New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

BACKGROUND: Pharmacologic control of Cre-mediated recombination using tamoxifen-dependent activation of a Cre-estrogen receptor ligand binding domain fusion protein [CreER(T)] is widely used to modify and/or visualize cells in the mouse. METHODS AND FINDINGS: We describe here two new mouse lines, constructed by gene targeting to the Rosa26 locus to facilitate Cre-mediated cell modification. These lines should prove particularly useful in the context of sparse labeling experiments. The R26rtTACreER line provides ubiquitous expression of CreER under transcriptional control by the tetracycline reverse transactivator (rtTA); dual control by doxycycline and tamoxifen provides an extended dynamic range of Cre-mediated recombination activity. The R26IAP line provides high efficiency Cre-mediated activation of human placental alkaline phosphatase (hPLAP), complementing the widely used, but low efficiency, Z/AP line. By crossing with mouse lines that direct cell-type specific CreER expression, the R26IAP line has been used to produce atlases of labeled cholinergic and catecholaminergic neurons in the mouse brain. The R26IAP line has also been used to visualize the full morphologies of retinal dopaminergic amacrine cells, among the largest neurons in the mammalian retina. CONCLUSIONS: The two new mouse lines described here expand the repertoire of genetically engineered mice available for controlled in vivo recombination and cell labeling using the Cre-lox system

Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP-seq with in vitro biochemistry to characterize a putative NsrR homologue in the model organism Streptomyces venezuelae. ChIP seq analysis revealed that rather than regulating the nitrosative stress response like NsrR, Sven6563 binds to a different, much larger regulon of genes with a diverse range of functions, including a range of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the target gene regulon are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator

Utilizing individual fish biomass and relative abundance models to map environmental niche associations of adult and juvenile targeted fishes

Many fishes undergo ontogenetic habitat shifts to meet their energy and resource needs as they grow. Habitat resource partitioning and patterns of habitat connectivity between conspecific fishes at different life-history stages is a significant knowledge gap. Species distribution models were used to examine patterns in the relative abundance, individual biomass estimates and environmental niche associations of different life stages of three iconic West Australian fishes. Continuous predictive maps describing the spatial distribution of abundance and individual biomass of the study species were created as well predictive hotspot maps that identify possible areas for aggregation of individuals of similar life stages of multiple species (i.e. spawning grounds, fisheries refugia or nursery areas). The models and maps indicate that processes driving the abundance patterns could be different from the body size associated demographic processes throughout an individual's life cycle. Incorporating life-history in the spatially explicit management plans can ensure that critical habitat of the vulnerable stages (e.g. juvenile fish, spawning stock) is included within proposed protected areas and can enhance connectivity between various functional areas (e.g. nursery areas and adult populations) which, in turn, can improve the abundance of targeted species as well as other fish species relying on healthy ecosystem functioning

The effect of tidal forcing on biogeochemical processes in intertidal salt marsh sediments

<p>Abstract</p> <p>Background</p> <p>Early diagenetic processes involved in natural organic matter (NOM) oxidation in marine sediments have been for the most part characterized after collecting sediment cores and extracting porewaters. These techniques have proven useful for deep-sea sediments where biogeochemical processes are limited to aerobic respiration, denitrification, and manganese reduction and span over several centimeters. In coastal marine sediments, however, the concentration of NOM is so high that the spatial resolution needed to characterize these processes cannot be achieved with conventional sampling techniques. In addition, coastal sediments are influenced by tidal forcing that likely affects the processes involved in carbon oxidation.</p> <p>Results</p> <p>In this study, we used in situ voltammetry to determine the role of tidal forcing on early diagenetic processes in intertidal salt marsh sediments. We compare ex situ measurements collected seasonally, in situ profiling measurements, and in situ time series collected at several depths in the sediment during tidal cycles at two distinct stations, a small perennial creek and a mud flat. Our results indicate that the tides coupled to the salt marsh topography drastically influence the distribution of redox geochemical species and may be responsible for local differences noted year-round in the same sediments. Monitoring wells deployed to observe the effects of the tides on the vertical component of porewater transport reveal that creek sediments, because of their confinements, are exposed to much higher hydrostatic pressure gradients than mud flats.</p> <p>Conclusion</p> <p>Our study indicates that iron reduction can be sustained in intertidal creek sediments by a combination of physical forcing and chemical oxidation, while intertidal mud flat sediments are mainly subject to sulfate reduction. These processes likely allow microbial iron reduction to be an important terminal electron accepting process in intertidal coastal sediments.</p

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

<p>Abstract</p> <p>Background</p> <p>Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in <it>Medicago truncatula</it>.</p> <p>Results</p> <p>This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in <it>M. truncatula</it>. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene.</p> <p>Conclusions</p> <p>This study shows that <it>Medicago truncatula </it>contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.</p

Anxiety Levels in Children with Autism Spectrum Disorder:A Meta-Analysis

The aim of the current study was to meta-analytically examine whether anxiety levels in children with autism spectrum disorders (ASD) are elevated. A total of 83 articles were selected from a systematic literature search and were included in the meta-analyses. Results demonstrated that children with ASD had higher anxiety levels compared to typically developing children, and this difference increased with IQ. Youth with ASD also tended to have higher anxiety levels compared to clinically referred children, and this difference increased with age. Children with ASD had higher anxiety levels compared to youth with externalizing or developmental problems, but not when compared to youth with internalizing problems. The study findings highlight the importance of more research in order to fully understand the nature and development of anxiety in children with ASD. More specifically, the results suggest that especially high-functioning adolescents with ASD may be at risk for developing anxiety disorders. Therefore, it seems important to carefully follow and monitor children with ASD transcending to adolescenc

Eccentric Exercise Activates Novel Transcriptional Regulation of Hypertrophic Signaling Pathways Not Affected by Hormone Changes

Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17β-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2

The relationship between manual coordination and mental health

Motor coordination impairments frequently co-occur with other developmental disorders and mental health problems in clinically referred populations. But does this reflect a broader dimensional relationship within the general population? A clearer understanding of this relationship might inform improvements in mental health service provision. However, ascertainment and referral bias means that there is limited value in conducting further research with clinically referred samples. We, therefore, conducted a cross-sectional population-based study investigating children’s manual coordination using an objective computerised test. These measures were related to teacher-completed responses on a behavioural screening questionnaire [the Strength and Difficulties Questionnaire (SDQ)]. We sampled 298 children (4–11 years old; 136 males) recruited from the general population. Hierarchical (logistic and linear) regression modelling indicated significant categorical and continuous relationships between manual coordination and overall SDQ score (a dimensional measure of psychopathology). Even after controlling for gender and age, manual coordination explained 15 % of the variance in total SDQ score. This dropped to 9 % after exclusion of participants whose SDQ responses indicated potential mental health problems. These results: (1) indicate that there is a clear relationship between children’s motor and mental health development in community-based samples; (2) demonstrate the relationship’s dimensional nature; and (3) have implications for service provision

Development of tissue-engineered models of oral dysplasia and early invasive oral squamous cell carcinoma

BACKGROUND: Current organotypic models of dysplasia and oral squamous cell carcinoma (OSCC) lack the complexity that mimics in vivo tissue. Here we describe a three-dimensional in vitro model of the oral epithelium that replicates tumour progression from dysplasia to an invasive phenotype. METHODS: The OSCC cell lines were seeded as a cell suspension (D20, Cal27) or as multicellular tumour spheroids (FaDu) with oral fibroblasts on to a de-epidermised acellular dermis to generate tissue-engineered models and compared with patient biopsies. RESULTS: The D20 and Cal27 cells generated a model of epithelial dysplasia. Overtime Cal27 cells traversed the basement membrane and invaded the connective tissue to reproduce features of early invasive OSCC. When seeded onto a model of the normal oral mucosa, FaDu spheroids produced a histological picture mimicking carcinoma in situ with severe cellular atypia juxtaposed to normal epithelium. CONCLUSION: It is possible to culture in vitro models with the morphological appearance and histological characteristics of dysplasia and tumour cell invasion seen in vivo using native dermis. Such models could facilitate study of the molecular processes involved in malignant transformation, invasion and tumour growth as well as in vitro testing of new treatments, diagnostic tests and drug delivery systems for OSCC