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PubMed2005

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria.

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_T1038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge.

Gammaproteobacteria↗

PubMed2003

Disseminated pythiosis in three horses.

Three cases of equine subcutaneous pythiosis with dissemination to the internal organs were investigated. The subcutaneous lesions were observed on the mammary gland, nostrils and limbs of the infected horses. Histopathological analysis of the infected tissues revealed a strong eosinophilic reaction, with macrophages, mast cells and giant cells. Sparsely septated hyphal filaments of 4-6 microm diameter were identified in the center of the eosinophilic areas. Specific fluorescent antibody against Pythium insidiosum confirmed the hyphae in the infected tissues in three examined horses. In one of the three cases, the DNA sequences amplified from the infected subcutaneous tissues and internal organs, revealed that P. insidiosum's 18S SSU rDNA amplicons shared 100% identity with those sequences deposit in GenBank. This is the first report confirming by immunochemical and genetic techniques that P. insidiosum can disseminated from superficial to deep structures.

Animals↗

PubMed1988

The structure of rat mast cell protease II at 1.9-A resolution.

The structure of rat mast cell protease II (RMCP II), a serine protease with chymotrypsin-like primary specificity, has been determined to a nominal resolution of 1.9 A by single isomorphous replacement, molecular replacement, and restrained crystallographic refinement to a final R-factor of 0.191. There are two independent molecules of RMCP II in the asymmetric unit of the crystal. The rms deviation from ideal bond lengths is 0.016 A and from ideal bond angles is 2.7 degrees. The overall structure of RMCP II is extremely similar to that of chymotrypsin, but the largest differences between the two structures are clustered around the active-site region in a manner which suggests that the unusual substrate specificity of RMCP II is due to these changes. Unlike chymotrypsin, RMCP II has a deep cleft around the active site. An insertion of three residues between residues 35 and 41 of chymotrypsin, combined with concerted changes in sequence and a deletion near residue 61, allows residues 35-41 of RMCP II to adopt a conformation not seen in any other serine protease. Additionally, the loss of the disulfide bridge between residues 191 and 220 of chymotrypsin leads to the formation of an additional substrate binding pocket that we propose to interact with the P3 side chain of bound substrate. RMCP II is a member of a homologous subclass of serine proteases that are expressed by mast cells, neutrophils, lymphocytes, and cytotoxic T-cells. Thus, the structure of RMCP II forms a basis for an explanation of the unusual properties of other members of this class.

Amino Acid Sequence↗

PubMed2000

A Definitive Optical Detection of a Supercluster at z approximately 0.91.

We present the results from a multiband optical imaging program that has definitively confirmed the existence of a supercluster at z approximately 0.91. Two massive clusters of galaxies, Cl 1604+4304 at z=0.897 and Cl 1604+4321 at z=0.924, were originally observed in the high-redshift cluster survey of Oke, Postman, & Lubin. They are separated by 4300 km s-1 in radial velocity and 17&arcmin; on the plane of the sky. Their physical and redshift proximity suggested a promising supercluster candidate. Deep BRi imaging of the region between the two clusters indicates a large population of red galaxies. This population forms a tight, red sequence in the color-magnitude diagram at (R-i&parr0; approximately 1.4. The characteristic color is identical to that of the spectroscopically confirmed early-type galaxies in the two member clusters. The red galaxies are spread throughout the 5 h-1 Mpc region between Cl 1604+4304 and Cl 1604+4321. Their spatial distribution delineates the entire large-scale structure with high concentrations at the cluster centers. In addition, we detect a significant overdensity of red galaxies directly between Cl 1604+4304 and Cl 1604+4321 which is the signature of a third, rich cluster associated with this system. The strong sequence of red galaxies and their spatial distribution clearly indicate that we have discovered a supercluster at z approximately 0.91.

Journal Article↗

PubMed1992

The effect of respiratory manoeuvres and pharmacological agents on the pharmacokinetics of nedocromil sodium after inhalation.

1. Eight healthy subjects inhaled nedocromil sodium from a metered-dose inhaler using a standardised inspiratory technique. Blood samples were taken for up to 270 min after inhalation for radioimmunoassay of plasma nedocromil sodium concentrations. 2. To investigate the possibility that respiratory manoeuvres can alter the absorption of the drug from the lungs, on the first (control) study day at 70 min after dosing, subjects performed nine forced expiratory manoeuvres over a 3 min period. At 110 min after dosing, subjects took a slow, full inspiration with a 30 s breath-hold, and at 150 min after dosing the subjects performed one single forced expiration. 3. On the second study day, subjects inhaled methoxamine, 0.15 mg kg-1 of a 20 mg ml-1 solution at 60 min after dosing, and the study continued as above. On the third day, subjects repeated the sequence of respiratory manoeuvres, after having taken phenoxymethyl penicillin and probenecid by mouth for 48 h. 4. Both multiple forced expirations and the deep inspiration with breath-hold produced significant increases in the absorption of nedocromil sodium. Inhaled methoxamine did not alter airway calibre or the response to the respiratory manoeuvres. Probenecid, but not penicillin, was detected in the subjects' plasma, and had the effect of increasing the rise in plasma nedocromil sodium concentrations after the multiple forced expirations when compared with the control day. 5. These data suggest that disruption of epithelial tight junctions induced by the respiratory manoeuvres leads to enhanced paracellular transport of nedocromil sodium into the draining circulation of the airways and alveoli.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗

PubMed2004

Optical coherence tomography in the diagnosis of juvenile X-linked retinoschisis.

PURPOSE: To describe the value of optical coherence tomography (OCT) as a diagnostic tool in the diagnosis of X-linked retinoschisis. METHODS: We report three boys aged between 8 and 17 years, diagnosed with X-linked retinoschisis. During investigations they were examined with OCT (Zeiss Humphrey OCT 1, upgraded version). Single scans of the central posterior pole and the region around the vascular arcades were obtained. Two of the boys underwent full-field ERG according to ISCEV standards. Genetic analysis was performed in all three boys, with sequencing of the XLRS gene. RESULTS: The OCT results revealed a pattern with a cleavage of the retina in two distinct planes, one deep (outer retina) and one superficial. This was very obvious in one patient and a similar but not as pronounced pattern was seen in the other two cases. The two layers were superficially connected with thin-walled, vertical palisades, separated by low reflective, cystoid spaces, confluent and most prominent in the foveal region. CONCLUSION: Full-field ERG and/or DNA analysis are well known methods used for diagnosis of X-linked juvenile retinoschisis. In this paper, we suggest that OCT can also be a helpful diagnostic tool.

Adolescent↗

PubMed1992

Iliotibial band friction syndrome: MR imaging findings.

Six patients with clinical histories and physical examination results consistent with iliotibial band friction syndrome (ITBFS) were examined with magnetic resonance (MR) imaging. Ill-defined decreased signal intensity on T1-weighted images and increased signal intensity on T2-weighted images was present deep to the iliotibial band, adjacent to the lateral femoral epicondyle. Axial fast imaging with steady-state precession (FISP) gradient-echo sequences were essential in differentiating the ill-defined signal intensity abnormality associated with ITBFS from fluid in the lateral knee joint. None of these patients were found to have lateral meniscal tears, and all responded to conservative measures directed at treating ITBFS. The authors conclude that MR imaging may be useful in confirming or establishing the diagnosis of ITBFS in patients with the appropriate clinical history and distal lateral thigh or lateral knee pain.

Adolescent↗

PubMedDate not supplied

Influence of irradiation sequence on dentin bond of resin inlays.

The relationship between the order in which a dual-cured resin cement is light activated and the bond strength of resin inlay materials to dentin was examined. Also evaluated was the setting time with various irradiation sequences. Lite-Fil CR/Imperva Bond (Shofu) and Clearfil CR/CR Cement (Kuraray) were employed. Ten specimens, 4 mm in diameter by 4 mm deep, were made with each material for each condition and bonded to bovine dentin with the respective bonding agent and cement. Order of light activation was: 1) no light activation; 2) premix activation of only the liquid prior to mix, 3 seconds for Lite-Fil and 25 seconds for Clearfil; 3) postplacement activation, 30 seconds for Lite-Fil and 40 seconds for Clearfil; and 4) premix and postplacement activation, 3 seconds and 30 seconds for Lite-Fil and 25 seconds and 40 seconds for Clearfil. Samples were stored in water for 24 hours and shear strength tested. The setting time with no activation and premix activation was measured according to the ISO #7489 standards. Bond strengths (MPa) were 1) 4.41, 2) 13.07, 3) 6.34, and 4) 14.81 for Lite-Fil, and 1) 0.37, 2) 2.44, 3) 0.52, and 4) 2.51 for Clearfil. No light activation or only postplacement activation resulted in lower bond strengths with a 4.0 mm-thick specimen. The setting time of the cement mix with premix activation was shorter than with no activation. Light activation of these dual-cure cements is essential. Premix activation of only the liquid resulted in bond strengths similar to those obtained with combined pre- and postplacement activating.

Analysis of Variance↗

PubMed1995

Comparison of phenotypical and molecular methods for the identification of bacterial strains isolated from a deep subsurface environment.

Seventy-four bacterial strains were freshly isolated from a mine gallery. Using these bacteria, we have investigated how a molecular identification based on the analysis of small subunit rDNA sequences would compare in terms of precision and reliability to a more classical comparison of phenotypical descriptions (100 morphological and physiological traits). Our data clearly showed that a phylogenetic analysis of small subunit rDNA sequences is more efficient than classical phenotypic methods for the identification of bacterial strains freshly isolated from a natural environment, because occurrences of misidentification are very much decreased by this method. The lack of rDNA sequences for many described species is probably the major cause of a few failures in molecular identification, as the completeness of the database of small subunit rDNA sequences holds much importance in the degree of uncertainty in such identifications.

Journal Article↗

PubMed2004

MtDNA sequencing from zooplankton after long-term preservation in buffered formalin.

Molecular genetic analysis of zooplankton has been slowed by the usual practice of preservation and storage of samples in dilute formalin solutions, which are not always adequately buffered for pH. We report here the determination of DNA sequences for Meganyctiphanes norvegica (Crustacea, Euphausiacea) preserved and stored in buffered formalin for up to 25 years. Specifically designed molecular protocols for DNA extraction and PCR amplification yielded valid sequence data for short (approximately 100-200 bp) regions of the mitochondrial cytochrome b (mtCYB) gene for individual euphausiids. Critical aspects of our approach include: extended extraction and proteinase-K digestion to maximize DNA yield; use of protocols requiring short DNA fragments; design of species-specific PCR primers to minimize risks of contamination by exogenous DNA; and comparison with published DNA sequences for the same gene and species. We conclude that the yield of DNA and the success of subsequent molecular analyses depend primarily on the length of time the tissue has been exposed to formalin and the pH of the solution. Zooplankton samples intended for molecular analysis should preferably be preserved and maintained in ethanol or deep-frozen, but long-term storage in buffered formalin does not preclude some types of molecular genetic analysis.

Animals↗

PubMedDate not supplied

Two-dimensional time-of-flight MR venography: assessment with detection of chronic deep venous thrombosis in combination with magnetization transfer contrast.

PURPOSE: The purpose of this study was to determine the optimum imaging parameters for 2D-TOF MR venography (MRV) of the pelvis and suprapopliteal deep venous system in combination with off-resonance magnetization transfer constant (MTC) and to evaluate the use of MRV in patients suspected of having chronic deep venous thrombosis (DVT) by comparing its accuracy with that of conventional venography (CV). METHOD: MRV was performed in 10 normal subjects to determine the optimum imaging parameters. Systematically varied scan parameters included flip angles ranging from 30 to 120 degrees by 10 degrees increments. Best TR was calculated by using the theoretical relation between the optimal TR and flow velocity. We then evaluated the use of MRV in 26 patients (total 52 deep venous systems) suspected of having chronic DVT by comparing its accuracy in evaluating the intrapelvic and suprapopliteal deep venous system with that of CV. RESULTS: The optimal imaging parameters for intrapelvic and suprapopliteal venous system MRV were found to be a flip angle of 60 degrees and a pulse sequence of 35/6.9 (TR ms/TE ms) combined with an off-resonance MTC technique. All the main veins of the intrapelvic and suprapopliteal venous systems were clearly demonstrated in each subject. The sensitivity of MRV was 100% and its specificity was 100% for diagnosis of pelvic venous stenosis. The collateral vessels were demonstrated with MRV in all patients. CONCLUSION: 2D-TOF off-resonance MTC MRV of the pelvis and the suprapopliteal lower extremities is suitable for evaluation of intrapelvic branches and suprapopliteal deep venous system and chronic DVT.

Chronic Disease↗

PubMed2004

Bilaterian phylogeny based on analyses of a region of the sodium-potassium ATPase beta-subunit gene.

Molecular investigations of deep-level relationships within and among the animal phyla have been hampered by a lack of slowly evolving genes that are amenable to study by molecular systematists. To provide new data for use in deep-level metazoan phylogenetic studies, primers were developed to amplify a 1.3-kb region of the alpha subunit of the nuclear-encoded sodium-potassium ATPase gene from 31 bilaterians representing several phyla. Maximum parsimony, maximum likelihood, and Bayesian analyses of these sequences (combined with ATPase sequences for 23 taxa downloaded from GenBank) yield congruent trees that corroborate recent findings based on analyses of other data sets (e.g., the 18S ribosomal RNA gene). The ATPase-based trees support monophyly for several clades (including Lophotrochozoa, a form of Ecdysozoa, Vertebrata, Mollusca, Bivalvia, Gastropoda, Arachnida, Hexapoda, Coleoptera, and Diptera) but do not support monophyly for Deuterostomia, Arthropoda, or Nemertea. Parametric bootstrapping tests reject monophyly for Arthropoda and Nemertea but are unable to reject deuterostome monophyly. Overall, the sodium-potassium ATPase alpha-subunit gene appears to be useful for deep-level studies of metazoan phylogeny.

Animals↗

PubMed2026

Chiron3D: an interpretable deep learning framework for understanding the DNA code of chromatin looping.

MOTIVATION: Three-dimensional folding of the genome into structures such as chromatin loops is essential for gene regulation. Current experimental methods for mapping these structures, like Hi-C and HiChIP, are labor-intensive and require repeated assays to test hypothesized mutation effects. This motivates the need for predictive approaches that reveal the sequence determinants of chromatin loops. RESULTS: In this work, we present a novel and interpretable computational pipeline for predicting CTCF-mediated chromatin loops. We propose Chiron3D, a DNA-only model trained in a cell-type specific manner to predict CTCF HiChIP contact maps. By leveraging pre-trained embeddings from a foundation model, our approach is competitive with baselines that take CTCF ChIP-seq as additional input, while enabling nucleotide-level attribution to the input DNA sequence. Using our framework, we provide likely mechanistic insights into the physical control of loop dynamics. Specifically, we find that the strength of the loop extrusion anchorage site is largely governed by the amount and binding affinity of CTCF sites at the boundaries. Furthermore, we reveal that loop stability is regulated by the amount of intra-loop CTCF binding sites, where fewer intra-loop sites are associated with greater loop stability. Using targeted, single-nucleotide edit simulations with Chiron3D, we show that both loop strength and stability can be precisely controlled. Together, these results provide novel mechanistic insights into the physical control of genome organization and highlight the potential of decoding the DNA sequence logic in silico. AVAILABILITY: The Chiron3D pipeline is made available at https://github.com/BoevaLab/Chiron3D.

Chromatin↗

PubMed2004

Molecular evidence for the evolution of metal homeostasis genes by lateral gene transfer in bacteria from the deep terrestrial subsurface.

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P(IB)-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P(IB)-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the beta subclass of the Proteobacteria (beta-proteobacterium) that may have predated the diversification of the genus Pseudomonas. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of beta- and gamma-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P(IB)-type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.

Bacteria↗

PubMed1998

Frequency of the 20210 G-->A mutation in the 3'-untranslated region of the prothrombin gene in 35 cases of cerebral venous thrombosis.

BACKGROUND AND PURPOSE: A novel sequence variation in the 3'-untranslated region of the prothrombin (factor II) gene (nucleotide 20210 G-->A) has been recently described as a risk factor for deep vein thrombosis and pulmonary embolism. It is found in approximately 1% to 4% of healthy subjects. We studied the frequency of this factor II variant in patients with cerebral venous thrombosis. METHODS: The 20210A allele of the prothrombin gene was studied after DNA extraction, polymerase chain reaction amplification, and HindIII digestion in 35 patients with magnetic resonance imaging or angiographically confirmed cerebral venous thrombosis (23 women and 12 men, aged 11 to 71 years). RESULTS: Two patients (5.7%) had the 20210A allele of the prothrombin gene. Both had other risk factors for thrombosis (use of oral contraceptives and of intrathecal steroids). CONCLUSIONS: The 20210A allele of the prothrombin gene in association with other prothrombic factors may increase the risk of cerebral venous thrombosis, but case-control studies will be necessary to clarify these associations.

Adolescent↗

PubMed2004

The colonization of land by animals: molecular phylogeny and divergence times among arthropods.

BACKGROUND: The earliest fossil evidence of terrestrial animal activity is from the Ordovician, approximately 450 million years ago (Ma). However, there are earlier animal fossils, and most molecular clocks suggest a deep origin of animal phyla in the Precambrian, leaving open the possibility that animals colonized land much earlier than the Ordovician. To further investigate the time of colonization of land by animals, we sequenced two nuclear genes, glyceraldehyde-3-phosphate dehydrogenase and enolase, in representative arthropods and conducted phylogenetic and molecular clock analyses of those and other available DNA and protein sequence data. To assess the robustness of animal molecular clocks, we estimated the deuterostome-arthropod divergence using the arthropod fossil record for calibration and tunicate instead of vertebrate sequences to represent Deuterostomia. Nine nuclear and 15 mitochondrial genes were used in phylogenetic analyses and 61 genes were used in molecular clock analyses. RESULTS: Significant support was found for the unconventional pairing of myriapods (millipedes and centipedes) with chelicerates (spiders, scorpions, horseshoe crabs, etc.) using nuclear and mitochondrial genes. Our estimated time for the divergence of millipedes (Diplopoda) and centipedes (Chilopoda) was 442 +/- 50 Ma, and the divergence of insects and crustaceans was estimated as 666 +/- 58 Ma. Our results also agree with previous studies suggesting a deep divergence (approximately 1100 - 900 Ma) for arthropods and deuterostomes, considerably predating the Cambrian Explosion seen in the animal fossil record. CONCLUSIONS: The consistent support for a close relationship between myriapods and chelicerates, using mitochondrial and nuclear genes and different methods of analysis, suggests that this unexpected result is not an artefact of analysis. We propose the name Myriochelata for this group of animals, which includes many that immobilize prey with venom. Our molecular clock analyses using arthropod fossil calibrations support earlier studies using vertebrate calibrations in finding that deuterostomes and arthropods diverged hundreds of millions of years before the Cambrian explosion. However, our molecular time estimate for the divergence of millipedes and centipedes is close to the divergence time inferred from fossils. This suggests that arthropods may have adapted to the terrestrial environment relatively late in their evolutionary history.

Animals↗

PubMed1987

Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp.

Comparison of partial 16S rRNA sequences from representative Campylobacter species indicates that the Campylobacter species form a previously undescribed basic eubacterial group, which is related to the other major groups only by very deep branching. This analysis was extended to include the spiral bacterium associated with human gastritis, Campylobacter pylori (formerly Campylobacter pyloridis). The distance between C. pylori and the other Campylobacter species is sufficient to exclude the pyloric organism from the Campylobacter genus. The results indicate that C. pylori is more closely related to Wolinella succinogenes than it is to the other Campylobacter species inspected. Another close relative of the campylobacters was found to be Thiovulum, a sulfide-dependent marine bacterium.

Base Sequence↗

PubMed1997

Deep fascial hyperintensity in soft-tissue abnormalities as revealed by T2-weighted MR imaging.

OBJECTIVE: The purpose of this study was to determine whether recently described criteria, including hyperintense T2-weighted signal or other abnormalities revealed by MR imaging within deep fascial planes, are specific for necrotizing soft-tissue infections. MATERIALS AND METHODS: We reviewed 22 MR imaging examinations that revealed abnormally high signal intensity within deep fascial planes on T2-weighted images. Twenty-one of the patients had clinical diagnoses other than necrotizing soft-tissue infection, including nonnecrotizing cellulitis (n = 4), abscess without evidence of necrotizing fasciitis (n = 5), and cellulitis with accompanying vascular thrombosis (n = 2). MR imaging was performed using T1-weighted spin-echo (range of TRs/range of TEs, 300-800/9-30) and fat-saturated T2-weighted fast spin-echo (3000-5000/76-108) sequences. Gadolinium-enhanced T1-weighted spin-echo MR images with fat saturation were obtained for 14 patients. Two reviewers, unaware of clinical diagnoses, evaluated each study for abnormalities in superficial and deep soft tissues and submitted a consensus diagnosis. RESULTS: Using only the described MR imaging criteria, we interpreted all cases as necrotizing cellulitis, including the cases of 21 patients who had nonnecrotizing conditions. CONCLUSION: Hyperintense T2-weighted signal within deep fascial planes and muscle, with or without contrast enhancement, is not specific for necrotizing soft-tissue infection. A variety of conditions exhibited similar findings and were indistinguishable from necrotizing soft-tissue infection.

Adult↗