Disrupted Iron Storage in Dental Fluorosis.

Enamel formation and quality are dependent on environmental conditions, including exposure to fluoride, which is a widespread natural element. Fluoride is routinely used to prevent caries. However, when absorbed in excess, fluoride may also lead to altered enamel structural properties associated with enamel gene expression modulations. As iron plays a determinant role in enamel quality, the aim of our study was to evaluate the iron metabolism in dental epithelial cells and forming enamel of mice exposed to fluoride, as well as its putative relation with enamel mechanical properties. Iron storage was investigated in dental epithelial cells with Perl's blue staining and secondary ion mass spectrometry imaging. Iron was mainly stored by maturation-stage ameloblasts involved in terminal enamel mineralization. Iron storage was drastically reduced by fluoride. Among the proteins involved in iron metabolism, ferritin heavy chain (Fth), in charge of iron storage, appeared as the preferential target of fluoride according to quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry analyses. Fluorotic enamel presented a decreased quantity of iron oxides attested by electron spin resonance technique, altered mechanical properties measured by nanoindentation, and ultrastructural defects analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. The in vivo functional role of Fth was illustrated with Fth+/- mice, which incorporated less iron into their dental epithelium and exhibited poor enamel quality. These data demonstrate that exposure to excessive fluoride decreases ameloblast iron storage, which contributes to the defective structural and mechanical properties in rodent fluorotic enamel. They raise the question of fluoride's effects on iron storage in other cells and organs that may contribute to its effects on population health.

The effect of estrogen-related receptor α on the regulation of angiogenesis after spinal cord injury.

Estrogen receptor-related receptor-α (ERRα) is an orphan member of the nuclear receptor superfamily that interacts with peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) to stimulate vascular endothelial growth factor (VEGF) expression and angiogenesis in a hypoxia-inducible factor-1α-independent pathway. Although it is not regulated by any natural ligand, the action of ERRα can be blocked by the synthetic molecule XCT790. In the present study, Sprague-Dawley rats were randomly allocated to a sham group, injury-saline group or injury-XCT90 group. A modified Allen's weight-drop method was applied to induce the acute traumatic spinal cord injury (SCI) model in these rats, and an injection of XCT790 was administered every 24h, starting half an hour after the SCI contusion. Histological analyses revealed that XCT790 significantly aggravated tissue damage and decreased the number of ERRα-positive cells at 1, 3 and 7 days after SCI. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses also indicated that XCT790 dramatically repressed the expression of ERRα, thus reducing the expression of VEGF and angiopoietin-2 (Ang-2) throughout the duration of the experiment, but the expression of PGC-1α was not affected. Immunofluorescence analyses indicated that vascular density and endothelial cell proliferation were decreased in the injury-XCT90 group compared with the injury-saline group. These results suggest that ERRα is involved in mediating angiogenesis after SCI in the rat traumatic SCI model.

Cyanobacterial diversity and activity in modern conical microbialites.

Modern conical microbialites are similar to some ancient conical stromatolites, but growth, behavior and diversity of cyanobacteria in modern conical microbialites remain poorly characterized. Here, we analyze the diversity of cyanobacterial 16S rRNA gene sequences in conical microbialites from 14 ponds fed by four thermal sources in Yellowstone National Park and compare cyanobacterial activity in the tips of cones and in the surrounding topographic lows (mats), respectively, by high-resolution mapping of labeled carbon. Cones and adjacent mats contain similar 16S rRNA gene sequences from genetically distinct clusters of filamentous, non-heterocystous cyanobacteria from Subsection III and unicellular cyanobacteria from Subsection I. These sequences vary among different ponds and between two sampling years, suggesting that coniform mats through time and space contain a number of cyanobacteria capable of vertical aggregation, filamentous cyanobacteria incapable of initiating cone formation and unicellular cyanobacteria. Unicellular cyanobacteria are more diverse in topographic lows, where some of these organisms respond to nutrient pulses more rapidly than thin filamentous cyanobacteria. The densest active cyanobacteria are found below the upper 50 µm of the cone tip, whereas cyanobacterial cells in mats are less dense, and are more commonly degraded or encrusted by silica. These spatial differences in cellular activity and density within macroscopic coniform mats imply a strong role for diffusion limitation in the development and the persistence of the conical shape. Similar mechanisms may have controlled the growth, morphology and persistence of small coniform stromatolites in shallow, quiet environments throughout geologic history.

Stimulation of the sacral nerve reduces gut bacterial translocation and endotoxemia caused by acute spinal cord injury in rabbits.

Study design:Disturbance of gastrointestinal motility following acute spinal cord injury complicated with paraplegia can lead to bacterial overgrowth in the gastrointestinal tract, and increase the incidence of bacterial translocation. Experiments in a New Zealand rabbit model of acute spinal cord injury were performed.Objective:This study was designed to determine if the electrical stimulation of the sacral nerve prevents gut-origin bacterial translocation and endotoxemia in an animal model of acute spinal cord injury.Settings:Xiangya Hospital, Central South University, Hunan, PR China.Methods:New Zealand rabbits were divided into three groups: Control group, Paraplegic Vehicle group without sacral nerve stimulation and Paraplegic Experiment group with sacral nerve stimulation. Blood and multiple organs were collected for bacterial cultures and endotoxin determination at 24, 48 and 72 h following spinal cord injury. The histology and ultra-structural features of the organs were studied.Results:Bacterial translocation and endotoxemia were observed in all animals with acute spinal cord injury. Sacral nerve stimulation increased defecation, decreased endotoxin levels and bacterial translocation and improved the morphology of the organs.Conclusion:After acute spinal cord injury, stimulation of the sacral nerve reduced gut bacterial translocation and endotoxemia.Spinal Cord advance online publication, 6 April 2010; doi:10.1038/sc.2010.35.

Kinetics of intracolloidal iodine in thyroid of iodine-deficient or equilibrated newborn rats. Direct imaging using secondary ion mass spectrometry.

The most significant impact of the Chernobyl accident is the increased incidence of thyroid cancers among children. In order to accurately estimate the radiation dose provided by radioiodines, it is important to examine how the distribution of newly incorporated iodine varies with time and if this distribution varies according to the iodine status. The kinetic distribution of intra colloidal newly organified iodine in the rat immature thyroid was recorded and analysed using the ionic nanoprobe NanoSims50. Our observations imply that in case of radioiodine contamination, the energy deposits vary (i) with time, (ii) from one follicle to another, and (iii) from one cell to another inside the same follicle regardless the iodine status. The kinetic heterogeneity of iodine distribution must be take in account in thyroid dose evaluation.

Study of the localization of iron, ferritin, and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level.

Previous studies of the structure of core nanocrystals of ferritin (Ft) in the brains of patients with Alzheimer's disease (AD) have shown differences in the mineral compound in comparison with physiological Ft. Both Ft cores have a polyphasic composition but whereas the major phase in physiological Ft is hexagonal ferric iron oxide (ferrihydrite), the major phases in brain AD Ft are two cubic mixed ferric-ferrous iron oxides (magnetite and wüstite). One of these (wüstite) is similar to what is detected in hemosiderin (Hm) cores in primary hemochromatosis (Quintana, C., Cowley, J.M, Marhic, C., 2004. Electron nanodiffraction and high resolution electron microscopy studies of the structure and composition of physiological and pathological ferritin. J. Struct. Biol. 147, 166-178). We have studied, herein, the distribution of iron, Ft, and Hm in sections of AD hippocampus using analytical microscopy. Iron present in Ft cores was directly mapped in a nanoSIMS microscope and the iron distribution has been correlated with the constituent elements N, P, and S. Ft and Hm cores were visualized at an ultrastructural level in an analytical transmission electron microscope. In senile plaques, Ft was observed in the coronal region associated with a non-beta-amyloid component and in the periphery of plaques, together with Hm, in sulfur-rich dense bodies of dystrophic neurites. Hm was also found in lysosomes and siderosomes of glial cells. Ft was observed in the cytoplasm and nucleus of oligodendrocytes. Ft was particularly abundant in myelinated axons in association with oligodendrocyte processes. These findings provide new arguments to support the hypothesis of a dysfunction of Ft (with eventual degradation to Hm) in AD resulting in an increase of toxic brain ferrous ions that may contribute to the production of free radicals that induce both cellular oxidative stress and aged-related myelin breakdown associated with cognitive decline and AD (Bartzokis, G., 2004. Age-related myelin breakdown: a developmental model of cognitive decline and Alzheimer's disease. Neurobiol. Aging 25, 5-18).

Expression of vascular endothelial growth factor, hypoxia inducible factor 1alpha, and carbonic anhydrase IX in human tumours.

AIMS: To measure vascular endothelial growth factor (VEGF-A) mRNA in a large, diverse cohort of tumours and to investigate whether VEGF-A expression is associated with markers of hypoxia, including hypoxia inducible factor 1alpha (HIF-1alpha) and carbonic anhydrase IX (CA9). METHODS: The expression of VEGF-A and CA9 was assessed in 5067 fresh frozen human tissue samples and 238 cell lines by DNA microarray analysis. In addition, tissue microarrays were constructed from 388 malignancies to investigate the expression of VEGF-A and HIF-1alpha by in situ hybridisation and immunohistochemistry, respectively. RESULTS: VEGF-A was significantly upregulated in primary malignancies of the breast, cervix, colon and rectum, oesophagus, head and neck, kidney, ovary, skin, urinary system, and white blood cells by DNA microarray analysis. However, VEGF-A expression only correlated with CA9 expression in renal tissues. In the tissue microarrays, HIF-1alpha positive cores showed a significant increase in VEGF-A expression in lung, ovary, soft tissue, and thyroid malignancies. CONCLUSIONS: The expression of VEGF-A is upregulated in a large proportion of human malignancies, and may be associated with markers of hypoxia. VEGF-A expression can be induced in the absence of hypoxia and hypoxia does not always provoke VEGF-A upregulation in tumours.

Analysing gene expression data from DNA microarrays to identify candidate genes.

Microarray data analysis can be divided into two tasks: grouping of genes to discover broad patterns of biological behaviour, and filtering of genes to identify specific genes of interest. Whereas the gene-grouping task is largely addressed by cluster analysis, the gene-filtering task relies primarily on hypothesis testing. This review article surveys analytical methods for the gene-filtering task. Various types of data analysis are discussed for four basic types of experimental protocols: a comparison of two biological samples; a comparison of two biological conditions; each represented by a set of replicate samples; a comparison of multiple biological conditions; and analysis of covariate information.

Fast probabilistic analysis of sequence function using scoring matrices.

MOTIVATION: We present techniques for increasing the speed of sequence analysis using scoring matrices. Our techniques are based on calculating, for a given scoring matrix, the quantile function, which assigns a probability, or p, value to each segmental score. Our techniques also permit the user to specify a p threshold to indicate the desired trade-off between sensitivity and speed for a particular sequence analysis. The resulting increase in speed should allow scoring matrices to be used more widely in large-scale sequencing and annotation projects. RESULTS: We develop three techniques for increasing the speed of sequence analysis: probability filtering, lookahead scoring, and permuted lookahead scoring. In probability filtering, we compute the score threshold that corresponds to the user-specified p threshold. We use the score threshold to limit the number of segments that are retained in the search process. In lookahead scoring, we test intermediate scores to determine whether they will possibly exceed the score threshold. In permuted lookahead scoring, we score each segment in a particular order designed to maximize the likelihood of early termination. Our two lookahead scoring techniques reduce substantially the number of residues that must be examined. The fraction of residues examined ranges from 62 to 6%, depending on the p threshold chosen by the user. These techniques permit sequence analysis with scoring matrices at speeds that are several times faster than existing programs. On a database of 12 177 alignment blocks, our techniques permit sequence analysis at a speed of 225 residues/s for a p threshold of 10-6, and 541 residues/s for a p threshold of 10-20. In order to compute the quantile function, we may use either an independence assumption or a Markov assumption. We measure the effect of first- and second-order Markov assumptions and find that they tend to raise the p value of segments, when compared with the independence assumption, by average ratios of 1.30 and 1.69, respectively. We also compare our technique with the empirical 99. 5th percentile scores compiled in the BLOCKSPLUS database, and find that they correspond on average to a p value of 1.5 x 10-5. AVAILABILITY: The techniques described above are implemented in a software package called EMATRIX. This package is available from the authors for free academic use or for licensed commercial use. The EMATRIX set of programs is also available on the Internet at http://motif.stanford.edu/ematrix.

Small functional adrenal cortical adenoma: treatment with CT-guided percutaneous acetic acid injection--report of three cases.

Two patients with Conn syndrome and one patient with Cushing syndrome underwent computed tomography (CT)-guided tumor ablation with a total of 5-11 mL of 50% acetic acid injected into their adrenal nodule (1.3-3.3 cm in diameter). No major complications were encountered during or after the procedure. All patients were symptom free with normal laboratory test results for at least 1-year follow-up. CT images showed complete cystic change with tumor size regression. Our preliminary results suggest that percutaneous acetic acid injection is a safe and effective alternative for treatment of small functional adrenal cortical adenoma.

Minimal-risk scoring matrices for sequence analysis.

We introduce a minimal-risk method for estimating the frequencies of amino acids at conserved positions in a protein family. Our method, called minimal-risk estimation, finds the optimal weighting between a set of observed amino acid counts and a set of pseudofrequencies, which represent prior information about the frequencies. We compute the optimal weighting by minimizing the expected distance between the estimated frequencies and the true population frequencies, measured by either a squared-error or a relative-entropy metric. Our method accounts for the source of the pseudofrequencies, which arise either from the background distribution of amino acids or from applying a substitution matrix to the observed data. Our frequency estimates therefore depend on the size and composition of the observed data as well as the source of the pseudofrequencies. We convert our frequency estimates into minimal-risk scoring matrices for sequence analysis. A large-scale cross-validation study, involving 48 variants of seven methods, shows that the best performing method is minimal-risk estimation using the squared-error metric. Our method is implemented in the package EMATRIX, which is available on the Internet at http://motif.stanford.edu/ematrix.

Regression analysis of multiple protein structures.

A general framework is presented for analyzing multiple protein structures using statistical regression methods. The regression approach can superimpose protein structures rigidly or with shear. Also, this approach can superimpose multiple structures explicitly, without resorting to pairwise superpositions. The algorithm alternates between matching corresponding landmarks among the protein structures and superimposing these landmarks. Matching is performed using a robust dynamic programming technique that uses gap penalties that adapt to the given data. Superposition is performed using either orthogonal transformations, which impose the rigid-body assumption, or affine transformations, which allow shear. The resulting regression model of a protein family measures the amount of structural variability at each landmark. A variation of our algorithm permits a separate weight for each landmark, thereby allowing one to emphasize particular segments of a protein structure or to compensate for variances that differ at various positions in a structure. In addition, a method is introduced for finding an initial correspondence, by measuring the discrete curvature along each protein backbone. Discrete curvature also characterizes the secondary structure of a protein backbone, distinguishing among helical, strand, and loop regions. An example is presented involving a set of seven globin structures. Regression analysis, using both affine and orthogonal transformations, reveals that globins are most strongly conserved structurally in helical regions, particularly in the mid-regions of the E, F, and G helices.

Modeling and superposition of multiple protein structures using affine transformations: analysis of the globins.

A novel approach for analyzing multiple protein structures is presented. A family of related protein structures may be characterized by an affine model, obtained by applying transformation matrices that permit both rotation and shear. The affine model and transformation matrices can be computed efficiently using a single eigen-decomposition. A novel method for finding correspondences is also introduced. This method matches curvatures along the protein backbone. The algorithm is applied to analyze a set of seven globin structures. Our method identifies 100 corresponding landmarks across all seven structures. Results show that most helices in globins can be identified by high curvature, with the exception of the C and D helices. Analysis of the superposition reveals that globins are most strongly conserved structurally in the mid-regions of the E and G helices.

Clinical course of gamma-hydroxybutyrate overdose.

STUDY OBJECTIVE: To describe the clinical characteristics and course of gamma-hydroxybutyrate (GHB) overdose. METHODS: We assembled a retrospective series of all cases of GHB ingestion see in an urban public-hospital emergency department and entered in a computerized database January 1993 through December 1996. From these cases we extracted demographic information, concurrent drug use, vital signs, Glasgow Coma Scale (GCS) score, laboratory values, and clinical course. RESULTS: Sixty-one (69%) of the 88 patients were male. The mean age was 28 years. Thirty-four cases (39%) involved coingestion of ethanol, and 25 (28%) involved coingestion of another drug, most commonly amphetamines. Twenty-five cases (28%) had a GCS score of 3, and 28 (33%) had scores ranging from 4 through 8. The mean time to regained consciousness from initial presentation among nonintubated patients with an initial GCS of 13 or less was 146 minutes (range, 16-389). Twenty-two patients (31%) had an initial temperature of 35 degrees C or less. Thirty-two (36%) had asymptomatic bradycardia; in 29 of these cases, the initial GCS score was 8 or less. Ten patients (11%) presented with hypotension (systolic blood pressure < or = 90 mm Hg); 6 of these patients also demonstrated concurrent bradycardia. Arterial blood gases were measured in 30 patients; 21 had a PCO2 of 45 or greater, with pH ranging from 7.24 to 7.34, consistent with mild acute respiratory acidosis. Twenty-six patients (30%) had an episode of emesis; in 22 of these cases, the initial GCS was 8 or less. CONCLUSION: In our study population, patients who overdosed on GHB presented with a markedly decreased level of consciousness. Coingestion of ethanol or other drugs is common, as are bradycardia, hypothermia, respiratory acidosis, and emesis. Hypotension occurs occasionally. Patients typically regain consciousness spontaneously within 5 hours of the ingestion.

Highly specific protein sequence motifs for genome analysis.

We present a method for discovering conserved sequence motifs from families of aligned protein sequences. The method has been implemented as a computer program called EMOTIF (http://motif. stanford.edu/emotif). Given an aligned set of protein sequences, EMOTIF generates a set of motifs with a wide range of specificities and sensitivities. EMOTIF also can generate motifs that describe possible subfamilies of a protein superfamily. A disjunction of such motifs often can represent the entire superfamily with high specificity and sensitivity. We have used EMOTIF to generate sets of motifs from all 7,000 protein alignments in the BLOCKS and PRINTS databases. The resulting database, called IDENTIFY (http://motif. stanford.edu/identify), contains more than 50,000 motifs. For each alignment, the database contains several motifs having a probability of matching a false positive that range from 10(-10) to 10(-5). Highly specific motifs are well suited for searching entire proteomes, while generating very few false predictions. IDENTIFY assigns biological functions to 25-30% of all proteins encoded by the Saccharomyces cerevisiae genome and by several bacterial genomes. In particular, IDENTIFY assigned functions to 172 of proteins of unknown function in the yeast genome.

Characterization of a desiccation-related protein in lily pollen during development and stress.

This work characterizes a lily (Lilium longiflorum Thunb. cv. Snow Queen) anther (LLA) protein associated with desiccation. Peptide mapping analysis revealed that the abundant LLA-23 doublet contained similar polypeptides, having an isoelectric point of 6.1. Immunoblots of pollen protein from developing anther/pollen confirmed that the LLA-23 protein accumulated only at the later stage of pollen maturation and that the levels remained steady in mature and vital pollen. The accumulation of LLA-23 proteins was correlated with desiccation that naturally occurred in pollen. Subcellular fractionation of pollen proteins revealed that the protein was located in the cytoplasmic fraction. Premature drying of developing pollen confirmed that the concomitant accumulation of LLA-23 was associated with desiccation. Peptide sequence analysis demonstrates similarities between the lily LLA-23 and a family of water-deficit/ripening-induced proteins including LP3 of pine, DS2 of potato, and Asr of tomato and pummelo. In addition, the concomitant accumulation of LLA-23 can be experimentally manipulated by methyl jasmonate (Me-JA) and salicylic acid (SA) as well as by mannitol and methyl viologen. The LLA-23 represents a novel member of the water-deficit/ripening-induced proteins.

Enumerating and ranking discrete motifs.

Discrete motifs that discriminate functional classes of proteins are useful for classifying new sequences, capturing structural constraints, and identifying protein subclasses. Despite the fact that the space of such motifs can grow exponentially with sequence length and number, we show that in practice it usually does not, and we describe a technique that infers motifs from aligned protein sequences by exhaustively searching this space. Our method generates sequence motifs over a wide range of recall and precision, and chooses a representative motif based on a score that we derive from both statistical and information-theoretic frameworks. Finally, we show that the selected motifs perform well in practice, classifying unseen sequences with extremely high precision, and infer protein subclasses that correspond to known biochemical classes.

A segment-based dynamic programming algorithm for predicting gene structure.

An algorithm called segment-based dynamic programming is described for predicting gene structure from a sequence of genomic DNA. The algorithm explores the space of gene structures that satisfy junctional and frame constraints and finds the gene structure that optimizes the sum of junctional and segmental scoring functions. Junctional constraints specify acceptable sites of initiation, termination, and splicing, whereas frame constraints ensure that the total exon length is a multiple of three and that no in-frame stop codons occur within exons or at exon-exon junctions. By computing over segments, segment-based dynamic programming maintains reading frame and phase information for each segment, it can assemble exons in-frame as well as score them in-frame. The algorithm is used to quantify the computational power of constraints. Experimental results show that frame constraints reduce the size of the search space by several orders of magnitude and that cardinality constraints place an asymptotic limit on the size of the search space. The algorithm is also used to compare the accuracy of different methods for assembly and scoring. A scoring scheme based on fifth-order Markov hexamer frequencies is presented and used in three objective functions, corresponding to in-frame, frame-independent, and frame-maximal scoring strategies. Experimental results show that in-frame assembly improves specificity only slightly over frame-independent assembly, whereas in-frame scoring improves specificity substantially over frame-independent and frame-maximal scoring.