High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today's annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic models. To overcome these problems, we have developed the PlantSEED, an integrated, metabolism-centric database to support subsystems-based annotation and metabolic model reconstruction for plant genomes. PlantSEED combines SEED subsystems technology, first developed for microbial genomes, with refined protein families and biochemical data to assign fully consistent functional annotations to orthologous genes, particularly those encoding primary metabolic pathways. Seamless integration with its parent, the prokaryotic SEED database, makes PlantSEED a unique environment for cross-kingdom comparative analysis of plant and bacterial genomes. The consistent annotations imposed by PlantSEED permit rapid reconstruction and modeling of primary metabolism for all plant genomes in the database. This feature opens the unique possibility of model-based assessment of the completeness and accuracy of gene annotation and thus allows computational identification of genes and pathways that are restricted to certain genomes or need better curation. We demonstrate the PlantSEED system by producing consistent annotations for 10 reference genomes. We also produce a functioning metabolic model for each genome, gapfilling to identify missing annotations and proposing gene candidates for missing annotations. Models are built around an extended biomass composition representing the most comprehensive published to date. To our knowledge, our models are the first to be published for seven of the genomes analyzed

Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes

Relation between viral fitness and immune escape within the hepatitis C virus protease

BACKGROUND: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non‐structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. AIMS: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073–1081 of the NS3 protease was limited by viral fitness. PATIENTS: Venous blood was obtained from six patients (four HLA‐A2+) with chronic HCV infection and from one HLA‐A2+ patient with acute HCV infection. METHODS: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA‐A2‐restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA‐A2‐transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. RESULTS: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073–1081 epitopes that were generally recognised by cross reactive human and murine HLA‐A2 restricted CTLs. Introduction of mutations at five positions of the 1073–1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. CONCLUSIONS: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans

Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5D mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes

VCAM-1 targeted alpha-particle therapy for early brain metastases

Background Brain metastases (BM) develop frequently in patients with breast cancer. Despite the use of external beam radiotherapy (EBRT), the average overall survival is short (6 months from diagnosis). The therapeutic challenge is to deliver molecularly targeted therapy at an early stage when relatively few metastatic tumor cells have invaded the brain. Vascular cell adhesion molecule 1 (VCAM-1), overexpressed by nearby endothelial cells during the early stages of BM development, is a promising target. The aim of this study was to investigate the therapeutic value of targeted alpha-particle radiotherapy, combining lead-212 (212Pb) with an anti–VCAM-1 antibody (212Pb-αVCAM-1). Methods Human breast carcinoma cells that metastasize to the brain, MDA-231-Br-GFP, were injected into the left cardiac ventricle of nude mice. Twenty-one days after injection, 212Pb-αVCAM-1 uptake in early BM was determined in a biodistribution study and systemic/brain toxicity was evaluated. Therapeutic efficacy was assessed using MR imaging and histology. Overall survival after 212Pb-αVCAM-1 treatment was compared with that observed after standard EBRT. Results 212Pb-αVCAM-1 was taken up into early BM with a tumor/healthy brain dose deposition ratio of 6 (5.52e108 and 0.92e108) disintegrations per gram of BM and healthy tissue, respectively. MRI analyses showed a statistically significant reduction in metastatic burden after 212Pb-αVCAM-1 treatment compared with EBRT (P &lt; 0.001), translating to an increase in overall survival of 29% at 40 days post prescription (P &lt; 0.01). No major toxicity was observed. Conclusions The present investigation demonstrates that 212Pb-αVCAM-1 specifically accumulates at sites of early BM causing tumor growth inhibition.</p

Early brain metastases treatment using VCAM-1 targeted alpha-particle therapy

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