Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.     

Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA^Gly, integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.     

GenomeFingerprinter: The Genome Fingerprint and the Universal Genome Fingerprint Analysis for Systematic Comparative Genomics

Background

No attention has been paid on comparing a set of genome sequences crossing genetic components and biological categories with far divergence over large size range. We define it as the systematic comparative genomics and aim to develop the methodology.

Results

First, we create a method, GenomeFingerprinter, to unambiguously produce a set of three-dimensional coordinates from a sequence, followed by one three-dimensional plot and six two-dimensional trajectory projections, to illustrate the genome fingerprint of a given genome sequence. Second, we develop a set of concepts and tools, and thereby establish a method called the universal genome fingerprint analysis (UGFA). Particularly, we define the total genetic component configuration (TGCC) (including chromosome, plasmid, and phage) for describing a strain as a systematic unit, the universal genome fingerprint map (UGFM) of TGCC for differentiating strains as a universal system, and the systematic comparative genomics (SCG) for comparing a set of genomes crossing genetic components and biological categories. Third, we construct a method of quantitative analysis to compare two genomes by using the outcome dataset of genome fingerprint analysis. Specifically, we define the geometric center and its geometric mean for a given genome fingerprint map, followed by the Euclidean distance, the differentiate rate, and the weighted differentiate rate to quantitatively describe the difference between two genomes of comparison. Moreover, we demonstrate the applications through case studies on various genome sequences, giving tremendous insights into the critical issues in microbial genomics and taxonomy.

Conclusions

We have created a method, GenomeFingerprinter, for rapidly computing, geometrically visualizing, intuitively comparing a set of genomes at genome fingerprint level, and hence established a method called the universal genome fingerprint analysis, as well as developed a method of quantitative analysis of the outcome dataset. These have set up the methodology of systematic comparative genomics based on the genome fingerprint analysis.     

Beegle: from literature mining to disease-gene discovery

Disease-gene identification is a challenging process that has multiple applications within functional genomics and personalized medicine. Typically, this process involves both finding genes known to be associated with the disease (through literature search) and carrying out preliminary experiments or screens (e.g. linkage or association studies, copy number analyses, expression profiling) to determine a set of promising candidates for experimental validation. This requires extensive time and monetary resources. We describe Beegle, an online search and discovery engine that attempts to simplify this process by automating the typical approaches. It starts by mining the literature to quickly extract a set of genes known to be linked with a given query, then it integrates the learning methodology of Endeavour (a gene prioritization tool) to train a genomic model and rank a set of candidate genes to generate novel hypotheses. In a realistic evaluation setup, Beegle has an average recall of 84% in the top 100 returned genes as a search engine, which improves the discovery engine by 12.6% in the top 5% prioritized genes. Beegle is publicly available at http://beegle.esat.kuleuven.be/.     

Salmonella paratyphi C: Genetic Divergence from Salmonella choleraesuis and Pathogenic Convergence with Salmonella typhi

Background

Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved.

Methodologies/Principal Findings

We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis.

Conclusions

S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

Description of third instar larvae of Ceratitis fasciventris,C. anonae,C. rosa (FAR complex) and C. capitata (Diptera,Tephritidae)

Third instar larvae of members of the Ceratitis FAR complex, including Ceratitisfasciventris (Bezzi), Ceratitisanonae Graham, and Ceratitisrosa Karsch are described and compared with those of Ceratitiscapitata (Wiedemann). Diagnostic characters, such as presence vs. absence of a secondary tooth on the mandibles, previously used to separate Ceratitiscapitata from Ceratitisrosa, are shown to vary in each species. Significant variation in diagnostic morphological characters among populations of Ceratitisrosa from east and south Africa is documented; however, the differences are not simply congruent with the R1 and R2 designations based on other studies. Quantitative measures of numerous morphological characters are consistently smaller in the larvae of Ceratitisfasciventris and distinguish them from other species of the FAR complex. Larvae of Ceratitiscapitata can be distinguished from those of the FAR complex by characters such as absence of accessory plates of the oral ridges, the shape of the anterior spiracle, and the pattern of dorsal spinules. Previous studies indicated that absence of accessory lobes separate the genus Ceratitis from Bactrocera, but this is shown to be incorrect, as accessory lobes are in fact present in several species of Ceratitis.     

Yy1 Gene Dosage Effect and Bi-Allelic Expression of Peg3

In the current study, we tested the in vivo effects of Yy1 gene dosage on the Peg3 imprinted domain with various breeding schemes utilizing two sets of mutant alleles. The results indicated that a half dosage of Yy1 coincides with the up-regulation of Peg3 and Zim1, suggesting a repressor role of Yy1 in this imprinted domain. This repressor role of Yy1 is consistent with the observations derived from previous in vitro studies. The current study also provided an unexpected observation that the maternal allele of Peg3 is also normally expressed, and thus the expression of Peg3 is bi-allelic in the specific areas of the brain, including the choroid plexus, the PVN (Paraventricular Nucleus) and the SON (Supraoptic Nucleus) of the hypothalamus. The exact roles of the maternal allele of Peg3 in these cell types are currently unknown, but this new finding confirms the previous prediction that the maternal allele may be functional in specific cell types based on the lethality associated with the homozygotes for several mutant alleles of the Peg3 locus. Overall, these results confirm the repressor role of Yy1 in the Peg3 domain and also provide a new insight regarding the bi-allelic expression of Peg3 in mouse brain.     

Complete Genome Sequences of Elephant Endotheliotropic Herpesviruses 1A and 1B Determined Directly from Fatal Cases

A highly lethal hemorrhagic disease associated with infection by elephant endotheliotropic herpesvirus (EEHV) poses a severe threat to Asian elephant husbandry. We have used high-throughput methods to sequence the genomes of the two genotypes that are involved in most fatalities, namely, EEHV1A and EEHV1B (species Elephantid herpesvirus 1, genus Proboscivirus, subfamily Betaherpesvirinae, family Herpesviridae). The sequences were determined from postmortem tissue samples, despite the data containing tiny proportions of viral reads among reads from a host for which the genome sequence was not available. The EEHV1A genome is 180,421 bp in size and consists of a unique sequence (174,601 bp) flanked by a terminal direct repeat (2,910 bp). The genome contains 116 predicted protein-coding genes, of which six are fragmented, and seven paralogous gene families are present. The EEHV1B genome is very similar to that of EEHV1A in structure, size, and gene layout. Half of the EEHV1A genes lack orthologs in other members of subfamily Betaherpesvirinae, such as human cytomegalovirus (genus Cytomegalovirus) and human herpesvirus 6A (genus Roseolovirus). Notable among these are 23 genes encoding type 3 membrane proteins containing seven transmembrane domains (the 7TM family) and seven genes encoding related type 2 membrane proteins (the EE50 family). The EE50 family appears to be under intense evolutionary selection, as it is highly diverged between the two genotypes, exhibits evidence of sequence duplications or deletions, and contains several fragmented genes. The availability of the genome sequences will facilitate future research on the epidemiology, pathogenesis, diagnosis, and treatment of EEHV-associated disease.     

发现微生物药物的新途径:从基因到新化合物

本文介绍一种基于基因组扫描(鸟枪法)和生物信息学相结合的寻找微生物新化合物的平台.基因组扫描发现新的合成基因,通过对新的次生代谢产物合成途径基因的分析,预测化合物的结构、物理化学性质参数,指导该化合物的分离与纯化.     

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B''-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.     

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.     

IL-10 Gene Polymorphisms and Susceptibility to Systemic Lupus Erythematosus: A Meta-Analysis

Background

A number of observational studies have been conducted to investigate the association of the IL-10 gene polymorphisms with systemic lupus erythematosus (SLE) susceptibility. However, their results are conflicting.

Method

We searched published case-control studies on the IL-10 polymorphisms and SLE in PubMed, EMBASE and Chinese Biomedical Literature Database. A meta-analysis was conducted using a fixed-effect or random-effect model based on between-study heterogeneity.

Results

A total of 42 studies with 7948 cases and 11866 controls were included in this meta-analysis. Among Caucasians, the CA27 allele of the IL10.G microsatellites (OR 2.38, 95% CI 1.01–5.62), the G allele of the IL-10 -1082G/A polymorphism (G vs. A: OR 1.21, 95% CI 1.02–1.44; GG vs. AA: OR 1.45, 95% CI 1.16–1.82; GG+GA vs. AA: OR 1.16, 95% CI 1.03–1.29) and its associated haplotype -1082G/−819C/−592C (OR 1.25, 95% CI 1.10–1.42) were associated with increased SLE susceptibility without or with unimportant between-study heterogeneity. Removing studies deviating from Hardy-Weinberg equilibrium (HWE) hardly changed these results. Among Asians, the CA21 allele of the IL-10.G microsatellites (OR 1.28, 95% CI 1.02–1.60) and the -1082G/−819C/−592C haplotype (OR 1.24, 95% CI 1.00–1.53) were associated with increased SLE susceptibility, but with substantial between-study heterogeneity or sensitive to HWE status. Removing studies deviating from HWE also produced statistically significant associations of the IL-10 -1082G/A (GG vs. AA: OR 3.21, 95% CI 1.24–8.28; GG vs. AA+GA: OR 2.85, 95% CI 1.19–6.79) and -592C/A polymorphisms (CC+CA vs. AA: OR 0.69, 95% CI 0.51–0.94) with SLE among Asians.

Conclusion

This meta-analysis showed that the IL10.G microsatellites, the IL-10 -1082G/A and -592C/A polymorphisms and the haplotype -1082G/−819C/−592C are associated with SLE susceptibility. Besides, this is the first time to report an association between the CA27 allele of the IL-10.G microsatellites and SLE among Caucasians. Further studies are needed to confirm these findings.