Neuroprotection by Exogenous and Endogenous Neuregulin-1 in Mouse Models of Focal Ischemic Stroke.

Identifying novel neuroprotectants that can halt or reverse the neurological effects of stroke is of interest to both clinicians and scientists. We and others previously showed the pre-clinical neuroprotective efficacy of neuregulin-1 (NRG-1) in rats following focal brain ischemia. In this study, we examined neuroprotection by exogenous and endogenous NRG-1 using a mouse model of ischemic stroke. C57BL6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion. NRG-1 or vehicle was infused intra-arterially (i.a.) or intravenously (i.v.) after MCAO and before the onset of reperfusion. NRG-1 treatment (16 μg/kg; i.a.) reduced cerebral cortical infarct volume by 72% in mice when delivered post-ischemia. NRG-1 also inhibited neuronal injury as measured by Fluoro Jade B labeling and rescued NeuN immunoreactivity in neurons. Neuroprotection by NRG-1 was also observed in mice when administered i.v. (100 μg/kg) in both male and female mice. We investigated whether endogenous NRG-1 was neuroprotective using male and female heterozygous NRG-1 knockout mice (NRG-1+/-) compared with wild-type mice (WT) littermates. NRG-1+/- and WT mice were subjected to MCAO for 45 min, and infarct size was measured 24 h following MCAO. NRG-1+/- mice displayed a sixfold increase in cortical infarct size compared with WT mice. These results demonstrate that NRG-1 treatment mitigates neuronal damage following cerebral ischemia. We further showed that reduced endogenous NRG-1 results in exacerbated neuronal injury in vivo. These findings suggest that NRG-1 represents a promising therapy to treat stroke in human patients

Conditioned Flavor Aversion: A Mechanism for Goats to Avoid Condensed Tannins in Blackbrush

It has been hypothesized that herbivores instinctively avoid tannin-containing plant parts in response to the adverse effects of tannins on forage digestion. However, we found that goats learned to avoid condensed tannins (CTs) from blackbrush current season\u27s growth by associating the flavor of foods containing CTs with aversive postingestive consequences. The aversive consequences experienced by goats apparently are not related to digestion inhibition and may depend on the structure of CTs and on how CTs are bound with other cell constituents. These observations suggest several areas of inquiry related to the interaction between CTs and herbivores. A better understanding of the physiological effects of CTs and how herbivores perceive these effects is essential to our knowledge of chemically mediated interactions between plants and mammalian herbivores. With few exceptions, the effects of food flavor have not been separated from those associated with postingestive consequences, even though our data show that postingestive consequences strongly influence palatability. We also need to know how herbivores learn which plant species to eat and which to avoid while foraging in areas that contain a variety of plant species and parts with different kinds and concentrations of CTs. Condensed tannins are pervasive in nature and can defend plants from herbivory, but since many important forages contain high levels of tannins, the presence or absence of tannins per se does not reliably indicate food quality. To predict the ability of a tannin-producing plant to deter herbivores requires a full understanding of how changes in CT structure and binding affect herbivores

RATA: A method for high-throughput identification of RNA bound transcription factors.

Long non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-bound transcription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest. RATA requires only ~5 million cells and standard molecular biology reagents for multiplexed identification of up to 96 transcription factors in 2-3 d. Thus, RATA offers significant advantages over other technologies for analysis of RNA-transcription factor interactions

The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition

<p>Abstract</p> <p>Background</p> <p>The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised <it>via </it>a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.</p> <p>Results</p> <p>To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by <it>in vivo </it>measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects <it>in vivo</it>.</p> <p>Conclusions</p> <p>Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.</p

Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode

The phylogenetic relationship of chemoautotrophic, sulfur-oxidizing, ectosymbiotic bacteria growing on a marine nematode, a Laxus sp. (formerly a Catanema sp.), to known endosymbionts and free-living bacteria was determined. Comparative 16S rRNA sequencing was used to investigate the unculturable nematode epibionts, and rRNA-targeted oligonucleotide hybridization probes were used to identify the ectosymbionts in situ. Both analyses revealed a remarkably specific and stable symbiosis. Unique hybridization of a specific probe to the ectosymbionts indicated that only one species of bacteria was present and growing on the cuticle of the nematode. Distance and parsimony methods used to infer phylogenetic trees both placed the nematode ectosymbionts at the base of a branch containing chemoautotrophic, sulfur-oxidizing endosymbionts of three bivalve families and of the tube worm Riftia pachyptila. The most closely related free-living bacteria were chemoautotrophic sulfur oxidizers belonging to the genus Thiomicrospira. Furthermore, our results suggested that a second, only distantly related group of thioautotrophic endosymbionts has as its deepest branch surface-colonizing bacteria belonging to the genus Thiothrix, some of which are capable of sulfur-oxidizing chemoautotrophic growth.Organismic and Evolutionary Biolog

A hypothetico-deductive approach to assessing the social function of chemical signalling in a non-territorial solitary carnivore

The function of chemical signalling in non-territorial solitary carnivores is still relatively unclear. Studies on territorial solitary and social carnivores have highlighted odour capability and utility, however the social function of chemical signalling in wild carnivore populations operating dominance hierarchy social systems has received little attention. We monitored scent marking and investigatory behaviour of wild brown bears Ursus arctos, to test multiple hypotheses relating to the social function of chemical signalling. Camera traps were stationed facing bear ‘marking trees’ to document behaviour by different age sex classes in different seasons. We found evidence to support the hypothesis that adult males utilise chemical signalling to communicate dominance to other males throughout the non-denning period. Adult females did not appear to utilise marking trees to advertise oestrous state during the breeding season. The function of marking by subadult bears is somewhat unclear, but may be related to the behaviour of adult males. Subadults investigated trees more often than they scent marked during the breeding season, which could be a result of an increased risk from adult males. Females with young showed an increase in marking and investigation of trees outside of the breeding season. We propose the hypothesis that females engage their dependent young with marking trees from a young age, at a relatively ‘safe’ time of year. Memory, experience, and learning at a young age, may all contribute towards odour capabilities in adult bears

Genome-wide analyses for personality traits identify six genomic loci and show correlations with psychiatric disorders

Personality is influenced by genetic and environmental factors1 and associated with mental health. However, the underlying genetic determinants are largely unknown. We identified six genetic loci, including five novel loci2,3, significantly associated with personality traits in a meta-analysis of genome-wide association studies (N = 123,132–260,861). Of these genomewide significant loci, extraversion was associated with variants in WSCD2 and near PCDH15, and neuroticism with variants on chromosome 8p23.1 and in L3MBTL2. We performed a principal component analysis to extract major dimensions underlying genetic variations among five personality traits and six psychiatric disorders (N = 5,422–18,759). The first genetic dimension separated personality traits and psychiatric disorders, except that neuroticism and openness to experience were clustered with the disorders. High genetic correlations were found between extraversion and attention-deficit– hyperactivity disorder (ADHD) and between openness and schizophrenia and bipolar disorder. The second genetic dimension was closely aligned with extraversion–introversion and grouped neuroticism with internalizing psychopathology (e.g., depression or anxiety)

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts

Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations

Radiosensitization by BRAF inhibitor therapy—mechanism and frequency of toxicity in melanoma patients

This study shows radiosensitization by BRAF inhibitors in clinical practice and ex vivo by fluorescence in situ hybridization of chromosomal breaks. Nevertheless, radiotherapy with concomitant BRAF inhibitor therapy is feasible with an acceptable increase in toxicity. Vemurafenib is a more potent radiosensitizer than dabrafenib in both the patient study and the ex vivo experiment