, we address recent advances made in the field and the ongoing challenges faced in translating experimental findings into clinically relevant treatments.

Summary: Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance. : Atkin-Smith et al. examine the role of Plexin B2, a membrane receptor, in the disassembly of apoptotic monocytes. Apoptosis induces caspase cleavage of Plexin B2 and cleaved Plexin B2 is enriched in apoptotic fragments. Genetic deletion of Plexin B2 impairs apoptotic monocyte disassembly and compromises apoptotic debris uptake by phagocytes. Keywords: apoptotic cell disassembly, plexin B2, apoptotic bodies, apoptotic cell clearance, apoptosis, apoptopodia

© 2016, Springer International Publishing. Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.

The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys(4) as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys(4) that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys(4) mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens.

Plant defensins are small (~5 kDa) basic cysteine-rich proteins that are being explored in important agricultural crops for their ability to confer enhanced disease resistance against fungal pathogens. NaD1, isolated from the flowers of the ornamental tobacco (Nicotiana alata), is a particularly well characterized antifungal defensin. Here, the crystallization and preliminary X-ray crystallographic analysis of NaD1 is reported. Crystals of NaD1 were crystallized using the sitting-drop vapour-diffusion method at 291 K. Data were collected from two crystal forms to 1.4 and 1.6 Å resolution, respectively. The crystals of form A belonged to the monoclinic space group P2(1), with unit-cell parameters a = 32.697, b = 32.685, c = 41.977 Å, α = 90, β = 100.828, γ = 90°, whereas crystals of form B belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 33.091, c = 128.77 Å, α = β = 90, γ = 120°.

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 μ m peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 μm in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

Russian aggression in 2014 caught U.S. policy and strategy off guard, forcing reactive measures and reevaluation of the U.S. approach toward Russia. Moscow employed nonlinear methodologies and operated just beneath traditional thresholds of conflict to take full advantage of U.S. and NATO policy and process limitations. In light of this strategic problem, the U.S. Army War College (USAWC), conducted a wargame that revealed four key considerations for future policy and strategy.

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a “cationic grip” conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species.

Plant defensins are small (c.a. 5 kDa), basic, cysteine-rich proteins with antimicrobial activities. They are ubiquitous in plants and form part of the innate immunity arsenal. Plant defensins are encoded by small multigene families and are expressed in various plant tissues, but are best characterized in seeds. They are typically produced as preproteins, however, a small subset are produced as larger precursors with C-terminal prodomains. To date, the three-dimensional solution structures of seven seed- and two floral-derived defensins have been elucidated by (1)H-NMR spectroscopy. Despite limited amino acid sequence identities, these defensins have comparable global folds with features that are characteristic of the cysteine-stabilized alphabeta (CSalphabeta) motif. Interestingly, their structures are remarkably similar to those of insect defensins and scorpion toxins. Functionally, these proteins exhibit a diverse array of biological activities, although they all serve a common function as defenders of their hosts. This review describes the distribution, biosynthesis, structure, function and mode of action of plant defensins and reflects on their potential in agribiotechnological applications.

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.

Bovine herpes virus 1 (BoHV-1) causes respiratory infections and abortions in cattle, and is an important component of Bovine respiratory disease complex (BRDC). BoHV-1 has also been isolated from sheep with respiratory disorder. Experimentally, sheep and goats are infected productively with BoHV-1 after intranasal inoculation and they generate immune response against BoHV-1. We have engineered a triple gene- mutated bovine herpes virus type 1 (BoHV-1 tmv) vaccine virus which is protective against BoHV-1 in calves. In the present study, we have tested BoHV-1 tmv infectivity and serum neutralizing antibody titers in lambs after intranasal inoculation. The results showed that BoHV-1 tmv replicated well in the nasal mucosa with moderate titers for 7 days and the infected lambs generated serum neutralizing titers.

We have carried out investigations into the photocapacitance property of domestically fabricated Cu/Cu2O/Au sandwich structures. For the fabrication, p-type cuprous oxide (Cu2O) was grown on a copper sheet, and a transparent gold metal contact was applied on the cuprous oxide layer under a pressure of 1 × 10−6 Torr. The capacitance characteristics versus light intensity for different wavelengths in the visible region and the current–voltage characteristics of the sandwich structure were measured at a temperature of 15 °C. It was observed that the capacitance of the Cu/Cu2O/Au structure was significantly dependent on both wavelength and intensity of the light. The photocapacitance property of Cu/Cu2O/Au is discussed in terms of the effect of light on the interface states filled by charges and the change occurring in the imaginary part of the dielectric constant of the cuprous oxide created due to interface states.