Effects of community-accessible biochar and compost on diesel-contaminated soil

Petroleum pollution is a global problem that requires effective and accessible remediation strategies that takes ecosystem functioning into serious consideration. Bioremediation can be an effective tool to address the challenge. In this study, we used a mesocosm experiment to evaluate the effects of locally sourced and community produced biochar and compost amendments on diesel-contaminated soil. At the end of the 90-day experiment, we quantified the effects of the amendments on total petroleum hydrocarbons (C9-C40) (TPH) and soil pH, organic matter, aggregate stability, soil respiration, extractable phosphorus, extractable potassium, and micronutrients (Mg, Fe, Mn, and Zn). We observed significantly higher TPH degradation in compost-amended soils than in controls and soils amended with biochar. We propose that the addition of compost improved TPH biodegradation by augmenting soil nutrient content and microbial activity. Our results suggest that community-accessible compost can improve TPH biodegradation, and that implementation is possible at the community level.     

Metabolomics identifies a biological response to chronic low-dose natural uranium contamination in urine samples

Because uranium is a natural element present in the earth’s crust, the population may be chronically exposed to low doses of it through drinking water. Additionally, the military and civil uses of uranium can also lead to environmental dispersion that can result in high or low doses of acute or chronic exposure. Recent experimental data suggest this might lead to relatively innocuous biological reactions. The aim of this study was to assess the biological changes in rats caused by ingestion of natural uranium in drinking water with a mean daily intake of 2.7 mg/kg for 9 months and to identify potential biomarkers related to such a contamination. Subsequently, we observed no pathology and standard clinical tests were unable to distinguish between treated and untreated animals. Conversely, LC–MS metabolomics identified urine as an appropriate biofluid for discriminating the experimental groups. Of the 1,376 features detected in urine, the most discriminant were metabolites involved in tryptophan, nicotinate, and nicotinamide metabolic pathways. In particular, N-methylnicotinamide, which was found at a level seven times higher in untreated than in contaminated rats, had the greatest discriminating power. These novel results establish a proof of principle for using metabolomics to address chronic low-dose uranium contamination. They open interesting perspectives for understanding the underlying biological mechanisms and designing a diagnostic test of exposure.     

Dynamin2 GTPase and Cortactin Remodel Actin Filaments

The large GTPase dynamin, best known for its activities that remodel membranes during endocytosis, also regulates F-actin-rich structures, including podosomes, phagocytic cups, actin comet tails, subcortical ruffles, and stress fibers. The mechanisms by which dynamin regulates actin filaments are not known, but an emerging view is that dynamin influences F-actin via its interactions with proteins that interact directly or indirectly with actin filaments. We show here that dynamin2 GTPase activity remodels actin filaments in vitro via a mechanism that depends on the binding partner and F-actin-binding protein, cortactin. Tightly associated actin filaments cross-linked by dynamin2 and cortactin became loosely associated after GTP addition when viewed by transmission electron microscopy. Actin filaments were dynamically unraveled and fragmented after GTP addition when viewed in real time using total internal reflection fluorescence microscopy. Cortactin stimulated the intrinsic GTPase activity of dynamin2 and maintained a stable link between actin filaments and dynamin2, even in the presence of GTP. Filaments remodeled by dynamin2 GTPase in vitro exhibit enhanced sensitivity to severing by the actin depolymerizing factor, cofilin, suggesting that GTPase-dependent remodeling influences the interactions of actin regulatory proteins and F-actin. The global organization of the actomyosin cytoskeleton was perturbed in U2-OS cells depleted of dynamin2, implicating dynamin2 in remodeling actin filaments that comprise supramolecular F-actin arrays in vivo. We conclude that dynamin2 GTPase remodels actin filaments and plays a role in orchestrating the global actomyosin cytoskeleton.Controlled assembly and disassembly of actin filaments underlies movement, shape, division, trafficking of lipids and proteins of the cell and pathogenesis by infectious bacteria and viruses. Several proteins and signaling circuits modulate actin filament dynamics, including proteins that nucleate formation of new filaments, filament cross-linking proteins that stabilize branched and bundled filament arrays, and depolymerizing factors that promote filament disassembly (1). Studies with reconstituted systems show that a single actin nucleating factor, such as the Arp2/3 complex together with a nucleation-promoting factor, a barbed end capping protein to preserve the actin monomer pool and promote nucleation, and a filament disassembly factor, such as ADF/cofilin, are sufficient to establish a dynamic dendritic actin network in vitro that mimics many properties of actin networks at the leading edge of migrating cells (2–4). However, the mechanisms for coordinating the organization and dynamics of actin filaments associated with higher-order cellular structures such as the subcortical F-actin network, F-actin at focal adhesions, and actomyosin arrays are not as well understood.Considerable evidence indicates that the large GTPase dynamin, a key mediator of membrane remodeling and fission, also influences actin filaments (reviewed in Refs. 5–7). Although the mechanisms are unknown, dynamin could influence actin filaments via its interactions with a number of proteins that directly or indirectly regulate actin filament assembly, filament stability, or filament organization. For example, several protein scaffolds biochemically link dynamin and the Arp2/3 complex activating factor, N-WASP, suggesting that the machinery for de novo actin assembly may be targeted or activated by dynamin (6, 8, 9). Dynamin2 is associated with several dynamic F-actin-containing structures in vivo, including podosomes, F-actin comet tails, phagocytic cups, dynamic cortical ruffles, and pedestal structures elaborated by enteropathogenic Escherichia coli (10–20). Cortactin, which directly binds both dynamin and actin filaments, is associated with many of the same dynamic actin structures as dynamin (5, 7) and is required for both clathrin-dependent and -independent endocytosis (21, 22). Thus, dynamin-cortactin interaction may be an important link between actin filaments and dynamin during formation or turnover of F-actin-rich structures.Considerable evidence supports the notion that GTP hydrolysis by dynamin catalyzes membrane fission activity via GTPase-dependent changes in conformation (23, 24) or via GTPase-dependent cycles of assembly and disassembly (25, 26). We hypothesize that GTPase-dependent changes in dynamin linked via its interacting proteins to actin filaments or actin regulators could similarly influence actin filaments. Overexpressed, dominant negative dynamin mutant proteins impaired in binding or hydrolyzing GTP (most often the dynamin-K44A mutation) perturb a variety of F-actin-rich cellular structures, including stress fibers and focal adhesions (27, 28), dendritic spines of neurons (29), podosomes (12, 30), actin comet tails (13, 14), phagocytic cups and bacteria-induced pedestal structures (16, 19), and dynamic cortical ruffles (15, 17). In addition, F-actin of stress fibers and overall cell morphology were perturbed in Clone9 cells expressing a mutant dynamin2 protein lacking the C-terminal proline-rich domain, the domain through which dynamin2 interacts with actin regulatory factors (11). Whereas existing data indicates that the specific effects of dynamin GTPase activity on F-actin structures are cell type- and structure-specific, a general conclusion is that dynamin GTPase activity influences the organization or turnover of a subset of actin filaments.To determine the mechanisms by which dynamin2 GTPase activity influences actin filaments, we developed biochemical and microscopic approaches to quantitatively assess and observe GTPase-dependent effects on actin filaments formed in vitro with Arp2/3 complex, cortactin, and dynamin2. The activities of dynamin2 on actin filaments in vivo were examined in cells with disrupted dynamin2 function using siRNA²-mediated suppression or pharmacologic inhibition. We report that dynamin2 GTPase, together with cortactin, functions as a dynamic actin filament remodeling complex that influences the global organization of the actomyosin cytoskeleton.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Is the caste-ratio of the oligolectic bumblebee Bombus gerstaeckeri Morawitz (Hymenoptera: Apidae) biased to queens?

Three bumblebee species, foraging on Aconitum spp. have been commonly observed in Eyne (France, East Pyrénées): Bombus gerstaeckeri, B. hortorum and B. wurflenii. We estimated the population of these three species. For B. hortorum and B. wurflenii, the total workers populations foraging on Aconitum spp. ranged from 101 to 523 and 156 to 270, respectively. These two species also forage on other plants while B. gerstaeckeri visits only Aconitum spp. The population of B. gerstaeckeri observed was extremely small, founded by 33 queens only in 2001. With a total number of workers estimated from 40 to 102, the observed workers/queens ratio, 1 to 3 workers for each queen, is very unusual for a eusocial species. Also we observed queens foraging during the whole life of the colony. This kind of social organisation is similar to that of some high arctic species. It could be interpreted as the result of an insularity syndrome.     

Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale.     

Age‐related variation in the trophic characteristics of a marsupial carnivore,the Tasmanian devil Sarcophilus harrisii

Age‐related changes in diet have implications for competitive interactions and for predator–prey dynamics, affecting individuals and groups at different life stages. To quantify patterns of variation and ontogenetic change in the diets of Tasmanian devils Sarcophilus harrisii, a threatened marsupial carnivore, we analyzed variation in the stable isotope composition of whisker tissue samples taken from 91 individual devils from Wilmot, Tasmania from December 2014 to February 2017. Both δ¹³C and δ¹⁵N decreased with increasing age in weaned Tasmanian devils, indicating that as they age devils rely less on small mammals and birds, and more on large herbivores. Devils <12 months old had broader group isotopic niches, as estimated by Bayesian standard ellipses (SEA_B mode = 1.042) than devils from 12 to 23 months old (mode = 0.541) and devils ≥24 months old (mode = 0.532). Devils <24 months old had broader individual isotopic niches (SEA_B mode range 0.492–1.083) than devils ≥24 months old (mode range 0.092–0.240). A decrease in δ¹⁵N from the older whisker sections to the more recently grown sections in devils <24 months old likely reflects the period of weaning in this species, as this pattern was not observed in devils ≥24 months old. Our data reveal changes in the isotopic composition of devil whiskers with increasing age, accompanied by a reduction in isotopic variation both among population age classes and within individuals, reflecting the effect of weaning in early life, and a likely shift from an initially diverse diet of small mammals, birds, and invertebrates towards increasing consumption of larger herbivores in adulthood.     

CD99 is a key mediator of the transendothelial migration of neutrophils

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.     

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.