A simple, versatile and sensitive cell-based assay for prions from various species

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.     

Deletion of C9ORF72 Results in Motor Neuron Degeneration and Stress Sensitivity in C. elegans

An expansion of the hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the Caenorhabditis elegans C9ORF72 orthologue, F18A1.6, also called alfa-1. alfa-1 mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. alfa-1 mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of alfa-1 were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of alfa-1/C9ORF72 expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.     

Assessment of contrafreeloading preferences in giraffe (Giraffa camelopardalis)

Contrafreeloading is an intriguing phenomenon in which animals will work to obtain resources, such as food, when the same resource is simultaneously freely available. Multiple hypotheses exist for why animals might choose to contrafreeload. In this study, we assessed preferences for contrafreeloading in giraffe at the Bronx Zoo to determine whether they actually preferred to contrafreeload or were simply demonstrating a willingness to contrafreeload. Food was presented in a range of distributions between an easily accessed feeding device and a more challenging one and the giraffes’ feeding behavior at these two types of feeding devices was recorded. As the experiments progressed, more giraffe used these more challenging feeders. There was significant individual variation in the expression of preference for contrafreeloading and willingness to contrafreeload. Individual, phase of the experiment, and an interaction between these factors were significant predictors of challenge feeder use. Three foraging strategies emerged among the giraffe that we termed “freeloaders,” “contrafreeloaders,” and “opportunists.” The results of this study demonstrate that multiple indices of preference are necessary when assessing contrafreeloading behavior, and that giraffe are affected to different degrees by the factors that stimulate contrafreeloading. These results may shed light on why different individuals use complex feeding enrichment devices to varying extents.     

Responses of soil carbon sequestration to climate‐smart agriculture practices: A meta‐analysis

Climate‐smart agriculture (CSA) management practices (e.g., conservation tillage, cover crops, and biochar applications) have been widely adopted to enhance soil organic carbon (SOC) sequestration and to reduce greenhouse gas emissions while ensuring crop productivity. However, current measurements regarding the influences of CSA management practices on SOC sequestration diverge widely, making it difficult to derive conclusions about individual and combined CSA management effects and bringing large uncertainties in quantifying the potential of the agricultural sector to mitigate climate change. We conducted a meta‐analysis of 3,049 paired measurements from 417 peer‐reviewed articles to examine the effects of three common CSA management practices on SOC sequestration as well as the environmental controlling factors. We found that, on average, biochar applications represented the most effective approach for increasing SOC content (39%), followed by cover crops (6%) and conservation tillage (5%). Further analysis suggested that the effects of CSA management practices were more pronounced in areas with relatively warmer climates or lower nitrogen fertilizer inputs. Our meta‐analysis demonstrated that, through adopting CSA practices, cropland could be an improved carbon sink. We also highlight the importance of considering local environmental factors (e.g., climate and soil conditions and their combination with other management practices) in identifying appropriate CSA practices for mitigating greenhouse gas emissions while ensuring crop productivity.     

Contribution of allochthonous resources to breeding in a high-arctic avian predator

Allochthonous input of resources (i.e., originating from a place other than where they are found) can have a significant impact on food availability for consumers. We assessed the impact of an allochthonous source of food (the sewage outfall stream of a military base) on an avian predator breeding in a low-productivity, high-arctic site (Alert, 83°N, 62°W, Ellesmere Island), the long-tailed skua (Stercorarius longicaudus). We collected blood samples throughout the breeding season during two contrasting years of lemming abundance to characterize diet composition of skuas and evaluated the contribution of the anthropogenic and lemming food sources using stable isotopes (carbon ¹³C and nitrogen ¹⁵N). The isotopic signature of skuas changed seasonally because they switch from a marine to a terrestrial diet when they come ashore to breed but also differed between the 2 years of the study. Anthropogenic food source accounted for 33 % of the summer diet but this proportion varied between years, from 41 % (5–95 ‰: 13–59 %) in a year of low lemming abundance to 16 % (5–95 ‰: 10–21 %) in a high year. Skua nest density recorded in years of low lemming abundance at Alert (0.15 nests/km²) was higher than at any other comparable arctic sites (0–0.02 nests/km²). Overall, the use of an anthropogenic food sources apparently subsidizes skua reproduction at this site, which could affect the food web of this low-productivity ecosystem.     

Targeting thrombin and factor VIIa: design,synthesis, and inhibitory activity of functionally relevant indolizidinones

Guided by molecular modeling, docking experiments, and available X-ray crystal structure data on the serine protease Factor VIIa and thrombin, a series of indolizidinone derivatives was designed and synthesized having diverse functionality at the P1, P2, and P3 sites.     

Synthesis of glutaminyl adenylate analogues that are inhibitors of glutaminyl-tRNA synthetase

Glutaminol adenylate 5 is a competitive inhibitor of glutaminyl-tRNA synthetase with respect to glutamine (Ki = 280 nM) and to ATP (Ki = 860 nM). The corresponding methyl phosphate ester 4 is a weaker inhibitor (Ki approximately 10 microM) with respect to glutamine.     

Mononuclear arene ruthenium complexes containing 5,6-diphenyl-3-(pyridine-2-yl)-1,2,4-triazine as chelating ligand: Synthesis and molecular structure

The mononuclear cations of the general formula [(η⁶-arene)RuCl(pdpt)]⁺ (pdpt = 5,6-diphenyl-3-(pyridine-2-yl)-1,2,4-triazine; arene = C₆H₆ (1); C₆H₅Me (2); p-PrⁱC₆H₄Me (3); C₆Me₆ (4)) have been synthesised from 5,6-diphenyl-3-(pyridine-2-yl)-1,2,4-triazine (pdpt) and the corresponding chloro complexes [(η⁶-C₆H₆)Ru(μ-Cl)Cl]₂, [(η⁶-C₆H₅Me)Ru(μ-Cl)Cl]₂, [(η⁶p-PrⁱC₆H₄Me)Ru(μ-Cl)Cl]₂ and [(η⁶-C₆Me₆)Ru(μ-Cl)Cl]₂, respectively. The X-ray crystal structure analyses of [1][PF₆] · (C₆H₆)_2.5 and [2][PF₆] · (CH₃CN)₂ reveal a typical piano-stool geometry around the metal centre and in the crystal packing a complexed networks of intermolecular interactions.     

Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.     

Mesenchymal‐like stem cells in canine ovary show high differentiation potential

Objectives

Recent studies have reported the existence of stem cells in ovarian tissue that show enhanced proliferative and differentiation potential compared to other adult tissues. Based on this evidence, we hypothesized that ovarian tissue contained mesenchymal‐like stem cells (MSC) that could be isolated using a novel rapid plastic adhesion technique.

Materials and methods

We established MSC lines derived from ovarian and adipose tissue based on their ability to rapidly adhere to plastic culture dishes in the first 3 hours after plating and studied their potentiality in terms of molecular markers and differentiation capacity.

Results

Morphological and kinetic properties of in vitro cultured ovarian MSC were similar to adipose‐derived MSC, and both reached senescence after similar passage numbers. Ovarian‐derived MSC expressed mesenchymal (CD90 and CD44) but not haematopoietic markers (CD34 and CD45), indicating similarity to adipose‐derived MSC. Moreover, ovarian‐derived MSC expressed NANOG, TERT, SOX2, OCT4 and showed extensive capacity to differentiate not only into adipogenic, osteogenic and chondrogenic tissue but also towards neurogenic and endodermal lineages and even precursors of primordial germ cells.

Conclusion

These results show for the first time the derivation of ovarian cells with the molecular properties of MSC as well as wide differentiation potential. Canine ovarian tissue is accessible, expandable, multipotent and has high plasticity, holding promise for applications in regenerative medicine.