Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region

A Randomized Controlled Study of Parent-assisted Children’s Friendship Training with Children having Autism Spectrum Disorders

This study evaluated Children’s Friendship Training (CFT), a manualized parent-assisted intervention to improve social skills among second to fifth grade children with autism spectrum disorders. Comparison was made with a delayed treatment control group (DTC). Targeted skills included conversational skills, peer entry skills, developing friendship networks, good sportsmanship, good host behavior during play dates, and handling teasing. At post-testing, the CFT group was superior to the DTC group on parent measures of social skill and play date behavior, and child measures of popularity and loneliness, At 3-month follow-up, parent measures showed significant improvement from baseline. Post-hoc analysis indicated more than 87% of children receiving CFT showed reliable change on at least one measure at post-test and 66.7% after 3 months follow-up

Accelerated optical projection tomography applied to in vivo imaging of zebrafish

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections-achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds

Influence of homogenization conditions on physical properties and antioxidant activity of fully biodegradable pea protein-alpha-tocopherol films

In this study, antioxidant biodegradable films based on pea protein and alpha-tocopherol were successfully developed by solution casting. The effect of both the homogenization conditions (rotor stator and microfluidizer) and the relative humidity (RH) on the microstructure and physical properties (transparency, tensile, oxygen and water vapour barrier properties) of pea protein/alpha-tocopherol-based films was evaluated. The addition of alpha-tocopherol produced minimal changes in the films transparency, while providing them with antioxidant properties and improved water vapour and oxygen barrier properties (up to 30 % in both water vapour and oxygen permeability) when films were at low and intermediate RH. The addition of alpha-tocopherol in microfluidized films gave rise to an increase in their resistance to break and extensibility (up to 27 % in E values) at intermediate and high RH. These results add a new insight into the potential of employing pea protein and alpha-tocopherol in the development of fully biodegradable antioxidant films which are of interest in food packagingThe authors acknowledge the financial support from the Spanish Ministerio de Educacion y Ciencia throughout the project AGL2010-20694, co-funded by FEDER. Author M.J.Fabra is a recipient of a Juan de la Cierva contract from the Spanish Ministerio de Economia y Competitividad.Fabra, MJ.; Jiménez, A.; Talens Oliag, P.; Chiralt, A. (2014). Influence of homogenization conditions on physical properties and antioxidant activity of fully biodegradable pea protein-alpha-tocopherol films. Food and Bioprocess Technology. 7(12):3569-3578. https://doi.org/10.1007/s11947-014-1372-0S35693578712ASTM (1995). Standard test methods for water vapor transmission of materials. Standards Desingnations: E96-95. 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Effect of lipid self-association on the microstructure and physical properties of hydroxypropylmethylcellulose edible films containing fatty acids. Carbohydrate Polymers, 82(3), 585–593.Jiménez, A., Fabra, M. J., Talens, P., & Chiralt, A. (2013). Physical properties and antioxidant capacity of starch-sodium caseinate films containing lipids. Journal of Food Engineering, 116(3), 695–702.Jung, M. Y., & Min, D. B. (1990). Effects of alpha-. γ-, and δ-tocopherols on oxidative stability of soybean oil. Journal of Food Science, 55(5), 1464–1465.López-de-Dicastillo, C., Alonso, J. M., Catalá, R., Gavara, R., & Hernández-Muñoz, P. (2010). Improving the antioxidant protection of packaged food by incorporating natural flavonoids into ethylene-vinyl alcohol copolymer (EVOH) films. Journal of Agricultural and Food Chemistry, 58, 10958–10964.Ma, W., Tang, C.-H., Yin, S.-W., Yang, X. Q., Qi, J. R., & Xia, N. (2012). Effect of homogenization conditions on properties of gelatin-olive oil composite films. Journal of Food Engineering, 113(1), 136–142.Mauer, L. J., Smith, D. E., & Labuza, T. P. (2000). Water vapor permeability, mechanical, and structural properties of edible β-casein films. International Dairy Journal, 10(5–6), 353–358.Mc Hugh, T. H., Avena-Bustillos, R., & Krochta, J. M. (1993). Hydrophobic edible films:modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science, 58(4), 899–903.McHugh, T. H., & Krochta, J. M. (1994). Dispersed phase particle size effects on water vapour permeability of whey protein–beeswax emulsion films. Journal of Food Processing and Preservation, 18, 173–188.Ozkan, G., Simsek, B., & Kuleasan, H. (2007). Antioxidant activities of Satureja cilicica essential oil in butter and in vitro. Journal of Food Engineering, 79, 1391–1396.Pereira de Abreu, D. A., Paseiro Losada, P., Maroto, J., & Cruz, J. M. (2011). 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Autolysis-assisted production of fish protein hydrolysates with antioxidant properties form Pacific hake (Merluccius productus). Food Chemistry, 107, 768–776.Souza, B. W. S., Cerqueira, A., Casariego, A., Lima, A. M. P., Teixeira, J. A., & Vicente, A. A. (2009). Effect of moderate electric fields in the permeation properties of chitosan coatings. Food Hydrocolloids, 23, 2110–2115

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. © 2013 Rogers et al

t2prhd: a tool to study the patterns of repeat evolution

BACKGROUND: The models developed to characterize the evolution of multigene families (such as the birth-and-death and the concerted models) have also been applied on the level of sequence repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the evolution of gene families and also sequence repeats in the light of these models. The characterization of the gene family evolution in view of the evolutionary models is done by the evaluation of the clustering of the sequences with the originating loci in mind. As the locus represents positional information, it is straightforward that in the case of the repeats the exact position in the sequence should be used, as the simple numbering according to repeat order can be misleading. RESULTS: We have developed a novel rapid visual approach to study repeat evolution, that takes into account the exact repeat position in a sequence. The "pairwise repeat homology diagram" visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script (t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences. CONCLUSION: Although pairwise repeat homology diagrams do not carry all the information provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution. We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This method can be particularly useful in cases of large datasets (such as large gene families), as the command line interface makes it possible to automate the generation of pairwise repeat homology diagrams with the aid of script

Anthrax Lethal Toxin Suppresses Murine Cardiomyocyte Contractile Function and Intracellular Ca2+ Handling via a NADPH Oxidase-Dependent Mechanism

OBJECTIVES: Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca(2+) properties. METHODS: Murine cardiomyocyte contractile function and intracellular Ca(2+) handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), intracellular Ca(2+) rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca(2+) decay rate. Stress signaling and Ca(2+) regulatory proteins were assessed using Western blot analysis. RESULTS: In vitro exposure to a lethal toxin (0.05-50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca(2+) properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca(2+) decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca(2+) responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca(2+) regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure. CONCLUSIONS: Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca(2+) through a NADPH oxidase-dependent mechanism

Systematic review of interventions for children with Fetal Alcohol Spectrum Disorders

<p>Abstract</p> <p>Background</p> <p>Children with Fetal Alcohol Spectrum Disorders (FASD) may have significant neurobehavioural problems persisting into adulthood. Early diagnosis may decrease the risk of adverse life outcomes. However, little is known about effective interventions for children with FASD. Our aim is to conduct a systematic review of the literature to identify and evaluate the evidence for pharmacological and non-pharmacological interventions for children with FASD.</p> <p>Methods</p> <p>We did an electronic search of the Cochrane Library, MEDLINE, EMBASE, PsychINFO, CINAHL and ERIC for clinical studies (Randomized controlled trials (RCT), quasi RCT, controlled trials and pre- and post-intervention studies) which evaluated pharmacological, behavioural, speech therapy, occupational therapy, physiotherapy, psychosocial and educational interventions and early intervention programs. Participants were aged under 18 years with a diagnosis of a FASD. Selection of studies for inclusion and assessment of study quality was undertaken independently by two reviewers. Meta-analysis was not possible due to diversity in the interventions and outcome measures.</p> <p>Results</p> <p>Twelve studies met the inclusion criteria. Methodological weaknesses were common, including small sample sizes; inadequate study design and short term follow up. Pharmacological interventions, evaluated in two studies (both RCT) showed some benefit from stimulant medications. Educational and learning strategies (three RCT) were evaluated in seven studies. There was some evidence to suggest that virtual reality training, cognitive control therapy, language and literacy therapy, mathematics intervention and rehearsal training for memory may be beneficial strategies. Three studies evaluating social communication and behavioural strategies (two RCT) suggested that social skills training may improve social skills and behaviour at home and Attention Process Training may improve attention.</p> <p>Conclusion</p> <p>There is limited good quality evidence for specific interventions for managing FASD, however seven randomized controlled trials that address specific functional deficits of children with FASD are underway or recently completed.</p

Viscosity affected by nanoparticle aggregation in Al2O3-water nanofluids

An investigation on viscosity was conducted 2 weeks after the Al2O3-water nanofluids having dispersants were prepared at the volume concentration of 1-5%. The shear stress was observed with a non-Newtonian behavior. On further ultrasonic agitation treatment, the nanofluids resumed as a Newtonian fluids. The relative viscosity increases as the volume concentrations increases. At 5% volume concentration, an increment was about 60% in the re-ultrasonication nanofluids in comparison with the base fluid. The microstructure analysis indicates that a higher nanoparticle aggregation had been observed in the nanofluids before re-ultrasonication

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region