Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols

Worldwide comparison of survival from childhood leukaemia for 1995–2009, by subtype, age, and sex (CONCORD-2): a population-based study of individual data for 89 828 children from 198 registries in 53 countries

Background Global inequalities in access to health care are reflected in differences in cancer survival. The CONCORD programme was designed to assess worldwide differences and trends in population-based cancer survival. In this population-based study, we aimed to estimate survival inequalities globally for several subtypes of childhood leukaemia. Methods Cancer registries participating in CONCORD were asked to submit tumour registrations for all children aged 0-14 years who were diagnosed with leukaemia between Jan 1, 1995, and Dec 31, 2009, and followed up until Dec 31, 2009. Haematological malignancies were defined by morphology codes in the International Classification of Diseases for Oncology, third revision. We excluded data from registries from which the data were judged to be less reliable, or included only lymphomas, and data from countries in which data for fewer than ten children were available for analysis. We also excluded records because of a missing date of birth, diagnosis, or last known vital status. We estimated 5-year net survival (ie, the probability of surviving at least 5 years after diagnosis, after controlling for deaths from other causes [background mortality]) for children by calendar period of diagnosis (1995-99, 2000-04, and 2005-09), sex, and age at diagnosis (< 1, 1-4, 5-9, and 10-14 years, inclusive) using appropriate life tables. We estimated age-standardised net survival for international comparison of survival trends for precursor-cell acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Findings We analysed data from 89 828 children from 198 registries in 53 countries. During 1995-99, 5-year agestandardised net survival for all lymphoid leukaemias combined ranged from 10.6% (95% CI 3.1-18.2) in the Chinese registries to 86.8% (81.6-92.0) in Austria. International differences in 5-year survival for childhood leukaemia were still large as recently as 2005-09, when age-standardised survival for lymphoid leukaemias ranged from 52.4% (95% CI 42.8-61.9) in Cali, Colombia, to 91.6% (89.5-93.6) in the German registries, and for AML ranged from 33.3% (18.9-47.7) in Bulgaria to 78.2% (72.0-84.3) in German registries. Survival from precursor-cell ALL was very close to that of all lymphoid leukaemias combined, with similar variation. In most countries, survival from AML improved more than survival from ALL between 2000-04 and 2005-09. Survival for each type of leukaemia varied markedly with age: survival was highest for children aged 1-4 and 5-9 years, and lowest for infants (younger than 1 year). There was no systematic difference in survival between boys and girls. Interpretation Global inequalities in survival from childhood leukaemia have narrowed with time but remain very wide for both ALL and AML. These results provide useful information for health policy makers on the effectiveness of health-care systems and for cancer policy makers to reduce inequalities in childhood survival

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

Marguerite E. Hunt is with UT Austin; Michael P. Scherrer is with UT Austin; Frank D. Ferrari is with the National Museum of Natural History at the Smithsonian Institution; Mikhail V. Matz is with UT Austin.Background -- Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. Results -- Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M−1 cm−) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. Conclusions -- The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.This work was supported by the Ocean Exploration program of the National Oceanic and Atmospheric Administration (“Operation Deep Scope 2007”), and the National Institutes of Health grant R01 GM078247 to M. V. M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

Differential Response of High-Elevation Planktonic Bacterial Community Structure and Metabolism to Experimental Nutrient Enrichment

Nutrient enrichment of high-elevation freshwater ecosystems by atmospheric deposition is increasing worldwide, and bacteria are a key conduit for the metabolism of organic matter in these oligotrophic environments. We conducted two distinct in situ microcosm experiments in a high-elevation lake (Emerald Lake, Sierra Nevada, California, USA) to evaluate responses in bacterioplankton growth, carbon utilization, and community structure to short-term enrichment by nitrate and phosphate. The first experiment, conducted just following ice-off, employed dark dilution culture to directly assess the impact of nutrients on bacterioplankton growth and consumption of terrigenous dissolved organic matter during snowmelt. The second experiment, conducted in transparent microcosms during autumn overturn, examined how bacterioplankton in unmanipulated microbial communities responded to nutrients concomitant with increasing phytoplankton-derived organic matter. In both experiments, phosphate enrichment (but not nitrate) caused significant increases in bacterioplankton growth, changed particulate organic stoichiometry, and induced shifts in bacterial community composition, including consistent declines in the relative abundance of Actinobacteria. The dark dilution culture showed a significant increase in dissolved organic carbon removal in response to phosphate enrichment. In transparent microcosms nutrient enrichment had no effect on concentrations of chlorophyll, carbon, or the fluorescence characteristics of dissolved organic matter, suggesting that bacterioplankton responses were independent of phytoplankton responses. These results demonstrate that bacterioplankton communities in unproductive high-elevation habitats can rapidly alter their taxonomic composition and metabolism in response to short-term phosphate enrichment. Our results reinforce the key role that phosphorus plays in oligotrophic lake ecosystems, clarify the nature of bacterioplankton nutrient limitation, and emphasize that evaluation of eutrophication in these habitats should incorporate heterotrophic microbial communities and processes

Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology

Sensitivity of Calcification to Thermal Stress Varies among Genera of Massive Reef-Building Corals

Reductions in calcification in reef-building corals occur when thermal conditions are suboptimal, but it is unclear how they vary between genera in response to the same thermal stress event. Using densitometry techniques, we investigate reductions in the calcification rate of massive Porites spp. from the Great Barrier Reef (GBR), and P. astreoides, Montastraea faveolata, and M. franksi from the Mesoamerican Barrier Reef (MBR), and correlate them to thermal stress associated with ocean warming. Results show that Porites spp. are more sensitive to increasing temperature than Montastraea, with calcification rates decreasing by 0.40 g cm−2 year−1 in Porites spp. and 0.12 g cm−2 year−1 in Montastraea spp. for each 1°C increase. Under similar warming trends, the predicted calcification rates at 2100 are close to zero in Porites spp. and reduced by 40% in Montastraea spp. However, these predictions do not account for ocean acidification. Although yearly mean aragonite saturation (Ωar) at MBR sites has recently decreased, only P. astreoides at Chinchorro showed a reduction in calcification. In corals at the other sites calcification did not change, indicating there was no widespread effect of Ωar changes on coral calcification rate in the MBR. Even in the absence of ocean acidification, differential reductions in calcification between Porites spp. and Montastraea spp. associated with warming might be expected to have significant ecological repercussions. For instance, Porites spp. invest increased calcification in extension, and under warming scenarios it may reduce their ability to compete for space. As a consequence, shifts in taxonomic composition would be expected in Indo-Pacific reefs with uncertain repercussions for biodiversity. By contrast, Montastraea spp. use their increased calcification resources to construct denser skeletons. Reductions in calcification would therefore make them more susceptible to both physical and biological breakdown, seriously affecting ecosystem function in Atlantic reefs

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented