570 Biowissenschaften; Biologie
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Species of rust fungi of the genus Milesina (Pucciiastraceae, Pucciniales) are distributed mainly in northern temperate regions. They host-alternate between needles of fir (Abies spp.) and fronds of ferns (species of Polypodiales). Milesina species are distinguished based on host taxonomy and urediniospore morphology. In this study, 12 species of Milesina from Europe were revised. Specimens were examined by light and scanning electron microscopy for urediniospore morphology with a focus on visualising germ pores (number, size and position) and echinulation. In addition, barcode loci (ITS, nad6, 28S) were used for species delimitation and for molecular phylogenetic analyses. Barcodes of 72 Milesina specimens were provided, including 11 of the 12 species. Whereas urediniospore morphology features were sufficient to distinguish all 12 Milesina species except for 2 (M. blechni and M. kriegeriana), ITS sequences separated only 4 of 11 species. Sequencing with 28S and nad6 did not improve species resolution. Phylogenetic analysis, however, revealed four phylogenetic groups within Milesina that also correlate with specific urediniospore characters (germ pore number and position and echinulation). These groups are proposed as new sections within Milesina (sections Milesina, Vogesiacae M. Scholler & Bubner, sect. nov., Scolopendriorum M. Scholler & Bubner, sect. nov. and Carpaticae M. Scholler & Bubner, sect. nov.). In addition, Milesina woodwardiana Buchheit & M. Scholler, sp. nov. on Woodwardia radicans, a member of the type section Milesina, is newly described. An identification key for European Milesina species, based on urediniospore features, is provided.
NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFN gamma production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under "steady state" conditions in perfusion mode. Our data demonstrate that expansion of CD3(+) T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anticancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFN gamma expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.
Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 angstrom resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an E15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.
Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo
(2019)
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
The short- and long-term thrombogenicity of implant materials is still unpredictable, which is a significant challenge for the treatment of cardiovascular diseases. A knowledge-based approach for implementing biofunctions in materials requires a detailed understanding of the medical device in the biological system. In particular, the interplay between material and blood components/cells as well as standardized and commonly acknowledged in vitro test methods allowing a reproducible categorization of the material thrombogenicity requires further attention. Here, the status of in vitro thrombogenicity testing methods for biomaterials is reviewed, particularly taking in view the preparation of test materials and references, the selection and characterization of donors and blood samples, the prerequisites for reproducible approaches and applied test systems. Recent joint approaches in finding common standards for a reproducible testing are summarized and perspectives for a more disease oriented in vitro thrombogenicity testing are discussed.
The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus. This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (25,35)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed. IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.
Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.
The relationships between sedentary lifestyle, sitting behaviour, and low back pain (LBP) remain controversial. In this study, we investigated the relationship between back pain and occupational sitting habits in 64 call-centre employees. A textile pressure mat was used to evaluate and parameterise sitting behaviour over a total of 400 h, while pain questionnaires evaluated acute and chronic LBP. Seventy-five percent of the participants reported some level of either chronic or acute back pain. Individuals with chronic LBP demonstrated a possible trend (t-test not significant) towards more static sitting behaviour compared to their pain-free counterparts. Furthermore, a greater association was found between sitting behaviour and chronic LBP than for acute pain/disability, which is plausibly due to a greater awareness of pain-free sitting positions in individuals with chronic pain compared to those affected by acute pain.
Winter is an important season for many limnological processes, which can range from biogeochemical transformations to ecological interactions. Interest in the structure and function of lake ecosystems under ice is on the rise. Although limnologists working at polar latitudes have a long history of winter work, the required knowledge to successfully sample under winter conditions is not widely available and relatively few limnologists receive formal training. In particular, the deployment and operation of equipment in below 0 degrees C temperatures pose considerable logistical and methodological challenges, as do the safety risks of sampling during the ice-covered period. Here, we consolidate information on winter lake sampling and describe effective methods to measure physical, chemical, and biological variables in and under ice. We describe variation in snow and ice conditions and discuss implications for sampling logistics and safety. We outline commonly encountered methodological challenges and make recommendations for best practices to maximize safety and efficiency when sampling through ice or deploying instruments in ice-covered lakes. Application of such practices over a broad range of ice-covered lakes will contribute to a better understanding of the factors that regulate lakes during winter and how winter conditions affect the subsequent ice-free period.
Predator-prey cycles rank among the most fundamental concepts in ecology, are predicted by the simplest ecological models and enable, theoretically, the indefinite persistence of predator and prey(1-4). However, it remains an open question for how long cyclic dynamics can be self-sustained in real communities. Field observations have been restricted to a few cycle periods(5-8) and experimental studies indicate that oscillations may be short-lived without external stabilizing factors(9-19). Here we performed microcosm experiments with a planktonic predator-prey system and repeatedly observed oscillatory time series of unprecedented length that persisted for up to around 50 cycles or approximately 300 predator generations. The dominant type of dynamics was characterized by regular, coherent oscillations with a nearly constant predator-prey phase difference. Despite constant experimental conditions, we also observed shorter episodes of irregular, non-coherent oscillations without any significant phase relationship. However, the predator-prey system showed a strong tendency to return to the dominant dynamical regime with a defined phase relationship. A mathematical model suggests that stochasticity is probably responsible for the reversible shift from coherent to non-coherent oscillations, a notion that was supported by experiments with external forcing by pulsed nutrient supply. Our findings empirically demonstrate the potential for infinite persistence of predator and prey populations in a cyclic dynamic regime that shows resilience in the presence of stochastic events.