Neutrophil extracellular traps: casting the NET over pathogenesis

Neutrophil extracellular traps (NETs) are considered to be part of the human innate immunity because they trap and kill pathogens. NETs are formed by activated neutrophils and consist of a DNA backbone with embedded antimicrobial peptides and enzymes. They are involved in host defense during pneumococcal pneumonia, streptococcal necrotizing fasciitis, appendicitis and insemination. Recently, bacterial virulence factors that counteract NETs have been identified. These include the degradation of the NET-backbone by DNases enabling the liberation of bacteria from NETs, as well as capsule formation, which reduces bacterial trapping. Furthermore, pathogens can resist NET-mediated killing by adding positive charge to their cell surface.     

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models

In this review we focus on pattern recognition receptors in plants that detect extracellular signals indicative for pathogen attack and injury. We start out with a discussion on FLS2, which binds and responds to bacterial flagellin, and then concentrate on ligand–receptor interactions as initial steps in the molecular receptor activation process. Comparison with other receptor kinases, whether involved in plant immunity or regulation of other cellular programs, might indicate common principles of receptor activation.     

Die Kinetik der Ionenaufnahme durch junge und alte Sprosse von Mnium cuspidatum

Zusammenfassung Die Kinetik der Ionenaufnahme durch junge und alte Sprosse von Mnium cuspidatum wurde untersucht. Die verschieden alten Sprosse unterscheiden sich vor allem durch das Vorhandensein einer aktiven Gipfelknospe bei den jungen Gametophyten, die bei den alten offenbar ihre Tätigkeit eingestellt hat. Im niedrigen Konzentrationsbereich (0–0,5 mM) haben jungen und alte Sprosse hyperbolische Isothermen der Ionenaufnahme, die sich etwas hinsichtlich der apparenten Michaeliskonstanten und der Maximalgeschwindigkeit unterscheiden. Im hohen Konzentrationsbereich (1–10 mM) ist der Unterschied qualitativ. Mit jungen Sprossen erhält man eine lineare oder exponentielle Isotherme, mit alten Sprossen eine hyperbolische Kurve. Der vermutete Einfluß der Gipfelknospe kann mit Effekten von Wuchsstoffen auf den Stofftransport zusammenhängen und läßt einen Einfluß dieser Regulationssysteme auf die Membranfunktion vermuten.

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The kinetics of ion uptake by young and old branches of mnium cuspidatum

Summary Isotherms of K(Rb)-, Cl- and SO₄-uptake by young and old branches of the moss Mnium cuspidatum were investigated. Old moss gametophytes from the 1966 vegetation period were collected in the forests surrounding Darmstadt from February to mid-April 1967 and from the 1967 season in late September 1967. Young plants were sampled from mid-April to the end of May 1967 and they were also grown by water culture of old plants.Both young and old branches have hyperbolic isotherms of ion uptake in the low concentration range (0–0.5 mM) (Fig. 1–3), which slightly differ in K _mand V _max (Table). Isotherms in the high range (1–10 mM), however, are drastically different, changing from linear or exponential with young moss branches to hyperbolic with old gametophytes (Figs. 1–3).The linear or exponential high-range isotherm obtained with young moss plants is compared with other examples reported in the literature (Fig. 4). As the leaflets of the moss plants, which constitute 2/3 of the fresh weight of the material used in the experiments, have well developed vacuoles, the correlation between hyperbolic isotherms and vacuolation does not apply here (Fig. 4a, Torii and Laties, 1966).The change in shape of the high-range moss isotherm with age resembles the change from exponential to hyperbolic kinetics in isolated potato discs during washing (Fig. 4b, Laties, Macdonald and Dainty, 1964). The events triggered by isolation of potato discs from the interior of the tuber may be similar to the changes in the moss material under the control of the terminal bud, which is only active in the young branches.The suggested influence of the active terminal bud of young moss plants on the ion absorption process of cells in the tissue may be related to effects of growth substances on translocation reported in the literature and may point to a direct effect of these regulatory systems on membrane function.In this respect the comparison of corn root stele and cortex is of interest. Isolated steles, both freshly isolated and after washing, have exponential isotherms in the high range (Fig. 4c), whereas cortex displays a hyperbolic isotherm which changes little with ageing (Lüttge and Laties, 1967). In contrast to the case in potato and moss materials, this phenomenon is not simply due to ageing but involves morphogenetic differences.Temperature is another factor which influences the shape of the high range isotherm. All examples discussed so far refer to experiments at room temperature. At low temperatures high-range isotherms for proximal root tissue or aged potato discs have an exponential shape (Torii and Laties, 1966; Laties, Macdonald and Dainty, 1964). It thus appears that the exponential isotherm of young moss branches indicates that as in freshly isolated potato discs or in corn root stele the metabolic high-range uptake system is not developed.

     

Polar-localised poplar K<Superscript>+</Superscript> channel capable of controlling electrical properties of wood-forming cells

In previous studies, we have shown that annual expression profiles of cambial and wood tissue with respect to the Shaker K⁺ channel PTORK correlate with cambial activity. To follow PTORK-gene activity on the cellular level, we isolated the respective promoter regions and generated transgenic Arabidopsis plants expressing the GUS gene under the control of the PTORK promoter. Cross-sections of petioles showed PTORK-driven signals predominantly in the xylem parenchyma surrounding the vessels and in the phloem. Antibodies raised against a unique N-terminal region of PTORK in histo-immunochemical analyses recognised this K⁺-release channel in growth-active poplar plants only. PTORK labelling was found in differentiating xylem cells (young fibres) and mature xylem (vessel-associated cells of the ray parenchyma). Patch-clamp measurements on fibre cell protoplasts, derived from young poplar twigs, identified outward-rectifying K⁺ channels as the major K⁺ conductance of this cell type, which resembled the biophysical properties of PTORK when expressed in Xenopus oocytes.Electronic Supplementary Material Supplementary material is available for this article at Matthias Arend and Andrea Stinzing contributed equally to this work     

In aged primary T cells,mitochondrial stress contributes to telomere attrition measured by a novel imaging flow cytometry assay

The decline of the immune system with age known as immune senescence contributes to inefficient pathogen clearance and is a key risk factor for many aged‐related diseases. However, reversing or halting immune aging requires more knowledge about the cell biology of senescence in immune cells. Telomere shortening, low autophagy and mitochondrial dysfunction have been shown to underpin cell senescence. While autophagy has been found to control mitochondrial damage, no link has been made to telomere attrition. In contrast, mitochondrial stress can contribute to telomere attrition and vice versa. Whereas this link has been investigated in fibroblasts or cell lines, it is unclear whether this link exists in primary cells such as human lymphocytes and whether autophagy contributes to it. As traditional methods for measuring telomere length are low throughput or unsuitable for the analysis of cell subtypes within a mixed population of primary cells, we have developed a novel sensitive flow‐FISH assay using the imaging flow cytometer. Using this assay, we show a correlation between age and increased mitochondrial reactive oxygen species in CD8⁺ T‐cell subsets, but not with autophagy. Telomere shortening within the CD8⁺ subset could be prevented in vitro by treatment with a ROS scavenger. Our novel assay is a sensitive assay to measure relative telomere length in primary cells and has revealed ROS as a contributing factor to the decline in telomere length.     

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Na?ve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.     

Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis) over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs) characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%). About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively) were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA) showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater), host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity.     

β-aminoisobutyric Acid,l-BAIBA,Is a Muscle-Derived Osteocyte Survival Factor

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A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Analytically, comprehensive and robust quantification of different eicosanoid species in a multi-method approach is problematic because most of these compounds are relatively unstable and may differ in their chemical properties. Here we describe a novel ultra-performance liquid chromatography-selected reaction monitoring mass spectroscopy (UPLC-SRM/MS) method for simultaneous quantification of key urinary eicosanoids, including the prostaglandins (PG) tetranor PGE-M, 8-iso-, and 2,3-dinor-8-iso-PGF_2α; the thromboxanes (TXs) 11-dehydro- and 2,3-dinor-TXB₂; leukotriene E₄; and 12-hydroxyeicosatetraenoic acid. In contrast to previous methods, which used time-consuming and complex solid phase extraction, we prepared samples with a simple liquid/liquid extraction procedure. Because collision-induced dissociation produced characteristic product ions for all analytes, no derivatization step for SRM/MS analysis was necessary. Analytes were separated with a short UPLC reversed-phase column (1.7 µm particles), allowing shorter run times than conventional HPLC columns. The method was validated and applied to human urine samples showing excellent precision, accuracy, detection limits, and robustness. In summary, the developed method allows robust and sensitive profiling of urinary eicosanoid species, making it a useful and valuable tool for biomarker profiling in clinical/toxicological studies.