Differential Proteomic Analysis of Mammalian Tissues Using SILAM

Differential expression of proteins between tissues underlies organ-specific functions. Under certain pathological conditions, this may also lead to tissue vulnerability. Furthermore, post-translational modifications exist between different cell types and pathological conditions. We employed SILAM (Stable Isotope Labeling in Mammals) combined with mass spectrometry to quantify the proteome between mammalian tissues. Using 15N labeled rat tissue, we quantified 3742 phosphorylated peptides in nuclear extracts from liver and brain tissue. Analysis of the phosphorylation sites revealed tissue specific kinase motifs. Although these tissues are quite different in their composition and function, more than 500 protein identifications were common to both tissues. Specifically, we identified an up-regulation in the brain of the phosphoprotein, ZFHX1B, in which a genetic deletion causes the neurological disorder Mowat–Wilson syndrome. Finally, pathway analysis revealed distinct nuclear pathways enriched in each tissue. Our findings provide a valuable resource as a starting point for further understanding of tissue specific gene regulation and demonstrate SILAM as a useful strategy for the differential proteomic analysis of mammalian tissues

Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae) Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids

The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively) to identify larvae (n = 188) collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology- based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei), albacore (Thunnus alalunga) and little tunny (Euthynnus alletteratus). We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.Versión del editor4,411

A framework for conceptualizing, representing, and analyzing distributed interaction.

The relationship between interaction and learning is a central concern of the learning sciences, and analysis of interaction has emerged as a major theme within the current literature on computersupported collaborative learning. The nature of technology-mediated interaction poses analytic challenges. Interaction may be distributed across actors, space, and time, and vary from synchronous, quasi-synchronous, and asynchronous, even within one data set. Often multiple media are involved and the data comes in a variety of formats. As a consequence, there are multiple analytic artifacts to inspect and the interaction may not be apparent upon inspection, being distributed across these artifacts. To address these problems as they were encountered in several studies in our own laboratory, we developed a framework for conceptualizing and representing distributed interaction. The framework assumes an analytic concern with uncovering or characterizing the organization of interaction in sequential records of events. The framework includes a media independent characterization of the most fundamental unit of interaction, which we call uptake. Uptake is present when a participant takes aspects of prior events as having relevance for ongoing activity. Uptake can be refined into interactional relationships of argumentation, information sharing, transactivity, and so forth. for specific analytic objectives. Faced with the myriad of ways in which uptake can manifest in practice, we represent data using graphs of relationships between events that capture the potential ways in which one act can be contingent upon another. These contingency graphs serve as abstract transcripts that document in one representation interaction that is distributed across multiple media. This paper summarizes the requirements that motivate the framework, and discusses the theoretical foundations on which it is based. It then presents the framework and its application in detail, with examples from our work to illustrate how we have used it to support both ideographic and nomothetic research, using qualitative and quantitative methods. The paper concludes with a discussion of the framework’s potential role in supporting dialogue between various analytic concerns and methods represented in CSCL

TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8 + T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor β (TGFβ) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8 + T cells from the tumour parenchyma that were instead found in the fibroblast-and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-Administration of TGFβ-blocking and anti-PD-L1 antibodies reduced TGFβ signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-Tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFβ shapes the tumour microenvironment to restrain anti-Tumour immunity by restricting T-cell infiltration

Misidentification of bluefin tuna larvae: a call for caution and taxonomic reform

The international effort to prevent the collapse of Atlantic bluefin tuna (BFT, Thunnus thynnus, Scombridae) stocks exemplifies the challenges associated with modern marine resource conservation. Rampant mismanagement, under-reporting and illegal, unreported and unregulated fishing led to decades of over-exploitation in the BFT fishery. Surveys of larval abundance in the Gulf of Mexico and the Mediterranean Sea have been used as a proxy for both spawning biomass and recruitment by researchers working to improve estimates of stock abundance. Recent genetic barcoding studies have revealed that species identification errors are common among larvae surveys that use morphology-based taxonomy alone. Misidentification of larvae can lead to uncertainty about the spatial distribution of a species, confusion over life history traits and population dynamics, and potentially disguise the collapse or recovery of localized spawning sites. In an effort to identify the source of these errors, we review several weaknesses in modern morphology-based taxonomy including demographic decline of expert taxonomists, flawed identification keys, reluctance of the taxonomic community to embrace advances in digital communications and a general scarcity of modern user-friendly materials. Recent advances in molecular techniques useful for specimen identification and population studies are discussed at length. We advocate a more constructive integration of morphology-based taxonomy and barcoding in order to add confidence to larval surveys and to strengthen associated fisheries managementVersión del editor2,270