Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1(ts) mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.     

Prevalence of Different Head-Neck Positions in Horses Shown at Dressage Competitions and Their Relation to Conflict Behaviour and Performance Marks

Much controversy exists among riders, and in particular among those practicing dressage, regarding what can be considered an “appropriate” Head-Neck-Position (HNP). The objective was to assess the prevalence of different HNPs in the field, the behavioural reactions of horses during warm-up and competition rides in relation to HNP and the relation between HNP and marks achieved in the competition. Horses (n = 171) were selected during dressage competitions according to their HNP (3 categories based on the degree of flexion), and their behaviour was recorded during 3 minutes each of riding in the warm-up area and in the competition. Scans were carried out on an additional 355 horses every 15 minutes to determine the proportion of each HNP in the warm-up area. Sixty-nine percent of the 355 horses were ridden with their nasal planes behind the vertical in the warm-up area, 19% were ridden at or behind the vertical and only 12% were ridden with their nasal plane in front of the vertical. Horses carrying their nasal plane behind the vertical exhibited significantly (P<0.0001) more conflict behaviours than horses with their nose held in front of the vertical. Horses were commonly presented with a less flexed HNP during competition compared to warm-up (P<0.05). A HNP behind the vertical was penalised with lower marks in the lower (P = 0.0434) but not in the higher (P = 0.9629) competition levels. Horses in higher classes showed more (P = 0.0015) conflict behaviour than those in lower classes. In conclusion, dressage horses are commonly ridden during warm-up for competitions with their nasal plane behind the vertical, and this posture seems to cause significantly more conflict behaviour than HNPs in front of the vertical.     

Assay of tramadol in urine by capillary electrophoresis using laser-induced native fluorescence detection

Capillary electrophoresis (CE) with UV laser-induced native fluorescence detection was developed as a sensitive and selective assay for the direct determination of tramadol in human urine without extraction or preconcentration. The main problem in CE is the small inner diameter of the capillary which causes a low sensitivity with instruments equipped with a UV detector. Laser-induced native fluorescence with a frequency doubled argon ion laser at an excitation wavelength of 257 nm was used for the direct assay of tramadol in urine to enhance the limit of detection about 1000-fold compared to UV absorption detection. The detection system consists of an imaging spectrograph and an intensified CCD camera, which views an illuminated 1.5 mm section of the capillary. This set-up is able to record the whole emission spectra of the analytes to achieve additionally wavelength-resolved electropherograms. In the concentration range of 20 ng/ml–5 μg/ml in human urine coefficients of correlation were better than 0.998. Within-day variation determined on four different concentrations showed accuracies ranging from 90.2 to 108.4%. The relative standard deviation (RSD) was determined to be less than 10%. Day-to-day variation presented accuracies ranging from 90.9 to 103.1% with an RSD less than 8%.     

NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.     

Temporal progression and extent of the return of sensation in the foot provided by the saphenous nerve after sciatic nerve transection and repair in the rat - implications for nociceptive assessments

Sensory testing, by providing stimuli for nociceptors of the foot, is a popular method of evaluating sensory regeneration after damage to the sciatic nerve in the rat. In the following study, 20 rats were submitted to double transection of the sciatic nerve. The subsequent 14 mm gap was repaired through guidance interponation. In order to evaluate nerve regeneration, sensory testing was performed additionally to other methods, which included motor testing, morphometry, and electron microscopic assessments of nerves. Somatosensory testing revealed that all animals exhibited next to the same amount of sensory reinnervation on their foot regardless of their experimental group. In motor tests, however, two out of the three experimental groups did not improve at all. These groups also failed to show neural regrowth in morphometric and electron microscopic assessments of the associated nerve. Retrograde tracing was able to prove the saphenous nerve as an alternative source of sensory reinnervation in animals with failed sciatic regeneration. This means that results of sensory testing in the rat should be treated with caution, taking into account the areas tested and the likelihood that in these areas saphenous sprouting could have taken place. Furthermore, it is strongly advised that somatosensory testing should be conducted only on toe 5.     

The HIF-1 response to simulated ischemia in mouse skeletal muscle cells neither enhances glycolysis nor prevents myotube cell death

Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1alpha protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1alpha accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization.     

An uncoupling screen for autonomous embryo mutants in Arabidopsis thaliana

Simple de novo screens in Arabidopsis thaliana have previously identified mutants that affect endosperm development but viable-embryo mutants have not been identified. Our strategy to identify autonomous embryo development was to uncouple embryo and endosperm fertilisation. This involved a male-sterile mutant population being crossed with a distinct pollen parent—the pollen was needed to initiate endosperm development and because it was distinct, the maternal progeny could be selected from the hybrid population. This process was refined over three stages, resulting in a viable approach to screen for autonomous embryo mutants. From 8,000 screened plants, a mutation was isolated in which the integument cells extended from the ovule and proliferated into a second complete twinned ovule. Some embryos from the mutant were normal but others developed fused cotyledons. In addition, a proportion of the progeny lacked paternal genes.     

Kohlenstoffpumpen im Ozean steuern das Klima. Globale Erwärmung

Here we describe how microscopic marine algae and animals, the plankton impact global climate and how the changing concentration of the climate relevant gas carbon dioxide (CO₂) result in shifts in the marine carbonate system, including a drop in the pH (ocean acidification). Three processes, summarily called the carbon pumps transfer carbon between the surface and the deep ocean: The solubility pump moves inorganic, dissolved carbon to depth. The biological pump transports organic, particulate carbon downward. The carbonate counter pump describes the formation and sedimentation of carbonate tests, whereby CO₂ is released into the surface ocean. On geological timescales the biological pump was strengthened during glacial times due to an increase in the iron supply, which lead to a (continued) decline in temperatures. Hence, the idea to fertilize the ocean with iron thereby strengthening the biological pump and mediating today's climate change has been discussed for the past 20 years.     

Selection of aquatic microbiota exposed to the herbicides flufenacet and metazachlor

Herbicides are important, ubiquitous environmental contaminants, but little is known about their interaction with bacterial aquatic communities. Here, we sampled a protected natural freshwater habitat and characterised its microbiome in interaction with herbicides. We evolved the freshwater microbiomes in a microcosm assay of exposure (28 days) to flufenacet and metazachlor at environmental concentrations of 0.5, 5 and 50 μg L⁻¹. Inhibitory effects of herbicides were exemplarily assessed in cultured bacteria from the same pond (Pseudomonas alcaligenes, Paenibacillus amylolyticus and Microbacterium hominis). Findings were compared to long-term concentrations as provided by local authorities. Here, environmental concentrations reached up to 11 μg L⁻¹ (flufenacet) and 76 μg L⁻¹ (metazachlor). Bacteria were inhibited at minimum inhibitory concentrations far above these values; however, concentrations of 50 μg L⁻¹ of flufenacet resulted in measurable growth impairment. While most herbicide-exposed microcosm assays did not differ from controls, Acidobacteria were selected at high environmental concentrations of herbicides. Alpha-diversity (e.g., taxonomic richness on phylum level) was reduced when aquatic microbiomes were exposed to 50 μg metazachlor or flufenacet. One environmental strain of P. alcaligenes showed resistance to high concentrations of flufenacet (50 g L⁻¹). In total, this study reveals that ecologic imbalance due to herbicide use significantly impacts aquatic microbiomes.