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1.  Thermo-Active Behavior of Ethylene-Vinyl Acetate | Multiwall Carbon Nanotube Composites Examined by in Situ near-Edge X-ray Absorption Fine-Structure Spectroscopy 
NEXAFS spectroscopy was used to investigate the temperature dependence of thermally active ethylene-vinyl acetate | multiwall carbon nanotube (EVA|MWCNT) films. The data shows systematic variations of intensities with increasing temperature. Molecular orbital assignment of interplaying intensities identified the 1s → π*C=C and 1s → π*C=O transitions as the main actors during temperature variation. Furthermore, enhanced near-edge interplay was observed in prestrained composites. Because macroscopic observations confirmed enhanced thermal-mechanical actuation in prestrained composites, our findings suggest that the interplay of C=C and C=O π orbitals may be instrumental to actuation.
PMCID: PMC3983319  PMID: 24803975
2.  Near-Edge X-ray Absorption Fine Structure Studies of Electrospun Poly(dimethylsiloxane)/Poly (methyl methacrylate)/Multiwall Carbon Nanotube Composites 
This work describes the near conduction band edge structure of electrospun mats of MWCNT-PDMS-PMMA by near edge X-Ray absorption fine structure (NEXAFS) spectroscopy. Effects of adding nanofillers of different sizes were addressed. Despite observed morphological variations and inhomogeneous carbon nanotube distribution, spun mats appeared homogeneous under NEXAFS analysis. Spectra revealed differences in emissions from glancing and normal spectra; which may evidence phase separation within the bulk of the micron-size fibers. Further, dichroic ratios show polymer chains did not align, even in the presence of nanofillers. Addition of nanofillers affected emissions in the C-H, C=O and C-C regimes, suggesting their involvement in interfacial matrix-carbon nanotube bonding. Spectral differences at glancing angles between pristine and composite mats suggest that geometric conformational configurations are taking place between polymeric chains and carbon nanotubes. These differences appear to be carbon nanotube-dimension dependent, and are promoted upon room temperature mixing and shear flow during electrospinning. CH-π bonding between polymer chains and graphitic walls, as well as H-bonds between impurities in the as-grown CNTs and polymer pendant groups are proposed bonding mechanisms promoting matrix conformation.
PMCID: PMC3925980  PMID: 24308286
NEXAFS; electrospinning; carbon nanotubes; PDMS/PMMA; polymer nanocomposites; CH-π bonding
3.  One Small Step for a Yeast - Microevolution within Macrophages Renders Candida glabrata Hypervirulent Due to a Single Point Mutation 
PLoS Pathogens  2014;10(10):e1004478.
Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures – a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.
Author Summary
Evolution is not limited to making new species emerge and others perish over the millennia. It is also a central force in shorter-term interactions between microbes and hosts. A good example can be found in fungi, which are an underestimated cause of human diseases. Some fungi exist as commensals, and have adapted well to life on human epithelia. But as facultative pathogens, they face a different, hostile environment. We tested the ability of C. glabrata, a pathogen closely related to baker's yeast, to adapt to macrophages. We found that by adaptation, it changed its growth type completely. This allowed the fungus to escape the phagocytes, and increased its virulence in a mouse model. Sequencing the complete genome revealed surprisingly few mutations. Further analyses allowed us to detect the single mutation responsible for the phenotype, and to recreate it in the parental strain. Our work shows that fungi can adapt to immune cells, and that this adaptation can lead to an increased virulence. Since commensals are continuously exposed to host cells, we suggest that this ability could lead to unexpected phenotype changes, including an increase in virulence potential.
PMCID: PMC4214790  PMID: 25356907
4.  In situ soft XAS study on nickel-based layered cathode material at elevated temperatures: A novel approach to study thermal stability 
Scientific Reports  2014;4:6827.
Tracking thermally induced reactions has always been challenging for electrode materials of electrochemical battery systems. Traditionally, a variety of calorimetric techniques and in situ XRD at elevated temperatures has been used to evaluate the thermal stability of electrode materials. These techniques are capable of providing variations in heat capacity, mass and average bulk composition of materials only. Herein, we report investigation of thermal characteristics of Li0.33Ni0.8Co0.15Al0.05O2 by using in situ soft XAS measurements in combination with XRD. Fluorescence yield and partial electron yield measurements are used simultaneously to obtain element selective surface and bulk information. Fluorescence yield measurements reveal no energy change of the absorption peak and thus no valence state change in the bulk. However, electron yield measurements indicate that NiO-type rock salt structure is formed at the surface at temperatures above 200°C while no evidence for a surface reaction near Co sites in investigated temperature range is found. These results clearly show that in situ soft XAS can give a unique understanding of the role of each element in the structural transformation under thermal abuse offering a useful guidance in developing new battery system with improved safety performance.
PMCID: PMC4212228  PMID: 25351344
5.  Heat shock protein 60: an endogenous inducer of dopaminergic cell death in Parkinson disease 
Increasing evidence suggests that inflammation associated with microglial cell activation in the substantia nigra (SN) of patients with Parkinson disease (PD) is not only a consequence of neuronal degeneration, but may actively sustain dopaminergic (DA) cell loss over time. We aimed to study whether the intracellular chaperone heat shock protein 60 (Hsp60) could serve as a signal of CNS injury for activation of microglial cells.
Hsp60 mRNA expression in the mesencephalon and the striatum of C57/BL6 mice treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and the Hsp60/TH mRNA ratios in the SN of PD patients and aged-matched subjects were measured. To further investigate a possible link between the neuronal Hsp60 response and PD-related cellular stress, Hsp60 immunoblot analysis and quantification in cell lysates from SH-SY5Y after treatment with 100 μM MPP+ (1-methyl-4-phenylpyridinium) at different time points (6, 12, 24 and 48 hours) compared to control cells were performed. Additional MTT and LDH assay were used. We next addressed the question as to whether Hsp60 influences the survival of TH+ neurons in mesencephalic neuron-glia cultures treated either with MPP+ (1 μM), hHsp60 (10 μg/ml) or a combination of both. Finally, we measured IL-1β, IL-6, TNF-α and NO-release by ELISA in primary microglial cell cultures following treatment with different hHsp60 preparations. Control cultures were exposed to LPS.
In the mesencephalon and striatum of mice treated with MPTP and also in the SN of PD patients, we found that Hsp60 mRNA was up-regulated. MPP+, the active metabolite of MPTP, also caused an increased expression and release of Hsp60 in the human dopaminergic cell line SH-SY5Y. Interestingly, in addition to being toxic to DA neurons in primary mesencephalic cultures, exogenous Hsp60 aggravated the effects of MPP+. Yet, although we demonstrated that Hsp60 specifically binds to microglial cells, it failed to stimulate the production of pro-inflammatory cytokines or NO by these cells.
Overall, our data suggest that Hsp60 is likely to participate in DA cell death in PD but via a mechanism unrelated to cytokine release.
PMCID: PMC4018945  PMID: 24886419
PD; Neuroinflammation; Hsp60; Neurodegeneration; Microglia; Innate immunity
6.  A Guide for Starting a Specialty Training Clinic: An Alcohol Treatment Program as an Example 
Since the Boulder conference more than 50 years ago, clinical psychology has been moving towards empirically based techniques and methods. Considerable research has been conducted and a multitude of studies have documented support for empirically supported treatments (ESTs). However, the literature on implementing ESTs in real-world settings is relatively limited. The absence of practical guidance poses a particular problem for students in clinical psychology training programs that emphasize training and competency in ESTs. This article describes the development of an alcohol specialty clinic within a clinical psychology training program from the first conceptualizations to establishment of a referral base and provision of services. At each step, integration of science and clinical practice is discussed. Future directions and suggestions for developing training clinics are provided.
PMCID: PMC3657837  PMID: 23704805
Alcohol treatment; Specialty clinic; EST; clinical training
7.  Synthetic Studies on Pseudo-Dimeric Lycopodium Alkaloids: Total Synthesis of Complanadine B 
Angewandte Chemie (International ed. in English)  2013;52(6):10.1002/anie.201208571.
PMCID: PMC3829996  PMID: 23307758
complanadine; borylative C-H functionalization; Lycopodium; dimeric natural products; pyridine oxidation
8.  ARLTS1 and Prostate Cancer Risk - Analysis of Expression and Regulation 
PLoS ONE  2013;8(8):e72040.
Prostate cancer (PCa) is a heterogeneous trait for which several susceptibility loci have been implicated by genome-wide linkage and association studies. The genomic region 13q14 is frequently deleted in tumour tissues of both sporadic and familial PCa patients and is consequently recognised as a possible locus of tumour suppressor gene(s). Deletions of this region have been found in many other cancers. Recently, we showed that homozygous carriers for the T442C variant of the ARLTS1 gene (ADP-ribosylation factor-like tumour suppressor protein 1 or ARL11, located at 13q14) are associated with an increased risk for both unselected and familial PCa. Furthermore, the variant T442C was observed in greater frequency among malignant tissue samples, PCa cell lines and xenografts, supporting its role in PCa tumourigenesis. In this study, 84 PCa cases and 15 controls were analysed for ARLTS1 expression status in blood-derived RNA. A statistically significant (p = 0.0037) decrease of ARLTS1 expression in PCa cases was detected. Regulation of ARLTS1 expression was analysed with eQTL (expression quantitative trait loci) methods. Altogether fourteen significant cis-eQTLs affecting the ARLTS1 expression level were found. In addition, epistatic interactions of ARLTS1 genomic variants with genes involved in immune system processes were predicted with the MDR program. In conclusion, this study further supports the role of ARLTS1 as a tumour suppressor gene and reveals that the expression is regulated through variants localised in regulatory regions.
PMCID: PMC3734304  PMID: 23940804
9.  DAP12 and CD11b contribute to the microglial-induced death of dopaminergic neurons in vitro but not in vivo in the MPTP mouse model of Parkinson’s disease 
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons (DN) in the substantia nigra (SN). Several lines of evidence suggest that apoptotic cell death of DN is driven in part by non-cell autonomous mechanisms orchestrated by microglial cell-mediated inflammatory processes. Although the mechanisms and molecular network underlying this deleterious cross-talk between DN and microglial cells remain largely unknown, previous work indicates that, upon DN injury, activation of the β2 integrin subunit CD11b is required for microglia-mediated DN cell death. Interestingly, during brain development, the CD11b integrin is also involved in microglial induction of neuronal apoptosis and has been shown to act in concert with the DAP12 immunoreceptor. Whether such a developmental CD11b/DAP12 pathway could be reactivated in a pathological context such as PD and play a role in microglia-induced DN cell death is a tantalizing hypothesis that we wished to test in this study.
To test the possibility that DAP12 could be involved in microglia-associated DN injury, we used both in vitro and in vivo toxin-based experimental models of PD recapitulating microglial-mediated non-cell autonomous mechanisms of DN cell death. In vitro, enriched mesencephalic neuronal/microglial co-cultures were exposed to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) whereas in vivo, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) according to acute or subchronic mode. Mice deficient for DAP12 or CD11b were used to determine the pathological function of the CD11b/DAP12 pathway in our disease models.
Our results show that DAP12 and CD11b partially contribute to microglia-induced DN cell death in vitro. Yet, in vivo, mice deficient for either of these factors develop similar neuropathological alterations as their wild-type counterparts in two different MPTP mouse models of PD.
Overall, our data suggest that DAP12 and CD11b contribute to microglial-induced DN cell death in vitro but not in vivo in the MPTP mouse model of PD. Therefore, the CD11b/DAP12 pathway may not be considered as a promising therapeutic target for PD.
PMCID: PMC3720270  PMID: 23844828
DAP12; CD11b; Microglia; Dopaminergic neuron; Parkinson’s disease; MPTP
10.  Direct Observation of Phenylalanine Orientations in Statherin Bound to Hydroxyapatite Surfaces 
Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and key protein side chains. This study demonstrates, for the first time, the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin, SN15, has been adsorbed onto HAP surfaces and the orientations of both phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.
PMCID: PMC3549518  PMID: 22563672
11.  Bee Venom and Its Component Apamin as Neuroprotective Agents in a Parkinson Disease Mouse Model 
PLoS ONE  2013;8(4):e61700.
Bee venom has recently been suggested to possess beneficial effects in the treatment of Parkinson disease (PD). For instance, it has been observed that bilateral acupoint stimulation of lower hind limbs with bee venom was protective in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In particular, a specific component of bee venom, apamin, has previously been shown to have protective effects on dopaminergic neurons in vitro. However, no information regarding a potential protective action of apamin in animal models of PD is available to date. The specific goals of the present study were to (i) establish that the protective effect of bee venom for dopaminergic neurons is not restricted to acupoint stimulation, but can also be observed using a more conventional mode of administration and to (ii) demonstrate that apamin can mimic the protective effects of a bee venom treatment on dopaminergic neurons. Using the chronic mouse model of MPTP/probenecid, we show that bee venom provides sustained protection in an animal model that mimics the chronic degenerative process of PD. Apamin, however, reproduced these protective effects only partially, suggesting that other components of bee venom enhance the protective action of the peptide.
PMCID: PMC3630120  PMID: 23637888
12.  Toll like receptor 4 mediates cell death in a mouse MPTP model of Parkinson disease 
Scientific Reports  2013;3:1393.
In mammalians, toll-like receptors (TLR) signal-transduction pathways induce the expression of a variety of immune-response genes, including inflammatory cytokines. It is therefore plausible to assume that TLRs are mediators in glial cells triggering the release of cytokines that ultimately kill DA neurons in the substantia nigra in Parkinson disease (PD). Accordingly, recent data indicate that TLR4 is up-regulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in a mouse model of PD. Here, we wished to evaluate the role of TLR4 in the acute mouse MPTP model of PD: TLR4-deficient mice and wild-type littermates control mice were used for the acute administration way of MPTP or a corresponding volume of saline. We demonstrate that TLR4-deficient mice are less vulnerable to MPTP intoxication than wild-type mice and display a decreased number of Iba1+ and MHC II+ activated microglial cells after MPTP application, suggesting that the TLR4 pathway is involved in experimental PD.
PMCID: PMC3589722  PMID: 23462811
13.  Acetylcholine content and viability of cholinergic neurons are influenced by the activity of protein histidine phosphatase 
BMC Neuroscience  2012;13:31.
The first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century. One of the known substrates of PHP is ATP-citrate lyase (ACL), which is responsible - amongst other functions - for providing acetyl-CoA for acetylcholine synthesis in neuronal tissues. It has been shown in previous studies that PHP downregulates the activity of ACL by dephosphorylation. According to this our present work focused on the influence of PHP activity on the acetylcholine level in cholinergic neurons.
The amount of PHP in SN56 cholinergic neuroblastoma cells was increased after overexpression of PHP by using pIRES2-AcGFP1-PHP as a vector. We demonstrated that PHP overexpression reduced the acetylcholine level and induced cell death. The acetylcholine content of SN56 cells was measured by fast liquid chromatography-tandem mass spectrometry method. Overexpression of the inactive H53A-PHP mutant also induced cell damage, but in a significantly reduced manner. However, this overexpression of the inactive PHP mutant did not change the acetylcholine content of SN56 cells significantly. In contrast, PHP downregulation, performed by RNAi-technique, did not induce cell death, but significantly increased the acetylcholine content in SN56 cells.
We could show for the first time that PHP downregulation increased the acetylcholine level in SN56 cells. This might be a potential therapeutic strategy for diseases involving cholinergic deficits like Alzheimer's disease.
PMCID: PMC3355010  PMID: 22436051
15.  Total Synthesis of (+)-Complanadine A Using an Iridium-Catalyzed Pyridine C-H Functionalization 
Total synthesis of the Lycopodium alkaloid complanadine A, which is an unsymmetrical dimer of lycodine, was achieved by exploiting a common tetracyclic precursor. Key to the success of the synthesis was the development of a late-stage site-selective C-H functionalization of a pyridine moiety to arrive at a key boronic ester intermediate.
PMCID: PMC2867450  PMID: 20387895
16.  Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease  
Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8+ and CD4+ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1–/– and Tcrb–/– mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1–/– mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1–/– mice reconstituted with IFN-γ–deficient splenocytes were not protected. These data indicate that T cell–mediated dopaminergic toxicity is almost exclusively arbitrated by CD4+ T cells and requires the expression of FasL but not IFNγ. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.
PMCID: PMC2613467  PMID: 19104149
17.  Identification and investigation of ORFans in the viral world 
BMC Genomics  2008;9:24.
Genome-wide studies have already shed light into the evolution and enormous diversity of the viral world. Nevertheless, one of the unresolved mysteries in comparative genomics today is the abundance of ORFans – ORFs with no detectable sequence similarity to any other ORF in the databases. Recently, studies attempting to understand the origin and functions of bacterial ORFans have been reported. Here we present a first genome-wide identification and analysis of ORFans in the viral world, with focus on bacteriophages.
Almost one-third of all ORFs in 1,456 complete virus genomes correspond to ORFans, a figure significantly larger than that observed in prokaryotes. Like prokaryotic ORFans, viral ORFans are shorter and have a lower GC content than non-ORFans. Nevertheless, a statistically significant lower GC content is found only on a minority of viruses. By focusing on phages, we find that 38.4% of phage ORFs have no homologs in other phages, and 30.1% have no homologs neither in the viral nor in the prokaryotic world. Phages with different host ranges have different percentages of ORFans, reflecting different sampling status and suggesting various diversities. Similarity searches of the phage ORFeome (ORFans and non-ORFans) against prokaryotic genomes shows that almost half of the phage ORFs have prokaryotic homologs, suggesting the major role that horizontal transfer plays in bacterial evolution. Surprisingly, the percentage of phage ORFans with prokaryotic homologs is only 18.7%. This suggests that phage ORFans play a lesser role in horizontal transfer to prokaryotes, but may be among the major players contributing to the vast phage diversity.
Although the current sampling of viral genomes is extremely low, ORFans and near-ORFans are likely to continue to grow in number as more genomes are sequenced. The abundance of phage ORFans may be partially due to the expected vast viral diversity, and may be instrumental in understanding viral evolution. The functions, origins and fates of the majority of viral ORFans remain a mystery. Further computational and experimental studies are likely to shed light on the mechanisms that have given rise to so many bacterial and viral ORFans.
PMCID: PMC2245933  PMID: 18205946
18.  Evidence for a Susceptibility Locus on Chromosome 10p15 in Early-Onset Obsessive-Compulsive Disorder 
Biological psychiatry  2007;62(8):856-862.
The goal of this study was to identify chromosomal regions likely to contain susceptibility loci for obsessive-compulsive disorder (OCD).
We conducted a genome-wide linkage scan, with average marker spacing less than 10 centimorgans (cM), in 121 subjects from 26 families ascertained through probands with early-onset OCD. Best-estimate lifetime psychiatric diagnoses were based on semi-structured interviews and all other available sources of information. Parametric and nonparametric linkage analyses were conducted with GENEHUNTER+ and Allegro. Family-based association analyses were done using 35 single nucleotide polymorphisms (SNPs) in the 10p15 region.
The maximum nonparametric LOD score was 2.43 on chromosome 10p15 at position 4.37. When data from our first genome scan were added to data from this scan, the maximum NLOD score in the 10p15 region was 1.79. Association was detected on 10p15 with three adjacent SNPs, including the amino acid variant rs2271275, in the 3′ region of adenosine deaminase acting on RNA 3 (ADAR3) (p < 0.05).
The results provide suggestive evidence for linkage on chromosome 10p15. Evidence for association was found with three markers in the 3′ end of ADAR3 in the linkage region. Limitations include the lack of significant linkage and association findings when corrected for multiple testing.
PMCID: PMC2040499  PMID: 17544380
obsessive-compulsive disorder; linkage analysis; genome scan; adenosine deaminase acting on RNA 3; microsatellite markers; single nucleotide polymorphisms
19.  FRalanyzer: a tool for functional analysis of fold-recognition sequence–structure alignments 
Nucleic Acids Research  2007;35(Web Server issue):W499-W502.
We describe FRalanyzer (Fold Recognition alignment analyzer), a new web tool to visually inspect sequence–structure alignments in order to predict functionally important residues in a query sequence of unknown function. This tool is aimed at helping to infer functional relationships between a query sequence and a template structure, and is particularly useful in analyzing fold recognition (FR) results. Because similar folds do not necessarily share the same function, it is not always straightforward to infer a function from an FR result alone. Manual inspection of the FR sequence-structure alignment is often required in order to search for conservation of functionally important residues. FRalanyzer automates parts of this time-consuming process. FRalanyzer takes as input a sequence–structure alignment, automatically searches annotated databases, displays functionally significant residues and highlights the functionally important positions that are identical in the alignment. FRalanyzer can also be used with sequence-structure alignments obtained by other methods, and with structure–structure alignments obtained from structural comparison of newly determined 3D-structures of unknown function. Fralanyzer is available at
PMCID: PMC1933221  PMID: 17537819
20.  Structural and functional insights into Mimivirus ORFans 
BMC Genomics  2007;8:115.
Mimivirus isolated from A. polyphaga is the largest virus discovered so far. It is unique among all the viruses in having genes related to translation, DNA repair and replication which bear close homology to eukaryotic genes. Nevertheless, only a small fraction of the proteins (33%) encoded in this genome has been assigned a function. Furthermore, a large fraction of the unassigned protein sequences bear no sequence similarity to proteins from other genomes. These sequences are referred to as ORFans. Because of their lack of sequence similarity to other proteins, they can not be assigned putative functions using standard sequence comparison methods. As part of our genome-wide computational efforts aimed at characterizing Mimivirus ORFans, we have applied fold-recognition methods to predict the structure of these ORFans and further functions were derived based on conservation of functionally important residues in sequence-template alignments.
Using fold recognition, we have identified highly confident computational 3D structural assignments for 21 Mimivirus ORFans. In addition, highly confident functional predictions for 6 of these ORFans were derived by analyzing the conservation of functional motifs between the predicted structures and proteins of known function. This analysis allowed us to classify these 6 previously unannotated ORFans into their specific protein families: carboxylesterase/thioesterase, metal-dependent deacetylase, P-loop kinases, 3-methyladenine DNA glycosylase, BTB domain and eukaryotic translation initiation factor eIF4E.
Using stringent fold recognition criteria we have assigned three-dimensional structures for 21 of the ORFans encoded in the Mimivirus genome. Further, based on the 3D models and an analysis of the conservation of functionally important residues and motifs, we were able to derive functional attributes for 6 of the ORFans. Our computational identification of important functional sites in these ORFans can be the basis for a subsequent experimental verification of our predictions. Further computational and experimental studies are required to elucidate the 3D structures and functions of the remaining Mimivirus ORFans.
PMCID: PMC1876218  PMID: 17490476
21.  On the origin of microbial ORFans: quantifying the strength of the evidence for viral lateral transfer 
The origin of microbial ORFans, ORFs having no detectable homology to other ORFs in the databases, is one of the unexplained puzzles of the post-genomic era. Several hypothesis on the origin of ORFans have been suggested in the last few years, most of which based on selected, relatively small, subsets of ORFans. One of the hypotheses for the origin of ORFans is that they have been acquired thru lateral transfer from viruses. Here we carry out a comprehensive, genome-wide study on the origins of ORFans to quantify the strength of current evidence supporting this hypothesis.
We performed similarity searches by querying all current ORFans against the public virus protein database. Surprisingly, we found that only 2.8% of all microbial ORFans have detectable homologs in viruses, while the percentage of non-ORFans with detectable homologs in viruses is 7.9%, a significantly higher figure. This suggests that the current evidence for the origin of ORFans from lateral transfer from viruses is at best weak. However, an analysis of individual genomes revealed a number of organisms with much higher percentages, many of them belonging to the Firmicutes and Gamma-proteobacteria. We provide evidence suggesting that the current virus database may be biased towards those viruses attacking Firmicutes and Gamma-proteobacteria.
We conclude that as more viral genomes are sequenced, more microbial ORFans will find homologs in viruses, but this trend may vary much for individual genomes. Thus, lateral transfer from viruses alone is unlikely to explain the origin of the majority of ORFans in the majority of prokaryotes and consequently, other, not necessarily exclusive, mechanisms are likely to better explain the origin of the increasing number of ORFans.
PMCID: PMC1559721  PMID: 16914045
22.  The ORFanage: an ORFan database 
Nucleic Acids Research  2004;32(Database issue):D281-D283.
As each newly sequenced genome contains a significant number of protein-coding ORFs that are species-, family- or lineage-specific, many interesting questions arise about the evolution and role of these ORFs and of the genomes they are part of. We refer to these poorly conserved ORFs as singleton or paralogous ORFans if they are unique to one genome, or as orthologous ORFans if they appear only in a family of closely related organisms and have no homolog in other genomes. In order to study and classify ORFans we have constructed the ORFanage, an ORFan database. This database consists of the predicted ORFs in fully sequenced microbial genomes, and enables searching for the three types of ORFans in any subset of the genomes chosen by the user. The ORFanage could help in choosing interesting targets for further genomic and evolutionary studies. The ORFanage is accessible via
PMCID: PMC308850  PMID: 14681413
23.  Unravelling the ORFan Puzzle 
ORFans are open reading frames (ORFs) with no detectable sequence similarity to any other sequence in the databases. Each newly sequenced genome contains a significant number of ORFans. Therefore, ORFans entail interesting evolutionary puzzles. However, little can be learned about them using bioinformatics tools, and their study seems to have been underemphasized. Here we present some of the questions that the existence of so many ORFans have raised and review some of the studies aimed at understanding ORFans, their functions and their origins. These works have demonstrated that ORFans are an untapped source of research, requiring further computational and experimental studies.
PMCID: PMC2447361  PMID: 18629076
24.  A study of quality measures for protein threading models 
BMC Bioinformatics  2001;2:5.
Prediction of protein structures is one of the fundamental challenges in biology today. To fully understand how well different prediction methods perform, it is necessary to use measures that evaluate their performance. Every two years, starting in 1994, the CASP (Critical Assessment of protein Structure Prediction) process has been organized to evaluate the ability of different predictors to blindly predict the structure of proteins. To capture different features of the models, several measures have been developed during the CASP processes. However, these measures have not been examined in detail before. In an attempt to develop fully automatic measures that can be used in CASP, as well as in other type of benchmarking experiments, we have compared twenty-one measures. These measures include the measures used in CASP3 and CASP2 as well as have measures introduced later. We have studied their ability to distinguish between the better and worse models submitted to CASP3 and the correlation between them.
Using a small set of 1340 models for 23 different targets we show that most methods correlate with each other. Most pairs of measures show a correlation coefficient of about 0.5. The correlation is slightly higher for measures of similar types. We found that a significant problem when developing automatic measures is how to deal with proteins of different length. Also the comparisons between different measures is complicated as many measures are dependent on the size of the target. We show that the manual assessment can be reproduced to about 70% using automatic measures. Alignment independent measures, detects slightly more of the models with the correct fold, while alignment dependent measures agree better when selecting the best models for each target. Finally we show that using automatic measures would, to a large extent, reproduce the assessors ranking of the predictors at CASP3.
We show that given a sufficient number of targets the manual and automatic measures would have given almost identical results at CASP3. If the intent is to reproduce the type of scoring done by the manual assessor in in CASP3, the best approach might be to use a combination of alignment independent and alignment dependent measures, as used in several recent studies.
PMCID: PMC55330  PMID: 11545673

Results 1-24 (24)