Not just fat: investigating the proteome of cetacean blubber tissue

Mammalian adipose tissue is increasingly being recognized as an endocrine organ involved in the regulation of a number of metabolic processes and pathways. It responds to signals from different hormone systems and the central nervous system, and expresses a variety of protein factors with important paracrine and endocrine functions. This study presents a first step towards the systematic analysis of the protein content of cetacean adipose tissue, the blubber, in order to investigate the kinds of proteins present and their relative abundance. Full depth blubber subsamples were collected from dead-stranded harbour porpoises (Phocoena phocoena) (n = 21). Three total protein extraction methods were trialled, and the highest total protein yields with the lowest extraction variability were achieved using a RIPA cell lysis and extraction buffer based protocol. Extracted proteins were separated using 1D Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry (nLC-ESI–MS/MS). A range of proteins were identified (n = 295) and classed into eight functional groups, the most abundant of which were involved in cell function and metabolism (45%), immune response and inflammation (15%) and lipid metabolism (11%). These proteins likely originate both from the various cell types within the blubber tissue itself, and from the circulation. They therefore have the potential to capture information on the cellular and physiological stresses experienced by individuals at the time of sampling. The importance of this proteomic approach is two-fold: Firstly, it could help to assign novel functions to marine mammal blubber in keeping with current understanding of the multi-functional role of adipose tissue in other mammals. Secondly, it could lead to the development of a suite of biomarkers to better monitor the physiological state and health of live individuals though remote blubber biopsy sampling

Overview of the Large Hadron Collider cryo-magnets logistics

More than 1700 superconducting cryo-magnets have to be installed in the Large Hadron Collider tunnel. The long, heavy and fragile LHC cryo-magnets are difficult to handle and transport in particular in the LEP tunnel environment originally designed for smaller, lighter LEP magnets. An installation rate of more than 20 cryo-magnets per week is needed to cope with the foreseen LHC installation end date. The paper gives an overview of the transport and installation sequence complexity, from the storage area at the surface to the cryo-magnet final position in the tunnel. The success of this task depends on a series of independent factors that have to be considered at the same time. The equipment needed for the transport and tunnel installation of the LHC cryo-magnets is briefly described. The manpower and equipment organisation as well as the challenges of logistics are then detailed. The paper includes conclusions and some of the lessons learned during the first phase of the LHC cryo-magnets installation

Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

BackgroundT cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s.MethodsPatients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 1010 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72.ResultsFourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72 binding domain of humanized CC49, reflecting an anti-CAR immune response. No radiologic tumor responses were observed.ConclusionThese findings demonstrate the relative safety of CART72 cells. The limited persistence supports the incorporation of co-stimulatory domains in the CAR design and the use of fully human CAR constructs to mitigate immunogenicity

Glycogen metabolic genes are involved in trehalose-6-phosphate synthase-mediated regulation of pathogenicity by the rice blast fungus Magnaporthe oryzae.

© 2013 Badaruddin et al.Editor - Peter N. Dodds, Commonwealth Scientific and Industrial Research Organisation (CSIRO), AustraliaThis work was funded by the Biotechnology and Biological Sciences Research Council and a European Research Council Advanced Investigator Award to NJT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a Δagl1Δgph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae.Biotechnology and Biological Sciences Research Council (BBSRC)European Research Council (ERC

Patterns of genetic diversity in southern and southeastern Araucaria angustifolia (Bert.) O. Kuntze relict populations

Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five “genetic clusters” (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three “genetic clusters”. These results are in agreement with the pattern of geographic distribution of the studied populations

High-resolution haplotype block structure in the cattle genome

<p>Abstract</p> <p>Background</p> <p>The Bovine HapMap Consortium has generated assay panels to genotype ~30,000 single nucleotide polymorphisms (SNPs) from 501 animals sampled from 19 worldwide taurine and indicine breeds, plus two outgroup species (Anoa and Water Buffalo). Within the larger set of SNPs we targeted 101 high density regions spanning up to 7.6 Mb with an average density of approximately one SNP per 4 kb, and characterized the linkage disequilibrium (LD) and haplotype block structure within individual breeds and groups of breeds in relation to their geographic origin and use.</p> <p>Results</p> <p>From the 101 targeted high-density regions on bovine chromosomes 6, 14, and 25, between 57 and 95% of the SNPs were informative in the individual breeds. The regions of high LD extend up to ~100 kb and the size of haplotype blocks ranges between 30 bases and 75 kb (10.3 kb average). On the scale from 1–100 kb the extent of LD and haplotype block structure in cattle has high similarity to humans. The estimation of effective population sizes over the previous 10,000 generations conforms to two main events in cattle history: the initiation of cattle domestication (~12,000 years ago), and the intensification of population isolation and current population bottleneck that breeds have experienced worldwide within the last ~700 years. Haplotype block density correlation, block boundary discordances, and haplotype sharing analyses were consistent in revealing unexpected similarities between some beef and dairy breeds, making them non-differentiable. Clustering techniques permitted grouping of breeds into different clades given their similarities and dissimilarities in genetic structure.</p> <p>Conclusion</p> <p>This work presents the first high-resolution analysis of haplotype block structure in worldwide cattle samples. Several novel results were obtained. First, cattle and human share a high similarity in LD and haplotype block structure on the scale of 1–100 kb. Second, unexpected similarities in haplotype block structure between dairy and beef breeds make them non-differentiable. Finally, our findings suggest that ~30,000 uniformly distributed SNPs would be necessary to construct a complete genome LD map in <it>Bos taurus </it>breeds, and ~580,000 SNPs would be necessary to characterize the haplotype block structure across the complete cattle genome.</p

Permian-Triassic boundary microbialites (PTBMs) in soutwest China: implications for paleoenvironment reconstruction

Permian–Triassic boundary microbialites (PTBMs) are commonly interpreted to be a sedimentary response to upwelling of anoxic alkaline seawater and indicate a harsh marine environment in the Permian–Triassic transition. However, recent studies propose that PTBMs may instead be developed in an oxic environment, therefore necessitating the need to reassess the paleoenvironment of formation of PTBMs. This paper is an integrated study of the PTBM sequence at Yudongzi, northwest Sichuan Basin, which is one of the thickest units of PTBMs in south China. Analysis of conodont biostratigraphy, mega- to microscopic microbialite structures, stratigraphic variations in abundance and size of metazoan fossils, and total organic carbon (TOC) and total sulfur (TS) contents within the PTBM reveals the following results: (1) the microbialites occur mainly in the Hindeodus parvus Zone but may cross the Permian–Triassic boundary, and are comprised of, from bottom to top: lamellar thrombolites, dendritic thrombolites and lamellar-reticular thrombolites; (2) most metazoan fossils of the microbialite succession increase in abundance upsection, so does the sizes of bivalve and brachiopod fossils; (3) TOC and TS values of microbialites account respectively for 0.07 and 0.31 wt% on average, both of which are very low. The combination of increase in abundance and size of metazoan fossils upsection, together with the low TOC and TS contents, is evidence that the Yudongzi PTBMs developed in oxic seawater. We thus dispute the previous view, at least for the Chinese sequences, of low-oxygen seawater for microbialite growth, and question whether it is now appropriate to associate PTBMs with anoxic, harsh environments associated with the end-Permian extinction. Instead, we interpret those conditions as fully oxygenated.13th Five-Year Plan National Scientific and Technology Major Project (2016ZX05004002-001); National Natural Science Foundation of China (41602166)

Two Plant Bacteria, S. meliloti and Ca. Liberibacter asiaticus, Share Functional znuABC Homologues That Encode for a High Affinity Zinc Uptake System

The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria

Age-Related Changes in the Epithelial and Stromal Compartments of the Mammary Gland in Normocalcemic Mice Lacking the Vitamin D3 Receptor

The vitamin D3 receptor (VDR) serves as a negative growth regulator during mammary gland development via suppression of branching morphogenesis during puberty and modulation of differentiation and apoptosis during pregnancy, lactation and involution. To assess the role of the VDR in the aging mammary gland, we utilized 12, 14, and 16 month old VDR knockout (KO) and wild type (WT) mice for assessment of integrity of the epithelial and stromal compartments, steroid hormone levels and signaling pathways. Our data indicate that VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary ductal branches and atrophy of the mammary fat pad. In association with loss of the white adipose tissue compartment, smooth muscle actin staining is increased in glands from VDR KO mice, suggesting a change in the stromal microenviroment. Activation of caspase-3 and increased Bax expression in mammary tissue of VDR KO mice suggests that enhanced apoptosis may contribute to loss of ductal branching. These morphological changes in the glands of VDR KO mice are associated with ovarian failure and reduced serum 17β-estradiol. VDR KO mice also exhibit progressive loss of adipose tissue stores, hypoleptinemia and increased metabolic rate with age. These developmental studies indicate that, under normocalcemic conditions, loss of VDR signaling is associated with age-related estrogen deficiency, disruption of epithelial ductal branching, abnormal energy expenditure and atrophy of the mammary adipose compartment