Time-Dependent Changes in Apoptosis Upon Autophagy Inhibition in Astrocytes Exposed to Oxygen and Glucose Deprivation

Recent studies have implicated the role of autophagy in brain ischemia pathophysiology. However, it remains unclear whether autophagy activation is protective or detrimental to astrocytes undergoing ischemic stress. This study evaluated the influence of ischemia-induced autophagy on cell death and the course of intrinsic and extrinsic apoptosis in primary cultures of rat cortical astrocytes exposed to combined oxygen-glucose deprivation (OGD). The role of autophagy was assessed by pharmacological inhibition with 3-methyladenine (3-MA). Cell viability was evaluated by measuring LDH release and through the use of the alamarBlue Assay. Apoptosis and necrosis were determined by fluorescence microscopy after Hoechst 33,342 and propidium iodide staining, respectively. The levels of apoptosis-related proteins were analyzed by immunoblotting. The downregulation of autophagy during OGD resulted in decreased cell viability and time-dependent changes in levels of apoptosis and necrosis. After short-term OGD (1, 4 h), cells treated with 3-MA showed higher level of cleaved caspase 3 compared with control cells. This result was consistent with an evaluation of apoptotic cell number by fluorescence microscopy. However, after prolonged exposure to OGD (8, 24 h), the number of apoptotic astrocytes (microscopically evaluated) did not differ or was even lower (as marked by caspase 3) in the presence of the autophagy inhibitor in comparison to the control. A higher level of necrosis was observed in 3-MA-treated cells compared to non-treated cells after 24 h OGD. The downregulation of autophagy caused time-dependent changes in both extrinsic (cleaved caspase 8, TNFα) and intrinsic (cleaved caspase 9) apoptotic pathways. Our results strongly indicate that the activation of autophagy in astrocytes undergoing ischemic stress is an adaptive mechanism, which allows for longer cell survival by delaying the initiation of apoptosis and necrosis.     

McComedy: A user-friendly tool for next-generation individual-based modeling of microbial consumer-resource systems

Individual-based modeling is widely applied to investigate the ecological mechanisms driving microbial community dynamics. In such models, the population or community dynamics emerge from the behavior and interplay of individual entities, which are simulated according to a predefined set of rules. If the rules that govern the behavior of individuals are based on generic and mechanistically sound principles, the models are referred to as next-generation individual-based models. These models perform particularly well in recapitulating actual ecological dynamics. However, implementation of such models is time-consuming and requires proficiency in programming or in using specific software, which likely hinders a broader application of this powerful method. Here we present McComedy, a modeling tool designed to facilitate the development of next-generation individual-based models of microbial consumer-resource systems. This tool allows flexibly combining pre-implemented building blocks that represent physical and biological processes. The ability of McComedy to capture the essential dynamics of microbial consumer-resource systems is demonstrated by reproducing and furthermore adding to the results of two distinct studies from the literature. With this article, we provide a versatile tool for developing next-generation individual-based models that can foster understanding of microbial ecology in both research and education.     

Recombinant baculoviruses as mammalian cell gene-delivery vectors

The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, recombinant baculovirus vectors engineered to contain mammalian cell-active promoter elements, have been used successfully for transient and stable gene delivery in a broad spectrum of primary and established mammalian cells. The application of modified baculoviruses for in vivo gene delivery has also been demonstrated. In contrast to other commonly used viral vectors, baculoviruses have the unique property of replicating in insect cells while being incapable of initiating a replication cycle and producing infectious virus in mammalian cells. The viruses can be readily manipulated, accommodate large insertions of foreign DNA, initiate little to no microscopically observable cytopathic effect in mammalian cells and have a good biosafety profile. These attributes will undoubtedly lead to the increased application and continued development of this system for efficient gene delivery into mammalian cells. Who said you can't teach an old dog new tricks?     

Morphologie,Anatomie und Systematik carotinoidhaltiger Blätterpilze

Während Carotinoide innerhalb mehrerer natürlicher Verwandtschaftsgruppen der Heterobasidiomyceten charakteristisch und regelmäßig auftreten, ist das Vorkommen dieser Sekundärstoffklasse innerhalb der Homobasidiomyceten auf wenige Arten der corticioiden, clavarioiden, cantharelloiden, agaricoiden und gateroiden Organisationsstufen beschränkt (Tab. 1). Die Anatomie der Fruchtkörper und die Morphologie der Hyphen und Basidien der agaricoiden Vertreter, Phyllotopsis nidulans, Gerronema chrysophyllum, Haasiella venustissima wurden mit verschiedenen lichtmikroskopischen Methoden untersucht. Die gewonnenen Daten wurden mit Mikrophotographien und maßstabsgetreuen Zeichnungen belegt. Die Merkmale von Phyllotopsis nidulans — dickwandige Hyphen in der Fruchtkörpertrama, Medaillonschnallen in der Lamellentrama, sehr schmale Basidien, allantoide Sporen und der Besitz von Zystiden — können bei den anderen Arten nicht gefunden werden. Merulius, Phlebia, Gloeoporus, Auriculariopsis, Dictyoploca u. a. müssen als nah verwandte Gattungen angesehen werden. Die beiden anderen omphalinoid organisierten Blätterpilze, Gerronema chrysophyllum und Haasiella venustissima zeigen trotz Ähnlichkeiten in der Fruchtkörperform zueinander kaum Übereinstimmungen. Gerronema chrysophyllum besitzt Parallelen im Merkmalsbestand zu Cantharellus-Arten, vor allem zu Arten der Untergattung Phaeocantharellus. Dies kann durch den Aufbau der Huthaut, des verdickenden Hymeniums, der Basidien und der Stieltrama belegt werden. Besonders durch die Ontogenie der Fruchtkörper und der Lamellen wird diese Annahme unterstützt. Haasiella venustissima bleibt trotz neu dargestellter Merkmale der Fruchtkörpermorphologie — wenig differenzierte Huthaut, mehrschichtige Lamellentrama, Kaulozystiden in hymenialer Anordnung — und der Karyologie der Basidie in seiner systematischen Stellung isoliert, zumal Vergleichsdaten anderer, für einen Vergleich notwendiger Arten vielfach fehlen.     

Evolutionary transition in symbiotic syndromes enabled diversification of phytophagous insects on an imbalanced diet

Evolutionary adaptations for the exploitation of nutritionally challenging or toxic host plants represent a major force driving the diversification of phytophagous insects. Although symbiotic bacteria are known to have essential nutritional roles for insects, examples of radiations into novel ecological niches following the acquisition of specific symbionts remain scarce. Here we characterized the microbiota across bugs of the family Pyrrhocoridae and investigated whether the acquisition of vitamin-supplementing symbionts enabled the hosts to diversify into the nutritionally imbalanced and chemically well-defended seeds of Malvales plants as a food source. Our results indicate that vitamin-provisioning Actinobacteria (Coriobacterium and Gordonibacter), as well as Firmicutes (Clostridium) and Proteobacteria (Klebsiella) are widespread across Pyrrhocoridae, but absent from the sister family Largidae and other outgroup taxa. Despite the consistent association with a specific microbiota, the Pyrrhocoridae phylogeny is neither congruent with a dendrogram based on the hosts'' microbial community profiles nor phylogenies of individual symbiont strains, indicating frequent horizontal exchange of symbiotic partners. Phylogenetic dating analyses based on the fossil record reveal an origin of the Pyrrhocoridae core microbiota in the late Cretaceous (81.2–86.5 million years ago), following the transition from crypt-associated beta-proteobacterial symbionts to an anaerobic community localized in the M3 region of the midgut. The change in symbiotic syndromes (that is, symbiont identity and localization) and the acquisition of the pyrrhocorid core microbiota followed the evolution of their preferred host plants (Malvales), suggesting that the symbionts facilitated their hosts'' adaptation to this imbalanced nutritional resource and enabled the subsequent diversification in a competition-poor ecological niche.     

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24–144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3′- or 5′-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.     

Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus

Overwintering adults of the spruce bark beetle, Ips typographus (L.) showed an unusually complex sugar/polyol cryoprotectant system. The major components of the multiple system were: glucose (177.6 mmolL(-1), March); trehalose (175.0 mmolL(-1), December); sorbitol (147.9 mmolL(-1), January); mannitol (81.2 mmolL(-1), March); and erythritol (40.7mmolL(-1), March) (in the parentheses, the maximum concentrations are shown and the month when they were reached). Other minor components were glycerol, fructose, threitol, myo-inositol, arabinitol and ribitol. Distinct seasonal patterns of accumulation/depletion in various components were found. Glycerol, trehalose and glucose started to accumulate first, during early autumn, when the air temperatures fluctuated between 20 and 0 degrees C, and diapause beetles continued in feeding. Glycerol was depleted, glucose remained stable and trehalose continued in accumulation during late autumn when the temperatures oscillated around 0 degrees C. During early winter severe frosts reaching -20 degrees C came, the beetles terminated their diapause and trehalose was partially depleted, while mannitol, sorbitol, fructose, threitol and erythritol started to accumulate. Cold weather continued also during late winter when the beetles remained quiescent. During this period, trehalose was re-accumulated, threitol and erythritol continued to increase, mannitol remained stable and sorbitol, fructose decreased. All cryoprotectans were finally cleared in the beetles which were spontaneously leaving bark during early spring. The seasonal maximum of total concentration of all cryoprotectants (578.2 mOsmol L(-1)) was reached in March. Such a concentration results in colligative depression of melting point of body fluids down by 1.08 degrees C only. It suggests that the potential cryoprotective effect of accumulated sugars and polyols was related rather to their non-colligative functions.     

Spatial control of Rho (Rac-Rop) signaling in tip-growing plant cells

Spatially restricted signaling by Rho GTPases is essential for the polarization of eukaryotic cells, which is required for the morphogenesis, mobility and division of single cells, and for the development of multicellular organisms. Rac-Rop GTPases, which constitute a plant-specific Rho GTPase subfamily, accumulate at the apical plasma membrane of pollen tubes and root hairs, where they control rapid polar cell expansion by a process known as tip growth. Here, recent insights into the spatial control of Rac-Rop-dependent signaling in tip-growing plant cells by regulatory proteins (i.e. Rho GTPase-activating proteins, Rho guanine nucleotide dissociation inhibitors, Rho guanine nucleotide-exchange factors and phosphoinositide-specific phospholipase C) and lipids [phosphatidylinositol (4,5)-bisphosphate and diacyl glycerol] are summarized. A model is presented, which integrates the current knowledge concerning the molecular mechanisms that maintain the polarization of Rho signaling in plant cells.     

KORRIGAN, an Arabidopsis endo-1,4-beta-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis

The formation of the cell plate, a unique structure in dividing plant cells, is pivotal for cytokinesis. A mutation in the Arabidopsis KORRIGAN (KOR) gene causes the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to severely abnormal seedling morphology. The mutant, designed kor1-2, was identified as a stronger allele than the previously identified kor1-1, which appears to be defective only in cell elongation. KOR1 encodes an endo-1,4-beta-d-glucanase with a transmembrane domain and two putative polarized targeting signals in the cytosolic tail. When expressed in tobacco BY2 cells, a KOR1-GFP (green fluorescence protein) fusion protein was localized to growing cell plates. Substitution mutations in the polarized targeting motifs of KOR1 caused the fusion proteins to localize to the plasma membrane as well. Expression of these mutant genes in kor1-2 plants complemented only the cell elongation defect but not the cytokinesis defect, indicating that polarized targeting of KOR1 to forming cell plates is essential for cytokinesis. Our results suggest that KOR1 plays a critical role during cytokinesis.     

Plasticity and epistasis strongly affect bacterial fitness after losing multiple metabolic genes

Many bacterial lineages lack seemingly essential metabolic genes. Previous work suggested selective benefits could drive the loss of biosynthetic functions from bacterial genomes when the corresponding metabolites are sufficiently available in the environment. However, the factors that govern this “genome streamlining” remain poorly understood. Here we determine the effect of plasticity and epistasis on the fitness of Escherichia coli genotypes from whose genome biosynthetic genes for one, two, or three different amino acids have been deleted. Competitive fitness experiments between auxotrophic mutants and prototrophic wild‐type cells in one of two carbon environments revealed that plasticity and epistasis strongly affected the mutants’ fitness individually and interactively. Positive and negative epistatic interactions were prevalent, yet on average cancelled each other out. Moreover, epistasis correlated negatively with the expected effects of combined auxotrophy‐causing mutations, thus producing a pattern of diminishing returns. Moreover, computationally analyzing 1,432 eubacterial metabolic networks revealed that most pairs of auxotrophies co‐occurred significantly more often than expected by chance, suggesting epistatic interactions and/or environmental factors favored these combinations. Our results demonstrate that both the genetic background and environmental conditions determine the adaptive value of a loss‐of‐biochemical‐function mutation and that fitness gains decelerate, as more biochemical functions are lost.