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PubMed2004

Unusual presentation of life-threatening Toscana virus meningoencephalitis.

This case report describes a brother and a sister with severe meningoencephalitis caused by Toscana virus (TOSv). The clinical presentation was characterized by stiff neck, deep coma, maculopapular rash, diffuse lymphadenopathy, hepatosplenomegaly, renal involvement, tendency to bleeding, and diffuse intravascular coagulation. The boy had epididymo-orchitis. Recovery with neurologic sequelae as hydrocephalus was observed. Microbiological diagnosis was obtained by serological tests and reverse transcriptase-polymerase chain reaction. Sequencing of polymerase chain reaction products from the S and M segments was carried out. TOSv may be a causative agent in severe meningoencephalitis.

Adolescent↗
PubMed2001

Structure-based phylogenetic analysis of short-chain alcohol dehydrogenases and reclassification of the 17beta-hydroxysteroid dehydrogenase family.

Short-chain alcohol dehydrogenases (SCAD) constitute a large and diverse family of ancient origin. Several of its members play an important role in human physiology and disease, especially in the metabolism of steroid substrates (e.g., prostaglandins, estrogens, androgens, and corticosteroids). Their involvement in common human disorders such as endocrine-related cancer, osteoporosis, and Alzheimer disease makes them an important candidate for drug targets. Recent phylogenetic analysis of SCAD is incomplete and does not allow any conclusions on very ancient divergences or on a functional characterization of novel proteins within this complex family. We have developed a 3D structure-based approach to establish the deep-branching pattern within the SCAD family. In this approach, pairwise superpositions of X-ray structures were used to calculate similarity scores as an input for a tree-building algorithm. The resulting phylogeny was validated by comparison with the results of sequence-based algorithms and biochemical data. It was possible to use the 3D data as a template for the reliable determination of the phylogenetic position of novel proteins as a first step toward functional predictions. We were able to discern new patterns in the phylogenetic relationships of the SCAD family, including a basal dichotomy of the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). These data provide an important contribution toward the development of type-specific inhibitors for 17beta-HSDs for the treatment and prevention of disease. Our structure-based phylogenetic approach can also be applied to increase the reliability of evolutionary reconstructions in other large protein families.

17-Hydroxysteroid Dehydrogenases↗
PubMed2006

Desulfurobacterium atlanticum sp. nov., Desulfurobacterium pacificum sp. nov. and Thermovibrio guaymasensis sp. nov., three thermophilic members of the Desulfurobacteriaceae fam. nov., a deep branching lineage within the Bacteria.

Three thermophilic, anaerobic, strictly chemolithoautotrophic, sulphur- and/or thiosulphate-reducing bacteria, designated SL17(T), SL19(T) and SL22(T), were isolated from deep-sea hydrothermal samples collected at 13 degrees N (East Pacific Rise), Guaymas Basin (Gulf of California) and 23 degrees N (Mid-Atlantic Ridge), respectively. These strains differed in their morphology, temperature range and optimum for growth, energy substrates and 16S rRNA gene sequences. The G+C content of the genomic DNA was 41 mol% (SL22(T)), 42 mol% (SL17(T)) and 46 mol% (SL19(T)). Comparative analysis of phenotypic and phylogenetic traits indicated that strains SL17(T) and SL22(T) represented two novel species of the genus Desulfurobacterium and that strain SL19(T) should be considered as a novel species of the genus Thermovibrio. The names Desulfurobacterium pacificum sp. nov. (type strain SL17(T)=DSM 15522(T)=JCM 12127(T)), Desulfurobacterium atlanticum sp. nov. (type strain SL22(T)=DSM 15668(T)=JCM 12129(T)) and Thermovibrio guaymasensis sp. nov. (type strain SL19(T)=DSM 15521(T)=JCM 12128(T)) are proposed for these organisms. Furthermore, phylogenetic data based on 16S rRNA gene sequence analyses correlated with the significant phenotypic differences between members of the lineage encompassing the genera Desulfurobacterium, Thermovibrio and Balnearium and that of the families Aquificaceae and Hydrogenothermaceae. It is therefore proposed that this lineage represents a new family, Desulfurobacteriaceae fam. nov., within the order Aquificales.

Bacterial Typing Techniques↗
PubMed2005

Universal primers and PCR of gut contents to study marine invertebrate diets.

Determining the diets of marine invertebrates by gut content analysis is problematic. Many consumed organisms become unrecognizable once partly digested, while those with hard remains (e.g. diatom skeletons) may bias the analysis. Here, we adapt DNA-based methods similar to those used for microbial diversity surveys as a novel approach to study the diets of macrophagous (the deep-sea amphipods Scopelocheirus schellenbergi and Eurythenes gryllus) and microphagous (the bivalve Lucinoma aequizonata) feeders in the deep sea. Polymerase chain reaction (PCR) in conjunction with 'universal' primers amplified portions of the mitochondrial cytochrome c oxidase I (COI) gene for animals ingested by S. schellenbergi and E. gryllus and the 18S rRNA gene for lesser eukaryotes ingested by L. aequizonata. Amplified sequences were combined with sequences from GenBank to construct phylogenetic trees of ingested organisms. Our analyses indicate that S. schellenbergi, E. gryllus and L. aequizonata diets are considerably more diverse than previously thought, casting new light on the foraging strategies of these species. Finally, we discuss the strengths and weaknesses of this technique and its potential applicability to diet analyses of other invertebrates.

Animals↗
PubMed1998

Preoperative staging of endometrial carcinoma: diagnostic effect of T2-weighted fast spin-echo MR imaging.

PURPOSE: To evaluate the usefulness of T2-weighted fast spin-echo magnetic resonance (MR) imaging with a 512 x 256 matrix for assessment of the preoperative stage of endometrial carcinoma. MATERIALS AND METHODS: Twenty-eight women with histopathologically proved endometrial carcinoma underwent preoperative T2-weighted fast spin-echo, dynamic T1-weighted fast spin-echo, and postcontrast T1-weighted spin-echo MR imaging with a phased-array surface coil. The uterine long-axis planes in each sequence were reviewed at separate sessions by three radiologists blinded to the histopathologic data. RESULTS: For the diagnosis of myometrial invasion, no statistically significant differences were found among T2-weighted imaging, dynamic imaging, and postcontrast T1-weighted imaging. For the diagnosis of deep myometrial invasion, T2-weighted and dynamic images showed higher specificity than postcontrast T1-weighted images (T2-weighted, 89%; dynamic, 88%; and postcontrast T1-weighted, 80%). For cervical invasion, T2-weighted and dynamic images showed larger areas under receiver operating characteristic curves than did postcontrast T1-weighted images (T2-weighted, 0.78; dynamic, 0.71; and postcontrast T1-weighted, 0.67). CONCLUSION: T2-weighted imaging is useful for identifying the stage of endometrial carcinoma.

Carcinoma↗
PubMed1999

The thyroid hormone receptor-beta gene mutation R383H is associated with isolated central resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) action is due to mutations in the beta-isoform of the thyroid hormone receptor (TR-beta). RTH patients display inappropriate central secretion of TRH from the hypothalamus and of TSH from the anterior pituitary despite elevated levels of thyroid hormone (T4 and T3). RTH mutations cluster in three hot spots in the C-terminal portion of the TR-beta. Most individuals with TR-beta mutations have generalized resistance to thyroid hormone, where most tissues in the body are hyporesponsive to thyroid hormone. The affected individuals are clinically euthyroid or even hypothyroid depending on the severity of the mutation. Whether TR-beta mutations cause a selective form of RTH that only leads to central thyroid hormone resistance is debated. Here, we describe an individual with striking peripheral sensitivity to graded T3 administration. The subject was enrolled in a protocol in which she received three escalating T3 doses over a 13-day period. Indexes of central and peripheral thyroid hormone action were measured at baseline and at each T3 dose. Although the patient's resting pulse rose only 11% in response to T3, her serum ferritin, alanine aminotransferase, aspartate transaminase, and lactate dehydrogenase rose 320%, 117%, 121%, and 30%, respectively. In addition, her serum cholesterol, creatinine phosphokinase, and deep tendon reflex relaxation time fell (25%, 36%, and 36%, respectively). Centrally, the patient was sufficiently resistant to T3 that her serum TSH was not suppressed with 200 microg T3, orally, daily for 4 days. The patient's C-terminal TR exons were sequenced revealing the mutation R383H in a region not otherwise known to harbor TR-beta mutations. Our clinical evaluation presented here represents the most thorough documentation to date of the central thyroid hormone resistance phenotype in an individual with an identified TR-beta mutation.

Body Weight↗
PubMed2003

On-line monitoring of apoptosis in insulin-secreting cells.

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.

Animals↗
PubMed1995

[Magnetic tomography in the examination of edema and pain in the leg].

Magnetic resonance imaging (MRI) was used to investigate 38 patients suffering from leg oedema or pain of various etiology. Spin echo series with 10 mm transverse slices of both legs were obtained. Soft tissue changes were visualized best by T2-weighted sequences. Characteristic changes could be observed by MR images of patients with closed compartment syndrome (n = 2), chronic lymph oedema (n = 10) and post-reconstructive leg oedema following vascular surgery (n = 14). MRI can also show typical soft tissue changes in patients with deep vein thrombosis (n = 5) or post-thrombotic syndrome (n = 3). For most of these conditions, the diagnosis can usually be established using simpler methods. However, MRI is an excellent supplementary method for showing soft tissue changes, and is a promising way of investigating conditions that may give rise to leg oedema and pain.

Adult↗
PubMed2003

Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.

Amino Acid Sequence↗
PubMed2004

The origin and radiation of Macaronesian beetles breeding in Euphorbia: the relative importance of multiple data partitions and population sampling.

Species-level phylogenies derived from many independent character sources and wide geographical sampling provide a powerful tool in assessing the importance of various factors associated with cladogenesis. In this study, we explore the relative importance of insular isolation and host plant switching in the diversification of a group of bark beetles (Curculionidae: Scolytinae) feeding and breeding in woody Euphor biaspurges. All species in the genus Aphanarthrumare each associated with only one species group of Euphorbia(succulents or one of three different arborescent groups), and the majority of species are endemic to one or several of the Macaronesian Islands. Hence, putative mechanisms of speciation could be assessed by identifying pairs of sister species in a phylogenetic analysis. We used DNA sequences from two nuclear and two mitochondrial genes, and morphological characters, to reconstruct the genealogical relationships among 92 individuals of 25 species and subspecies of Aphanarthrumand related genera. A stable tree topology was highly dependent on multiple character sources, but much less so on wide population sampling. However, multiple samples per species demonstrated one case of species paraphyly, as well as deep coalescence among three putative subspecies pairs. The phylogenetic analyses consistently placed the arborescent breeding and West African--Lanzarote-distributed species A. armatumin the most basal position in Aphanarthrum, rendering this genus paraphyletic with respect to Coleobothrus. Two major radiations followed, one predominantly African lineage of succulent feeding species, and one island radiation associated with arborescent host plants. Sister comparisons showed that most recent divergences occurred in allopatry on closely related hosts, with subsequent expansions obscuring more ancient events. Only 6 out of 24 cladogenetic events were associated with host switching, rendering geographical factors more important in recent diversification.

Animals↗
PubMed1998

Complete sequence of the mitochondrial DNA of Chlamydomonas eugametos.

The complete nucleotide sequence of the Chlamydomonas eugametos (Chlamydomonadales, Chlorophyceae, sensu Mattox and Stewart) mitochondrial genome has been determined (22,897 bp, 34.6% G + C). The genes identified in this circular-mapping genome include those for apocytochrome b, subunit 1 of the cytochrome oxidase complex, subunits 1, 2, 4, 5, and 6 of the NADH dehydrogenase complex, discontinuous large and small subunit ribosomal rRNAs and three tRNAs whose anticodons CAU, CCA and UUG are specific for methionine, tryptophan and glutamine, respectively. The C. eugametos mitochondrial DNA (mtDNA), therefore, shares almost the same reduced set of coding functions and similar unusual features of rRNA gene organization with the linear 15.8 kb mtDNA of Chlamydomonas reinhardtii, the only other completely sequenced chlamydomonadalean mtDNA. However, sequence analysis of the C. eugametos mtDNA has revealed the following distinguishing features relative to those of C. reinhardtii: (1) the absence of a reverse transcriptase-like gene homologue, (2) the presence of an additional gene for tRNA(met) that may be a pseudogene, (3) a completely different gene order, (4) transcription of all genes from the same mtDNA strand, (5) a lower G + C content, (6) less pronounced bias in codon usage, and (7) nine group I introns, several of which contain open reading frames coding for potential maturases/endonucleases and two have a nucleotide at the 5' or 3' splice site of the deduced precursor RNAs that deviates from highly conserved nucleotides reported in other group I introns. The features of mitochondrial genome organization and gene content shared by C. eugametos and C. reinhardtii contrast with those of other green algal mtDNAs that have been characterized in detail. The deep evolutionary divergence between these two Chlamydomonas taxa within the Chlamydomonadales suggests that their shared features of mitochondrial genome organization evolved prior to the origin of this group.

Animals↗
PubMed2005

Substantial genetic substructuring in southeastern and alpine Australia revealed by molecular phylogeography of the Egernia whitii (Lacertilia: Scincidae) species group.

Palaeoclimatic events and biogeographical processes since the mid-Tertiary are believed to have strongly influenced the evolution and distribution of the terrestrial vertebrate fauna of southeastern Australia. We examined the phylogeography of the temperate-adapted members of the Egernia whitii species group, a group of skinks that comprise both widespread low- to mid-elevation (E. whitii) and montane-restricted species (Egernia guthega, Egernia montana), in order to obtain important insights into the influence of past biogeographical processes on the herpetofauna of southeastern Australia. Sequence data were obtained from all six temperate-adapted species within the E. whitii species group, and specifically from across the distributional ranges of E. whitii, E. guthega and E. montana. We targeted a fragment of the ND4 mitochondrial gene (696 bp) and analysed the data using maximum likelihood and Bayesian methods. Our data reveal a deep phylogeographical break in the east Gippsland region of Victoria between 'northern' (Queensland, New South Wales, Australian Capital Territory) and 'southern' (Victoria, Tasmania, South Australia) populations of E. whitii. This divergence appears to have occurred during the late Miocene-Pliocene, with the Gippsland basin possibly forming a geographical barrier to dispersal. Substantial structuring within both the 'northern' and the 'southern' clades is consistent with the effects of Plio-Pleistocene glacial-interglacial cycles. Pleistocene glacial cycles also appear to have shaped the phylogeographical patterns observed in the alpine species, E. guthega and E. montana. We used our results to examine the biogeographical process that led to the origin and subsequent diversification of the lowland and alpine herpetofauna of southeastern Australia.

Altitude↗
PubMed2025

Increased yield of genetic diagnoses in inherited heart diseases using expanded genome and RNA-splicing analyses.

PURPOSE: The Australian Genomics Cardiovascular Disorders Flagship investigated genome sequencing as a first-line genetic test in 600 individuals with cardiomyopathy, primary arrhythmia syndromes, or congenital heart disease. Analysis of disease-specific virtual gene panels achieved a genetic diagnosis in 38% of participants. We sought to increase genetic diagnosis yields by analyzing lesser-evidenced disease genes, the mitochondrial genome, and by functional analysis of predicted splice-altering variants. METHODS: Genome sequences of 520 participants with cardiomyopathy or primary arrhythmia syndromes were reanalyzed in 572 cardiac genes and the mitochondrial genome. Participants with congenital heart disease were excluded. Variants predicted in silico to disrupt splicing were assessed with blood RNA and minigenes. RESULTS: A new genetic diagnosis was achieved in 4% (19/520) of participants, including deep intronic and mitochondrial genome variants. Ten participants had diagnostic variants in lesser evidenced disease genes; 9 had splicing variant pathogenicity functionally validated. Eleven participants had a newly identified variant of uncertain significance with high suspicion of pathogenicity, warranting clinical review. Our data supported the gene-disease association of 1 new cardiomyopathy gene, TBX20. CONCLUSION: Identifying new gene-disease relationships, maintaining contemporary gene panels, and integrating functional studies to refine splicing variant classifications increase genetic diagnoses for cardiomyopathies and primary arrhythmia syndromes.

Humans↗
PubMed2006

Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades.

Inference of evolutionary relationships between nematodes is severely hampered by their conserved morphology, the high frequency of homoplasy, and the scarcity of phylum-wide molecular data. To study the origin of nematode radiation and to unravel the phylogenetic relationships between distantly related species, 339 nearly full-length small-subunit rDNA sequences were analyzed from a diverse range of nematodes. Bayesian inference revealed a backbone comprising 12 consecutive dichotomies that subdivided the phylum Nematoda into 12 clades. The most basal clade is dominated by the subclass Enoplia, and members of the order Triplonchida occupy positions most close to the common ancestor of the nematodes. Crown Clades 8-12, a group formerly indicated as "Secernentea" that includes Caenorhabditis elegans and virtually all major plant and animal parasites, show significantly higher nucleotide substitution rates than the more basal Clades 1-7. Accelerated substitution rates are associated with parasitic lifestyles (Clades 8 and 12) or short generation times (Clades 9-11). The relatively high substitution rates in the distal clades resulted in numerous autapomorphies that allow in most cases DNA barcode-based species identification. Teratocephalus, a genus comprising terrestrial bacterivores, was shown to be most close to the starting point of Secernentean radiation. Notably, fungal feeding nematodes were exclusively found basal to or as sister taxon next to the 3 groups of plant parasitic nematodes, namely, Trichodoridae, Longidoridae, and Tylenchomorpha. The exclusive common presence of fungivorous and plant parasitic nematodes supports a long-standing hypothesis that states that plant parasitic nematodes arose from fungivorous ancestors.

Amino Acid Substitution↗
PubMed1979

Solar-driven chemical energy source for a Martian biota.

Microorganisms deep in the Martian soil could derive energy indirectly from the sun via chemical reactions involving atmospheric products of the solar ultraviolet flux. The Viking discovery of a chemically uniform regolith which, though poor in organics, is rich in sulfur-containing compounds suggests reaction sequences in which sulfur is recycled through reduced and oxidized states by biologically catalyzed reactions with photochemically-produced atmospheric constituents. One candidate reaction, reduction of soil ssufate minerals by molecular hydrogen, is already exploited on earth by bacteria of the ubiquitous and tenacious Desulfovibrio genus.

Desulfovibrio↗
PubMed2001

Archaeology of Archaea: geomicrobiological record of Pleistocene thermal events concealed in a deep-sea subseafloor environment.

A record of the history of the Earth is hidden in the Earth's crust, like the annual rings of an old tree. From very limited records retrieved from deep underground, one can infer the geographical, geological, and biological events that occurred throughout Earth's history. Here we report the discovery of vertically shifted community structures of Archaea in a typical oceanic subseafloor core sample (1410 cm long) recovered from the West Philippine Basin at a depth of 5719 m. Beneath a surface community of ubiquitous deep-sea archaea (marine crenarchaeotic group I; MGI), an unusual archaeal community consisting of extremophilic archaea, such as extreme halophiles and hyperthermophiles, was present. These organisms could not be cultivated, and may be microbial relicts more than 2 million years old. Our discovery of archaeal rDNA in this core sample, probably associated with the past terrestrial volcanic and submarine hydrothermal activities surrounding the West Philippine Basin, serves as potential geomicrobiological evidence reflecting novel records of geologic thermal events in the Pleistocene period concealed in the deep-sea subseafloor.

Archaea↗
PubMed2004

Evolutionary computer programming of protein folding and structure predictions.

In order to understand the mechanism of protein folding and to assist the rational de-novo design of fast-folding, non-aggregating and stable artificial enzymes it is very helpful to be able to simulate protein folding reactions and to predict the structures of proteins and other biomacromolecules. Here, we use a method of computer programming called "evolutionary computer programming" in which a program evolves depending on the evolutionary pressure exerted on the program. In the case of the presented application of this method on a computer program for folding simulations, the evolutionary pressure exerted was towards faster finding deep minima in the energy landscape of protein folding. Already after 20 evolution steps, the evolved program was able to find deep minima in the energy landscape more than 10 times faster than the original program prior to the evolution process.

Amino Acid Sequence↗
PubMed2002

Crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

The structure of Pyrococcus furiosus carboxypeptidase (PfuCP) has been determined to 2.2 A resolution using multiwavelength anomalous diffraction (MAD) methods. PfuCP represents the first structure of the new M32 family of carboxypeptidases. The overall structure is comprised of a homodimer. Each subunit is mostly helical with its most pronounced feature being a deep substrate binding groove. The active site lies at the bottom of this groove and contains an HEXXH motif that coordinates the metal ion required for catalysis. Surprisingly, the structure is similar to the recently reported rat neurolysin. Comparison of these structures as well as sequence analyses with other homologous proteins reveal several conserved residues. The roles for these conserved residues in the catalytic mechanism are inferred based on modeling and their location.

Amino Acid Sequence↗