A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of the kidney and in the trafficking and signaling of growth factor receptors in the brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway, consistent with the predominant localization of its preferred substrates, PI(4,5)P(2) and PI(3,4,5)P(3), at the cell surface.     

White adipose tissue reference network: a knowledge resource for exploring health-relevant relations

Optimal health is maintained by interaction of multiple intrinsic and environmental factors at different levels of complexity—from molecular, to physiological, to social. Understanding and quantification of these interactions will aid design of successful health interventions. We introduce the reference network concept as a platform for multi-level exploration of biological relations relevant for metabolic health, by integration and mining of biological interactions derived from public resources and context-specific experimental data. A White Adipose Tissue Health Reference Network (WATRefNet) was constructed as a resource for discovery and prioritization of mechanism-based biomarkers for white adipose tissue (WAT) health status and the effect of food and drug compounds on WAT health status. The WATRefNet (6,797 nodes and 32,171 edges) is based on (1) experimental data obtained from 10 studies addressing different adiposity states, (2) seven public knowledge bases of molecular interactions, (3) expert’s definitions of five physiologically relevant processes key to WAT health, namely WAT expandability, Oxidative capacity, Metabolic state, Oxidative stress and Tissue inflammation, and (4) a collection of relevant biomarkers of these processes identified by BIOCLAIMS (http://bioclaims.uib.es). The WATRefNet comprehends multiple layers of biological complexity as it contains various types of nodes and edges that represent different biological levels and interactions. We have validated the reference network by showing overrepresentation with anti-obesity drug targets, pathology-associated genes and differentially expressed genes from an external disease model dataset. The resulting network has been used to extract subnetworks specific to the above-mentioned expert-defined physiological processes. Each of these process-specific signatures represents a mechanistically supported composite biomarker for assessing and quantifying the effect of interventions on a physiological aspect that determines WAT health status. Following this principle, five anti-diabetic drug interventions and one diet intervention were scored for the match of their expression signature to the five biomarker signatures derived from the WATRefNet. This confirmed previous observations of successful intervention by dietary lifestyle and revealed WAT-specific effects of drug interventions. The WATRefNet represents a sustainable knowledge resource for extraction of relevant relationships such as mechanisms of action, nutrient intervention targets and biomarkers and for assessment of health effects for support of health claims made on food products.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0439-x) contains supplementary material, which is available to authorized users.     

Guidelines for evaluating performance of oyster habitat restoration

Restoration of degraded ecosystems is an important societal goal, yet inadequate monitoring and the absence of clear performance metrics are common criticisms of many habitat restoration projects. Funding limitations can prevent adequate monitoring, but we suggest that the lack of accepted metrics to address the diversity of restoration objectives also presents a serious challenge to the monitoring of restoration projects. A working group with experience in designing and monitoring oyster reef projects was used to develop standardized monitoring metrics, units, and performance criteria that would allow for comparison among restoration sites and projects of various construction types. A set of four universal metrics (reef areal dimensions, reef height, oyster density, and oyster size–frequency distribution) and a set of three universal environmental variables (water temperature, salinity, and dissolved oxygen) are recommended to be monitored for all oyster habitat restoration projects regardless of their goal(s). In addition, restoration goal‐based metrics specific to four commonly cited ecosystem service‐based restoration goals are recommended, along with an optional set of seven supplemental ancillary metrics that could provide information useful to the interpretation of prerestoration and postrestoration monitoring data. Widespread adoption of a common set of metrics with standardized techniques and units to assess well‐defined goals not only allows practitioners to gauge the performance of their own projects but also allows for comparison among projects, which is both essential to the advancement of the field of oyster restoration and can provide new knowledge about the structure and ecological function of oyster reef ecosystems.     

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.     

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.     

HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy

HMGA proteins are not translated in normal human somatic cells, but are present in high copy numbers in pluripotent embryonic stem cells and most neoplasias. Correlations between the degree of malignancy, patient prognostic index and HMGA levels have been firmly established. Intriguingly, HMGA2 is also found in rare tumor-inducing cells which are resistant to chemotherapy. Here, we demonstrate that HMGA1a/b and HMGA2 possess intrinsic dRP and AP site cleavage activities, and that lysines and arginines in the AT-hook DNA-binding domains function as nucleophiles. We also show that HMGA2 can be covalently trapped at genomic abasic sites in cancer cells. By employing a variety of cell-based assays, we provide evidence that the associated lyase activities promote cellular resistance against DNA damage that is targeted by base excision repair (BER) pathways, and that this protection directly correlates with the level of HMGA2 expression. In addition, we demonstrate an interaction between human AP endonuclease 1 and HMGA2 in cancer cells, which supports our conclusion that HMGA2 can be incorporated into the cellular BER machinery. Our study thus identifies an unexpected role for HMGA2 in DNA repair in cancer cells which has important clinical implications for disease diagnosis and therapy.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Improved approach for transferring and cultivating Methanosarcina acetivorans C2A (DSM 2834)

Aim:  A method for cultivating Methanosarcina acetivorans was further developed to handle these anaerobic archaea without special equipment such as an anaerobic chamber.
Methods and Results:  Medium was filtered and oxygen removed under a nitrogen gas-phase. A dithiothreitol-filled syringe was used to transfer cells from high density grown cultures to new medium. Growth time and cell mass were determined, as well as cell viability was proven by light microscopy.
Conclusion:  Cell transfer and growth was successful using this approach.
Significance and Impact of the Study:  This updated technique allows almost every laboratory the opportunity to grow these methanogenic organisms for further studies. The described method could be used for proteomic analysis and is also interesting for further protein structure determination.     

Characterisation of Phenolics in Flor‐Essence®—a Compound Herbal product and its Contributing Herbs

Introduction – Commercially available herbal mixture FE, a proprietary natural health product manufactured by Flora Manufacturing and Distributing Ltd (Flora), is a unique North American traditional herbal product. FE is a chemically complex mixture of eight herbs and has not been subjected to phytochemical analysis. Objective – To develop analytical methods to undertake detailed phytochemical analyses of FE, and its eight contributing herbs, including burdock (Arctium lappa L.), sheep sorrel (Rumex acetosella L.), Turkish rhubarb (Rheum palmatum L.), slippery elm Muhl. (Ulmus rubra), watercress (Nasturtium officinale R. Br.), red clover (Trifolium pratense L.), blessed thistle (Cnicus benedictus L.) and kelp (Laminaria digitata Lmx.). Methodology – The identification was undertaken by a combination of reversed phase high performance liquid chromatography–diode array detection–atmospheric pressure chemical ionisation–mass selective detection (RP‐HPLC‐DAD‐APCI‐MSD) analysis and phenolics metabolomic library matching. Results – New separation methods facilitated the identification of 43 markers in the individual herbs which constitute FE. Sixteen markers could be identified in FE originating from four contributing herbs including four caffeoyl quinic acids, three dicaffeoyl quinic acids and two caffeic acid derivatives from A. lappa, luteolin‐7‐O‐glucoside, luteolin, five apigenin glycosides and apigenin from R. acetocella and N. officinale and sissostrin from T. pretense. A validated method for quantitative determination of three markers is reported with good intraday, interday and interoperator repeatability using a reliable alcohol based extraction technique. Conclusion – FE and its contributing herbs predominantly contain phenolics. This methodology can be applied to further develop full‐scale validation of this product. Copyright © 2009 John Wiley & Sons, Ltd.