Blow flies as urban wildlife sensors

Wildlife detection in urban areas is very challenging. Conventional monitoring techniques such as direct observation are faced with the limitation that urban wildlife is extremely elusive. It was recently shown that invertebrate‐derived DNA (iDNA) can be used to assess wildlife diversity in tropical rainforests. Flies, which are ubiquitous and very abundant in most cities, may also be used to detect wildlife in urban areas. In urban ecosystems, however, overwhelming quantities of domestic mammal DNA could completely mask the presence of wild mammal DNA. To test whether urban wild mammals can be detected using fly iDNA, we performed DNA metabarcoding of pools of flies captured in Berlin, Germany, using three combinations of blocking primers. Our results show that domestic animal sequences are, as expected, very dominant in urban environments. Nevertheless, wild mammal sequences can often be retrieved, although they usually only represent a minor fraction of the sequence reads. Fly iDNA metabarcoding is therefore a viable approach for quick scans of urban wildlife diversity. Interestingly, our study also shows that blocking primers can interact with each other in ways that affect the outcome of metabarcoding. We conclude that the use of complex combinations of blocking primers, although potentially powerful, should be carefully planned when designing experiments.     

Microdissection-derived murine mcb probes from somatic cell hybrids.

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes.     

In EXOG‐depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain

Depletion of mitochondrial endo/exonuclease G‐like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G‐like‐depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G‐like depletion. Interestingly, PE‐induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G‐like‐depleted cells, suggesting a reverse signaling function of endo/exonuclease G‐like. Endo/exonuclease G‐like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G‐like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G‐like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G‐like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death.     

Behavioral Responses to Mammalian Blood Odor and a Blood Odor Component in Four Species of Large Carnivores

Only little is known about whether single volatile compounds are as efficient in eliciting behavioral responses in animals as the whole complex mixture of a behaviorally relevant odor. Recent studies analysing the composition of volatiles in mammalian blood, an important prey-associated odor stimulus for predators, found the odorant trans-4,5-epoxy-(E)-2-decenal to evoke a typical “metallic, blood-like” odor quality in humans. We therefore assessed the behavior of captive Asian wild dogs (Cuon alpinus), African wild dogs (Lycaon pictus), South American bush dogs (Speothos venaticus), and Siberian tigers (Panthera tigris altaica) when presented with wooden logs that were impregnated either with mammalian blood or with the blood odor component trans-4,5-epoxy-(E)-2-decenal, and compared it to their behavior towards a fruity odor (iso-pentyl acetate) and a near-odorless solvent (diethyl phthalate) as control. We found that all four species displayed significantly more interactions with the odorized wooden logs such as sniffing, licking, biting, pawing, and toying, when they were impregnated with the two prey-associated odors compared to the two non-prey-associated odors. Most importantly, no significant differences were found in the number of interactions with the wooden logs impregnated with mammalian blood and the blood odor component in any of the four species. Only one of the four species, the South American bush dogs, displayed a significant decrease in the number of interactions with the odorized logs across the five sessions performed per odor stimulus. Taken together, the results demonstrate that a single blood odor component can be as efficient in eliciting behavioral responses in large carnivores as the odor of real blood, suggesting that trans-4,5-epoxy-(E)-2-decenal may be perceived by predators as a “character impact compound” of mammalian blood odor. Further, the results suggest that odorized wooden logs are a suitable manner of environmental enrichment for captive carnivores.     

The Vip1 Inositol Polyphosphate Kinase Family Regulates Polarized Growth and Modulates the Microtubule Cytoskeleton in Fungi

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.     

The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network of Arabidopsis thaliana

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, γ-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate–treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.     

Breeding habitat preference and foraging of the Cyprus Wheatear Oenanthe cypriaca and niche partitioning in comparison with migrant Oenanthe species on Cyprus

Niche partitioning has been examined in breeding bird communities and in winter quarters, but has received less attention when comparing a resident breeder and migrants during spring. Here, such an assemblage of species of the same genus (Oenanthe) and guild were analysed on the Mediterranean island of Cyprus. Northern Wheatear O. oenanthe and Eastern Black-Eared Wheatear O. hispanica melanoleuca were migrants, while Cyprus Wheatear O. cypriaca was resident. Migrant wheatears were more common in open habitats without trees, with a lower proportion of vegetation in the 10 cm layer, less tree and bush cover, a higher proportion of herbaceous layer, and a higher amount of bare areas. By using a discriminant function, we found that O. oenanthe was least tolerant towards a high proportion of bush/tree cover, and O. cypriaca was most tolerant, with O. h. melanoleuca in between. Also, O. oenanthe tolerated the least proportion of vegetation in the lowest layer, and O. h. melanoleuca the highest. O. oenanthe hunted more often by hop-and-peck and O. cypriaca more often used sallying and perch-and-pounce. O. oenanthe was the most ground-dwelling species with low perch heights and highest number of hops per minute and hops per movement, while O. cypriaca was the most arboreal species with the highest perches. Mean foraging rate did not differ between the species. A principal component analysis followed by a discriminant function showed that O. cypriaca has a high amount of aerial sallying and perch-pounce hunting behaviour with fewer hops, while O. oenanthe represents the contrary with hop-and-peck movements on the ground and with fewer flights. The data further indicate a clearer separation between O. oenanthe and O. cypriaca while O. h. melanoleuca lies in between utilising both foraging modes.     

The Plk1 inhibitor BI 2536 temporarily arrests primary cardiac fibroblasts in mitosis and generates aneuploidy in vitro

BI 2536 is a new anti-mitotic drug that targets polo-like kinase 1 (Plk1) and is currently under clinical development for cancer therapy. The effect of this drug on cancer cells has been extensively investigated, but information about the effects on primary dividing cells and differentiated non-dividing cells is scarce. We have investigated the effects of this drug on primary neonatal rat cardiac fibroblasts and on differentiated cardiomyocytes and explored the possibility to use this drug to enrich differentiated cell populations in vitro. BI 2536 had a profound effect on cardiac fibroblast proliferation in vitro and arrested these cells in mitosis with an IC50 of about 43 nM. Similar results were observed with primary human cells (HUVEC, IC50 = 30 nM), whereas the cancer cell line HeLa was more sensitive (IC50 of 9 nM). Further analysis revealed that prolonged mitotic arrest resulted in cell death for about 40% of cardiac fibroblasts. The remaining cells showed an interphase morphology with mostly multi- and micro-nucleated nuclei. This indicates that a significant number of primary fibroblasts are able to escape BI 2536 induced mitotic arrest and apparently become aneuploid. No effects were observed on cardiomyocytes and hypertrophic response (growth) upon endothelin-1 and phenylephrine stimulation was normal in the presence of BI 2536. This indicates that BI 2536 has no adverse effects on terminally differentiated cells and still allows proliferation independent growth induction in these cells. In conclusion, cardiomyocytes could be enriched using BI 2536, but the formation of aneuploidy in proliferating cells most likely limits this in vitro application and does not allow its use in putative cell based therapies.