Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide−alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc

Upper crustal structure and axial topography at intermediate spreading ridges : seismic constraints from the southern Juan de Fuca Ridge

Author Posting. © American Geophysical Union, 2005. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 110 (2005): B12104, doi:10.1029/2005JB003630.We use multichannel seismic reflection data to image the upper crustal structure of 0-620 ka crust along the southern Juan de Fuca Ridge (JdFR). The study area comprises two segments spreading at intermediate rate with an axial high morphology with narrow (Cleft) and wide (Vance) axial summit grabens (ASG). Along most of the axis of both segments we image the top of an axial magma chamber (AMC). The AMC along Cleft deepens from south to north, from 2.0 km beneath the RIDGE Cleft Observatory and hydrothermal vents near the southern end of the segment, to 2.3 km at the northern end near the site of the 1980’s eruptive event. Along the Vance segment, the AMC also deepens from south to north, from 2.4 km to 2.7 km. Seismic layer 2A, interpreted as the basaltic extrusive layer, is 250-300 m thick at the ridge axis along the Cleft segment, and 300-350 m thick along the axis of the Vance segment. However off-axis layer 2A is similar in both segments (500-600 m), indicating ~90% and ~60% off-axis thickening at the Cleft and Vance segments, respectively. Half of the thickening occurs sharply at the walls of the ASG, with the remaining thickening occurring within 3-4 km of the ASG. Along the full length of both segments, layer 2A is thinner within the ASG, compared to the ridge flanks. Previous studies argued that the ASG is a cyclic feature formed by alternating periods of magmatism and tectonic extension. Our observations agree with the evolving nature of the ASG. However, we suggest that its evolution is related to large changes in axial morphology produced by small fluctuations in magma supply. Thus the ASG, rather than being formed by excess volcanism, is a rifted flexural axial high. The changes in axial morphology affect the distribution of lava flows along the ridge flanks, as indicated by the pattern of layer 2A thickness. The fluctuations in magma supply may occur at all spreading rates, but its effects on crustal structure and axial morphology are most pronounced along intermediate spreading rate ridges.This study was supported by the National Science Foundation grants OCE-0002551 to Woods Hole Oceanographic Institution, OCE-0002488 to Lamont-Doherty Earth Observatory, and OCE-0002600 to Scripps Institution of Oceanography

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage

Background: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.Results: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.Conclusions: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders

Towards the ecological intensification of european aquaculture: the GAIN project

Aquaculture Europe, Berlin, 7-10 October 2019The Green Aquaculture INtensification in Europe (GAIN) project is a 42 month long collaborative research programme funded by the European Union (EU), through Horizon 2020 (Grant Agreement 773330) (www.unive.it/gainh2020eu). GAIN started in May 2018 and is designed to support the ecological intensification of aquaculture in the EU and European Economic Area (EEA), with the dual objectives of increasing production and competitiveness of the industry, while ensuring sustainability and compliance with EU regulations on food safety and environment. Eco-intensification of European aquaculture is a challenge that requires the integration of scientific and technical innovations, new policies and economic instruments, as well as addressing social considerations, in order to promote the implementation of the principles of circular economy in aquaculturePeer reviewe