Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of approximately 84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.     

A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome

The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.     

Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.     

Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking

Background

Age-related macular degeneration (AMD) is the major cause of blindness in the elderly. Those with the neovascular end-stage of disease have irreversible loss of central vision. AMD is a complex disorder in which genetic and environmental factors play a role. Polymorphisms in the complement factor H (CFH) gene, LOC387715, and the HTRA1 promoter are strongly associated with AMD. Smoking also contributes to the etiology. We aimed to provide a model of disease risk based on these factors.

Methods and Findings

We genotyped polymorphisms in CFH and LOC387715/HTRA1 in a case–control study of 401 patients with neovascular AMD and 266 controls without signs of disease, and used the data to produce genetic risk scores for the European-descent population based on haplotypes at these loci and smoking history. CFH and LOC387715/HTRA1 haplotypes and smoking status exerted large effects on AMD susceptibility, enabling risk scores to be generated with appropriate weighting of these three factors. Five common haplotypes of CFH conferred a range of odds ratios (ORs) per copy from 1 to 4.17. Most of the effect of LOC387715/HTRA1 was mediated through one detrimental haplotype (carriage of one copy: OR 2.83; 95% confidence interval [CI] 1.91–4.20), with homozygotes being at particularly high risk (OR 32.83; 95% CI 12.53–86.07). Patients with neovascular macular degeneration had considerably higher scores than those without disease, and risk of blinding AMD rose to 15.5% in the tenth of the population with highest predicted risk.

Conclusions

An individual''s risk of developing AMD in old age can be predicted by combining haplotype data with smoking status. Until there is effective treatment for AMD, encouragement to avoid smoking in those at high genetic risk may be the best option. We estimate that total absence of smoking would have reduced the prevalence of severe AMD by 33%. Unless smoking habits change or preventative treatment becomes available, the prevalence of AMD will rise as a consequence of the increasing longevity of the population.     

Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium

2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.     

Classification and nomenclature of all human homeobox genes

Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.     

Long-term changes in temperate marine fish assemblages are driven by a small subset of species

The species composition of plant and animal assemblages across the globe has changed substantially over the past century. How do the dynamics of individual species cause this change? We classified species into seven unique categories of temporal dynamics based on the ordered sequence of presences and absences that each species contributes to an assemblage time series. We applied this framework to 14,434 species trajectories comprising 280 assemblages of temperate marine fishes surveyed annually for 20 or more years. Although 90% of the assemblages diverged in species composition from the baseline year, this compositional change was largely driven by only 8% of the species' trajectories. Quantifying the reorganization of assemblages based on species shared temporal dynamics should facilitate the task of monitoring and restoring biodiversity. We suggest ways in which our framework could provide informative measures of compositional change, as well as leverage future research on pattern and process in ecological systems.