Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

Affinity precipitation an option for early capture in bioprocessing

Affinity precipitation is a bioseparation technique where the affinity ligand is coupled to a stimuliresponsive polymer. Stimuli-responsive polymers show abrupt, yet reversible, phase transition (precipitation) in response to a small change in an environmental parameter. The corresponding ligand conjugates can be used to co-precipitate and thereby capture and isolate target molecules from complex solutions such as culture supernatants and cell lysates. The approach is compatible with a 'discardibles only' type of downstream process and can be scaled over several orders of magnitude. This report discusses the set-up and development of affinity precipitation procedures, the related instrumentation and scale up, as well as applications for the isolation of proteins and polynucleotides.     

Highly heterogeneous residual malaria risk in western Thailand

Over the past decades, the malaria burden in Thailand has substantially declined. Most infections now originate from the national border regions. In these areas, the prevalence of asymptomatic infections is still substantial and poses a challenge for the national malaria elimination program. To determine epidemiological parameters as well as risk factors for malaria infection in western Thailand, we carried out a cohort study in Kanchanaburi and Ratchaburi provinces on the Thailand-Myanmar border. Blood samples from 999 local participants were examined for malaria infection every 4 weeks between May 2013 and Jun 2014. Prevalence of Plasmodium falciparum and Plasmodium vivax was determined by quantitative PCR (qPCR) and showed a seasonal variation with values fluctuating from 1.7% to 4.2% for P. vivax and 0% to 1.3% for P. falciparum. Ninety percent of infections were asymptomatic. The annual molecular force of blood-stage infection (_molFOB) was estimated by microsatellite genotyping to be 0.24 new infections per person-year for P. vivax and 0.02 new infections per person-year for P. falciparum. The distribution of infections was heterogenous, that is, the vast majority of infections (>80%) were found in a small number of individuals (<8% of the study population) who tested positive at multiple timepoints. Significant risk factors were detected for P. vivax infections, including previous clinical malaria, occupation in agriculture and travel to Myanmar. In contrast, indoor residual spraying was associated with a protection from infection. These findings provide a recent landscape of malaria epidemiology and emphasize the importance of novel strategies to target asymptomatic and imported infections.     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.     

Behavioral patterns of two fiddler crab species Uca rapax and Uca tangeri in a seminatural mangrove system

Zoos increasingly transform their exhibitions from traditional one‐species enclosures to more natural exhibits, that is, environments that capture part of an ecosystem including a selection of animals and plants that occur there. Thus, enhancing the experience of its human visitors while also allowing its residents to possibly show more natural behavior. In 2017 Royal Burger's Zoo (Arnhem, The Netherlands) created and opened a mangrove‐like environment containing fiddler crabs. Fiddler crabs display a broad range of behaviors, and this research examines which wild‐type behavior and behavioral patterns can be observed on a seminatural mudflat. The behavior shown by Uca rapax and Uca tangeri on the mudflat was counted each hour between 07:00 and 17:00. An asymmetric tidal regime was present in the enclosure including two high water periods. Various known fiddler crab behaviors, including waving and combat, were observed but no copulation. A clear pattern in exposed crabs on the mudflat was found, with low numbers visible in the early morning and the highest numbers present in the early afternoon, while number of visitors did not have a significant effect on this pattern. Interestingly, the highest abundances were not observed around the ebbing tide (07:00–09:00), as observed in the wild, but somewhat later, possibly due to the asymmetric tidal scheme or the interaction of tidal and daily rhythms. This study shows that in captivity, fiddler crabs indeed show a range of natural behaviors which is linked to the tidal and possibly daily rhythm as well.     

Elevated α-Linolenic Acid Content in Extra-plastidial Membranes of Tomato Accelerates Wound-Induced Jasmonate Generation and Improves Tolerance to the Herbivorous Insects Heliothis peltigera and Spodoptera littoralis

Journal of Plant Growth Regulation - In tomato, desaturation of linoleic acid (18:2) to α-linolenic acid (18:3) is mediated in the plastidial membranes by the ω-3 fatty acid desaturases 7...     

Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera

BackgroundEnzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform.Methodology/Principal findingsDAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV.Conclusions/SignificanceDAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.     

Flight potential and oxygen uptake during early dormancy in Coccinella septempunctata

Two flight parameters (take-off and duration) and respiration level were measured, in two years in summer and early autumn, in dormant Coccinella septempunctata L. (Coleoptera: Coccinellidae) collected while hidden in grass tussocks in hibernation sites (HID) and in beetles collected on plants (PLA). The duration of tethered flight of HID beetles measured in the laboratory in late August and September 1995 (range of geometric means 190–440 s) was slightly longer than the flight of PLA beetles (80–310 s), both being much longer than trivial flight recorded in beetles foraging for prey during the breeding season (35 s). In general, the flight performance had a tendency to increase in September and to decrease in October.The oxygen consumption in HID beetles increased throughout September 1994 from 430 to 780 l g^–1 h^–1 and throughout October 1995 from 710 to 1060 l g^–1 h^–1. This increase is ascribed to a concomitant decrease in diapause intensity. A similar increase was observed also in PLA beetles in 1994 and oxygen consumption was always higher than in HID beetles, most probably due to feeding and digestion in PLA beetles.Laboratory feeding of HID beetles on aphids induced maturation of ovaries and increased oxygen uptake (from 680 to 1110 l g^–1 h^–1). Feeding on honey and pollen left their oxygen uptake unchanged. Effect of feeding on the flight parameters was mostly not significant. In agreement with its less suitable body shape and usually less distant dormancy sites, C. septempunctata was found a less apt flier than long-distance migrating coccinellid species.     

Contributo alla conoscenza dell'ecologia delle biocenosi sulle cascade travertinose nella regione Carstica Jugoslava

Riassunto Il nostro lavoro di molti anni sulle cascade travertinose dei fiumi carstici jugoslavi era diretto verso le esplorazioni delle condizioni di vita su questi specifici biotopi. A causa di questa specificità loro erano potuti popolati solo da determinati specie di piante e di animali, i queli sono adattati su queste condizioni speciali. Esse creano le associazioni, ai queli fa la base la vigorosa vegetazione di muschi e di alghe, e un po'di meno anche di altri piante. Questa vegetazione e accompagnata da numerosi rappresentanti dai diversi gruppi di animali, come queli di Turbellaria, Gastropoda, Oligochaeta, Hirudinea, Amphipoda, Insecta ed a.Dai fattori ecologici erano esplorati le condizioni di vita fisicochimiche e biologiche. In estate la temperatura dell'aqua era sempre oltre la temperatura-limite di deposizione (oltre 14° C), e durante l'inverno generalmente erano le condizioni inconvenienti per i processi di deposizione.Nella parte superficiale delle cascade abiamo constatato due specie di abitazione in relazione della luce. Forte illuminate cascade erano popolate dalle associazioni della luce, ed a causa di meno calcificata vegetazione erano molto più. ricche di animali. Meno illuminate cascade avevano più favorevoli condizioni per il sviluppo della vegetazione travertinosa, la quale era molto calcificata, the cosa non porgeva favorevoli condizioni per il sviluppo del mondo animale.Abiamo constatato the la più favorevole velocità dell'aqua per lo sviluppo dei organismi, i quali popolano i posti delle cascade travertinose, è tra 0,5 e l,5 m/sec., sebbene sui singoli abitazioni la velocita dell'aqua era fra 0,5–3,5 m/sec.A causa di una favorevole alcalità, la quale nelle aque esplorate ammonta tra 2,8 e 5,2, i processi di deposizione sono molto attivi. E costattato un graduale abassamento dell'alcalità in direzione scendente del flume, the cosa si mette in relazione colla perdita del carbonato durante il processo di deposizione.La quantità del ossigeno ammontava da 3,5–9 ccm/l. e perciò generalmente tutte queste aque si possono considerare come poliossitipiche.Per le biocenosi delle cascade travertinose e caratteristica una grande dinamica nel Toro sviluppo. Le più primitive forme delle associazioni sono composte principalmente dai idrofiti. Con un avanzato sviluppo delle formazioni travertinose appariscono nuove stazioni con nuove associazioni. Il climax presenta la vegetazione fanerogama, la quala crew stazioni ombreggiate, dove si sviluppa speciale vegetazione travertinosa dell' ombra. Questi cambiamenti di vegetazione vengono accompagnati da numerose specie di animali dei gruppi Amphipoda, Ephemerida, Trichoptera, Plecoptera, Coleoptera ed a.In fine sono dimostrate le principali caratteristiche di vita in relazione colle condizioni fisico-chimiche e biologiche.

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Summary For a number of years, our work on travertine cascades of Yugoslav Karstic rivers has been directed towards the study of living conditions in these specific biotypes. Owing to their specificity, they could have been inhabited only by certain plant and animal species which have been adapted to these specific conditions. Their communities have been created on a rich moss and seaweed vegetation basis and, to a somewhat smaller extent, also on that of other plants. This vegetation is accompanied by numerous representatives of various animal groups such as: turbellaria, gastropods, oligochaeta, hirudinoids, amphipods, insects et al.As far as ecologic factors are concerned, physical, chemical and biological living conditions have been studied. In summer, water temperature is always above the deposition boundary (above 14°C), while in winter, conditions are generally unfavourable for deposition processes.In connection with light, two habitats were observed on the surface parts of the cascades. The strongly lighted cascades were inhabited by light communities which were considerably richer in animal population, owing to poorly calcified vegetation. As to the development of travertine vegetation, poorly lighted cascades which had been amply calcified, were in a much more favourable position and thus provided unfavourable conditions for the development of animal life.The water speed most favourable for the development of organisms inhabiting travertine cascades has been found to lie between 0.5 and 1.5 meters per second, although in individual habitats water speed amounted to between 0.5 and 3.5 meters per second.Owing to favourable alkalinity, amounting, in the waters investigated, to between 2.8 and 5.2, depositing processes have been very intense. The gradual lowering of alkalinity in the downstream course of rivers, which is brought into connection with the losing of carbonates during the deposition process, has also been established.As the quantity of oxygen amounted to between 3.5–9 ccm/l, these waters can be considered to be polyoxitypical.The biocenosis on travertine cascades are characterized by their very dynamic development. The most primitive forms of communities are mainly composed of hydrophytes. The further development of travertine forms brings about new habitats with new communities. The climax is represented by phanerogamic vegetation, creating rocky habitats where special travertine shade vegetation develops. These changes in vegetation have been accompanied by numerous animal species of the afore-mentioned groups.Finaly, the main characteristics of life in terms of physical, chemical and biological conditions have been presented.

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Zusammenfassung Die mehrjärige Arbeit der Autoren auf den Kalktuffbildenden Wasserfällen der jugoslawischen Karstgewässer war den Erforschungen der Lebensbedingungen auf diesen spezifischen Biotopen gewidmet. Wegen ihrer Eigentümlichkeit konnten die Kalktuffwasserfälle nur von bestimmten Pflanzen- und Tierarten besiedelt werden, die an these besonderen Bedingungen angepasst sind. Sie bilden Gemeinschaften auf eine üppige Moos- und Algenvegetation und etwas weniger auch auf anderen Pflanzen. Diese Vegetation wird von zahlreichen Vertretern verschiedener Tiergruppen begleitet, wie Turbellaria, Gastropoda, Oligochaeta, Hirudinea, Amphipoda, Insecta u.a.Von ökologischen Faktoren wurden die physikalisch-chemischen und biologischen Bedingungen untersucht. Im Sommer war die Wassertemperatuur immer über der Grenztemperatur der Ablagerung (über 14°C), während die Ablagerungsbedingungen im Winter im allgemeinen ungünstig waren.In den oberflächenlichen Teilen der Wässer\:falle wurden, im Verhältnis zu dem Licht, zwei Arten von Standorten festgestellt. Die stark belichteten Wasserfälle sind von Lichtgerneinschaften besiedelt, die wegen der weniger kalzifizierten Vegetation viel mehr Tiere enthalten. Weniger belichtete Wasserfälle zeigen günstigere Bedingungen für eine Entwicklung der Kalktuffvegetation. Diese Vegetation wird sehr kalzifiziert und ist für der Entwicklung der Tierwelt ungünstig.Es wurde festgestellt, dass die günstige Wassergeschwindigkeit für die Entwicklung von Organismen, die die Standorte der Kalktuffwasserfälle besiedeln, zwischen 0,5 and 1,5 m-Sek ist, wiewohl die Wassergeschwindigkeit an einzelnen Standorten zwischen 0,5 and 3,5 m-Sek betrug.Wegen der günstigen Alkalität (2,8–5,2) sind die Ablagerungsvorgänge sehr aktiv. Flussabwärts wurde ein allmählicher Fall der Alkalitätwerte festgestellt, was auf den Verlust des Karbonates während der Ablagerungsvorgänge zurückgeführt wird.Die Sauerstoffmenge beträgt zwischen 3,5–9 ccm/l; im allgemeinen können wir deshalb all these Gewässer als polyoxytypisch betrachten.Für die Gemeinschaften der Kalktuffwasserfälle ist eine grosse Dynamik in ihrer Entwicklung charakteristisch. Die primitivsten Formen der Lebensgemeinschaften sind hauptsächlich aus Hydrophyten zusammengesetzt. Mit der weiteren Entwicklung der Kalktufformen erscheinen neue Standorte mit neuen Gemeinschaften. Das Klimax stellt die Vegetation der höheren Pflanzen tar, die Schattenstandorte macht, wo sich eine besondere Schattenkalktuf-vegetation entwickelt. Diese Vegetationsänderungen werden von zahlreichen Tierarten der genanten Gruppen begleitet.Zum Schluss wird die Hauptcharakteristik des Lebens in Bezug auf die physikalisch-chemischen und biologischen Bedingungen dargestellt.

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Botaniki institut Univerziteta Zagreb

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Bioloki institut Univerziteta Zagreb     

Physiologo-biochemical properties of a strain of Beijerinckia mobilis 1phi Phn+--a degrader of polycyclic aromatic hydrocarbons

Beijerinckia mobilis 1f capable of degrading polycyclic aromatic hydrocarbons (PAHs) was isolated from a soil contaminated with creosote. Strain 1f could utilize phenanthrene and naphthalene as the sole sources of carbon. The mean rate of phenanthrene degradation during culture growth was 7-8 micrograms/(ml h). After cultivation under nonselective conditions, strain 1f retained its ability to degrade phenanthrene. Cometabolism considerably widened the range of PAHs that could be transformed by strain 1f. The strain was able to grow in a mineral medium with creosote as the sole source of carbon. After 30 days of cultivation in this medium, the total concentration of PAHs decreased from 665.5 mg/l to 170 mg/l.