Fitness, risk taking, and spatial behavior covary with boldness in experimental vole populations.

Individuals of a population may vary along a pace-of-life syndrome from highly fecund, short-lived, bold, dispersive "fast" types at one end of the spectrum to less fecund, long-lived, shy, plastic "slow" types at the other end. Risk-taking behavior might mediate the underlying life history trade-off, but empirical evidence supporting this hypothesis is still ambiguous. Using experimentally created populations of common voles (Microtus arvalis)-a species with distinct seasonal life history trajectories-we aimed to test whether individual differences in boldness behavior covary with risk taking, space use, and fitness. We quantified risk taking, space use (via automated tracking), survival, and reproductive success (via genetic parentage analysis) in 8 to 14 experimental, mixed-sex populations of 113 common voles of known boldness type in large grassland enclosures over a significant part of their adult life span and two reproductive events. Populations were assorted to contain extreme boldness types (bold or shy) of both sexes. Bolder individuals took more risks than shyer ones, which did not affect survival. Bolder males but not females produced more offspring than shy conspecifics. Daily home range and core area sizes, based on 95% and 50% Kernel density estimates (20 ± 10 per individual, n = 54 individuals), were highly repeatable over time. Individual space use unfolded differently for sex-boldness type combinations over the course of the experiment. While day ranges decreased for shy females, they increased for bold females and all males. Space use trajectories may, hence, indicate differences in coping styles when confronted with a novel social and physical environment. Thus, interindividual differences in boldness predict risk taking under near-natural conditions and have consequences for fitness in males, which have a higher reproductive potential than females. Given extreme inter- and intra-annual fluctuations in population density in the study species and its short life span, density-dependent fluctuating selection operating differently on the sexes might maintain (co)variation in boldness, risk taking, and pace-of-life.

Bcl-XL but Not Bcl-2 Is a Potential Target in Medulloblastoma Therapy.

Medulloblastoma (MB) is the most common solid tumour in children and, despite current treatment with a rather aggressive combination therapy, accounts for 10% of all deaths associated with paediatric cancer. Breaking the tumour cells' intrinsic resistance to therapy-induced cell death should lead to less aggressive and more effective treatment options. In other tumour entities, this has been achieved by modulating the balance between the various pro- and anti-apoptotic members of the Bcl-2 family with small molecule inhibitors. To evaluate the therapeutic benefits of ABT-199 (Venetoclax), a Bcl-2 inhibitor, and ABT-263 (Navitoclax), a dual Bcl-XL/Bcl-2 inhibitor, increasingly more relevant model systems were investigated. Starting from established MB cell lines, progressing to primary patient-derived material and finally an experimental tumour system imbedded in an organic environment were chosen. Assessment of the metabolic activity (a surrogate readout for population viability), the induction of DNA fragmentation (apoptosis) and changes in cell number (the combined effect of alterations in proliferation and cell death induction) revealed that ABT-263, but not ABT-199, is a promising candidate for combination therapy, synergizing with cell death-inducing stimuli. Interestingly, in the experimental tumour setting, the sensitizing effect of ABT-263 seems to be predominantly mediated via an anti-proliferative and not a pro-apoptotic effect, opening a future line of investigation. Our data show that modulation of specific members of the Bcl-2 family might be a promising therapeutic addition for the treatment of MB.

The limitations of targeting MEK signalling in Glioblastoma therapy.

Glioblastoma (GB) is a highly aggressive, difficult to treat brain tumour. Successful treatment, consisting of maximal safe tumour de-bulking, followed by radiotherapy and treatment with the alkylating agent Temozolomide (TMZ), can extend patient survival to approximately 15 months. Combination treatments based on the inhibition of the PI3K pathway, which is the most frequently activated signalling cascade in GB, have so far only shown limited therapeutic success. Here, we use the clinically approved MEK inhibitor Trametinib to investigate its potential use in managing GB. Trametinib has a strong anti-proliferative effect on established GB cell lines, stem cell-like cells and their differentiated progeny and while it does not enhance anti-proliferative and cell death-inducing properties of the standard treatment, i.e. exposure to radiation or TMZ, neither does MEK inhibition block their effectiveness. However, upon MEK inhibition some cell populations appear to favour cell-substrate interactions in a sprouting assay and become more invasive in the Chorioallantoic Membrane assay, which assesses cell penetration into an organic membrane. While this increased invasion can be modulated by additional inhibition of the PI3K signalling cascade, there is no apparent benefit of blocking MEK compared to targeting PI3K.

Loss of function of CRT1a (calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape (Brassica napus).

Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up-regulation of so-called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up-regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild-type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas-induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele-specific sequencing suggests a major effect of mutations in the CRT1a A-genome copy, while the C-genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

A behavioural syndrome, but less evidence for a relationship with cognitive traits in a spatial orientation context.

BACKGROUND: Animals show consistent individual behavioural differences in many species. Further, behavioural traits (personality traits) form behavioural syndromes, characterised by correlations between different behaviours. Mechanisms maintaining these correlations could be constrained due to underlying relationships with cognitive traits. There is growing evidence for the non-independence of animal personality and general cognitive abilities in animals, but so far, studies on the direction of the relationship between them revealed contradictory results. Still, it is hypothesised that individuals may exhibit consistent learning and decision styles. Fast behavioural types (consistently bolder and more active individuals) are expected to show faster learning styles. Slow behavioural types in contrast are assumed to learn slower but more accurately. This can be caused by a speed-accuracy trade-off that individuals face during decision making. We measured the repeatability of three personality and four spatial cognitive traits in adult Eurasian harvest mice (Micromys minutus). We analysed correlations among personality traits (behavioural syndrome). We further investigated the relationships between personality and spatial cognitive traits as a first step exploring the potential connection between personality and cognition in this species. RESULTS: Our results showed that exploration, activity and boldness were repeatable in adult mice. Spatial recognition measured in a Y Maze was also significantly repeatable, as well as spatial learning performance and decision speed. We found no repeatability of decision accuracy. Harvest mice showed a behavioural syndrome as we observed strong positive correlations between personality traits. The speed-accuracy trade-off was not apparent within, nor between individuals. Nevertheless, we found weak evidence for a relationship between personality and spatial cognitive traits as fast behavioural types learned a spatial orientation task faster than slow types, and shyer harvest mice made decisions quicker than bolder mice. CONCLUSIONS: Given these correlations, our data provided some first insights into the relationship between personality and spatial cognitive traits in harvest mice and will hopefully stimulate more studies in this field.

Repeatability and consistency of individual behaviour in juvenile and adult Eurasian harvest mice.

Knowledge on animal personality has provided new insights into evolutionary biology and animal ecology, as behavioural types have been shown to affect fitness. Animal personality is characterized by repeatable and consistent between-individual behavioural differences throughout time and across different situations. Behavioural repeatability within life history stages and consistency between life history stages should be checked for the independence of sex and age, as recent data have shown that males and females in some species may differ in the repeatability of behavioural traits, as well as in their consistency. We measured the repeatability and consistency of three behavioural and one cognitive traits in juvenile and adult Eurasian harvest mice (Micromys minutus). We found that exploration, activity and boldness were repeatable in juveniles and adults. Spatial recognition measured in a Y Maze was only repeatable in adult mice. Exploration, activity and boldness were consistent before and after maturation, as well as before and after first sexual contact. Data on spatial recognition provided little evidence for consistency. Further, we found some evidence for a litter effect on behaviours by comparing different linear mixed models. We concluded that harvest mice express animal personality traits as behaviours were repeatable across sexes and consistent across life history stages. The tested cognitive trait showed low repeatability and was less consistent across life history stages. Given the rising interest in individual variation in cognitive performance, and in its relationship to animal personality, we suggest that it is important to gather more data on the repeatability and consistency of cognitive traits.

Evidence for selection maintaining MHC diversity in a rodent species despite strong density fluctuations.

Strong spatiotemporal variation in population size often leads to reduced genetic diversity limiting the adaptive potential of individual populations. Key genes of adaptive variation are encoded by the immune genes of the major histocompatibility complex (MHC) playing an essential role in parasite resistance. How MHC variation persists in rodent populations that regularly experience population bottlenecks remains an important topic in evolutionary genetics. We analysed the consequences of strong population fluctuations on MHC class II DRB exon 2 diversity in two distant common vole (Microtus arvalis) populations in three consecutive years using a high-throughput sequencing approach. In 143 individuals, we detected 25 nucleotide alleles translating into 14 unique amino acid MHC alleles belonging to at least three loci. Thus, the overall allelic diversity and amino acid distance among the remaining MHC alleles, used as a surrogate for the range of pathogenic antigens that can be presented to T-cells, are still remarkably high. Both study populations did not show significant population differentiation between years, but significant differences were found between sites. We concluded that selection processes seem to be strong enough to maintain moderate levels of MHC diversity in our study populations outcompeting genetic drift, as the same MHC alleles were conserved between years. Differences in allele frequencies between populations might be the outcome of different local parasite pressures and/or genetic drift. Further understanding of how pathogens vary across space and time will be crucial to further elucidate the mechanisms maintaining MHC diversity in cyclic populations.

Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor ß1 (TGFß1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Delivery of siRNA to the mouse lung via a functionalized lipopolyamine.

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.