Influence of MP 29-02 on ciliary beat frequency in human epithelial cells in vitro.

PURPOSE: MP 29-02, which contains fluticasone propionate and azelastine hydrochloride, is used as a topical nasal application for the treatment of seasonal and perennial allergic rhinitis. Although a multitude of data is available on the clinical symptom reduction and treatment safety of MP 29-02, the effect of MP 29-02 on ciliary beat frequency (CBF) has not been evaluated thus far. METHODS: MP 29-02-containing solution was applied at concentrations of 2.5, 5, 10, and 20% to 14 healthy subjects, and nasal ciliated epithelial cells were then visualized using a phase-contrast microscope. CBF was measured after the application of MP 29-02. For a comparison, fluticasone propionate was used. CBF measurements were then performed for 15 min at 22 °C. Ringer's solution was applied as a negative control. RESULTS: MP 29-02 significantly reduced CBF at all the tested concentrations compared with that of the control group within the observation time. At a 2.5% concentration, MP 29-02 significantly reduced CBF from 6.81 Hz (SD ± 1.35 Hz) at baseline to 4.88 Hz (SD ± 1.52 Hz, p < 0.001) after 15 min. In contrast, for fluticasone propionate, a significant reduction was observed only with the 20% concentration after 5, 10, and 15 min. CONCLUSIONS: MP 29-09 significantly reduced CB, with an almost linear relationship between the MP 29-09 concentration and reduction in CBF. For fluticasone propionate, a significant reduction of CBF was observed only at the highest analyzed concentration. The findings have implications for the long-term use of the MP 29-02. Yet, further clinical studies are needed to confirm these results in vivo, especially in patients with seasonal or perennial allergic rhinits.

The UCSC Genome Browser Database: update 2009.

The UCSC Genome Browser Database (GBD, http://genome.ucsc.edu) is a publicly available collection of genome assembly sequence data and integrated annotations for a large number of organisms, including extensive comparative-genomic resources. In the past year, 13 new genome assemblies have been added, including two important primate species, orangutan and marmoset, bringing the total to 46 assemblies for 24 different vertebrates and 39 assemblies for 22 different invertebrate animals. The GBD datasets may be viewed graphically with the UCSC Genome Browser, which uses a coordinate-based display system allowing users to juxtapose a wide variety of data. These data include all mRNAs from GenBank mapped to all organisms, RefSeq alignments, gene predictions, regulatory elements, gene expression data, repeats, SNPs and other variation data, as well as pairwise and multiple-genome alignments. A variety of other bioinformatics tools are also provided, including BLAT, the Table Browser, the Gene Sorter, the Proteome Browser, VisiGene and Genome Graphs.

The UCSC Genome Browser Database: 2008 update.

The University of California, Santa Cruz, Genome Browser Database (GBD) provides integrated sequence and annotation data for a large collection of vertebrate and model organism genomes. Seventeen new assemblies have been added to the database in the past year, for a total coverage of 19 vertebrate and 21 invertebrate species as of September 2007. For each assembly, the GBD contains a collection of annotation data aligned to the genomic sequence. Highlights of this year's additions include a 28-species human-based vertebrate conservation annotation, an enhanced UCSC Genes set, and more human variation, MGC, and ENCODE data. The database is optimized for fast interactive performance with a set of web-based tools that may be used to view, manipulate, filter and download the annotation data. New toolset features include the Genome Graphs tool for displaying genome-wide data sets, session saving and sharing, better custom track management, expanded Genome Browser configuration options and a Genome Browser wiki site. The downloadable GBD data, the companion Genome Browser toolset and links to documentation and related information can be found at: http://genome.ucsc.edu/.

Radioreceptor assay for 1 alpha,25-dihydroxyvitamin D3.

A competitive protein binding assay with a sensitivity of 80 picograms has been developed for 1alpha,25-dihydroxyvitamin D(3), the hormonal form of vitamin D(3). lalpha,25-Dihydroxyvitamin D(3) displaced tritiated hormone from a cytosol-chromatin receptor preparation isolated from chick small intestine, providing a simple assay for the hormone. The concentration of lalpha, 25-dihydroxyvitamin D(3) in human plasma, as determined by this assay, is approximately 6 nanograms per 100 milliliters; in patients with renal disease the concentration of this kidney-produced hormone is significantly lower.

The UCSC genome browser database: update 2007.

The University of California, Santa Cruz Genome Browser Database contains, as of September 2006, sequence and annotation data for the genomes of 13 vertebrate and 19 invertebrate species. The Genome Browser displays a wide variety of annotations at all scales from the single nucleotide level up to a full chromosome and includes assembly data, genes and gene predictions, mRNA and EST alignments, and comparative genomics, regulation, expression and variation data. The database is optimized for fast interactive performance with web tools that provide powerful visualization and querying capabilities for mining the data. In the past year, 22 new assemblies and several new sets of human variation annotation have been released. New features include VisiGene, a fully integrated in situ hybridization image browser; phyloGif, for drawing evolutionary tree diagrams; a redesigned Custom Track feature; an expanded SNP annotation track; and many new display options. The Genome Browser, other tools, downloadable data files and links to documentation and other information can be found at http://genome.ucsc.edu/.

The UCSC Genome Browser Database: update 2006.

The University of California Santa Cruz Genome Browser Database (GBD) contains sequence and annotation data for the genomes of about a dozen vertebrate species and several major model organisms. Genome annotations typically include assembly data, sequence composition, genes and gene predictions, mRNA and expressed sequence tag evidence, comparative genomics, regulation, expression and variation data. The database is optimized to support fast interactive performance with web tools that provide powerful visualization and querying capabilities for mining the data. The Genome Browser displays a wide variety of annotations at all scales from single nucleotide level up to a full chromosome. The Table Browser provides direct access to the database tables and sequence data, enabling complex queries on genome-wide datasets. The Proteome Browser graphically displays protein properties. The Gene Sorter allows filtering and comparison of genes by several metrics including expression data and several gene properties. BLAT and In Silico PCR search for sequences in entire genomes in seconds. These tools are highly integrated and provide many hyperlinks to other databases and websites. The GBD, browsing tools, downloadable data files and links to documentation and other information can be found at http://genome.ucsc.edu/.

Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel.

RNA and DNA strands produce ionic current signatures when driven through an alpha-hemolysin channel by an applied voltage. Here we combine this nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale. Measurable properties include duplex stem length, base pair mismatches, and loop length. This nanopore instrument can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.

The UCSC Genome Browser Database.

The University of California Santa Cruz (UCSC) Genome Browser Database is an up to date source for genome sequence data integrated with a large collection of related annotations. The database is optimized to support fast interactive performance with the web-based UCSC Genome Browser, a tool built on top of the database for rapid visualization and querying of the data at many levels. The annotations for a given genome are displayed in the browser as a series of tracks aligned with the genomic sequence. Sequence data and annotations may also be viewed in a text-based tabular format or downloaded as tab-delimited flat files. The Genome Browser Database, browsing tools and downloadable data files can all be found on the UCSC Genome Bioinformatics website (http://genome.ucsc.edu), which also contains links to documentation and related technical information.

Hidden Markov models in computational biology. Applications to protein modeling.

Hidden Markov Models (HMMs) are applied to the problems of statistical modeling, database searching and multiple sequence alignment of protein families and protein domains. These methods are demonstrated on the globin family, the protein kinase catalytic domain, and the EF-hand calcium binding motif. In each case the parameters of an HMM are estimated from a training set of unaligned sequences. After the HMM is built, it is used to obtain a multiple alignment of all the training sequences. It is also used to search the SWISS-PROT 22 database for other sequences that are members of the given protein family, or contain the given domain. The HMM produces multiple alignments of good quality that agree closely with the alignments produced by programs that incorporate three-dimensional structural information. When employed in discrimination tests (by examining how closely the sequences in a database fit the globin, kinase and EF-hand HMMs), the HMM is able to distinguish members of these families from non-members with a high degree of accuracy. Both the HMM and PROFILESEARCH (a technique used to search for relationships between a protein sequence and multiply aligned sequences) perform better in these tests than PROSITE (a dictionary of sites and patterns in proteins). The HMM appears to have a slight advantage over PROFILESEARCH in terms of lower rates of false negatives and false positives, even though the HMM is trained using only unaligned sequences, whereas PROFILESEARCH requires aligned training sequences. Our results suggest the presence of an EF-hand calcium binding motif in a highly conserved and evolutionary preserved putative intracellular region of 155 residues in the alpha-1 subunit of L-type calcium channels which play an important role in excitation-contraction coupling. This region has been suggested to contain the functional domains that are typical or essential for all L-type calcium channels regardless of whether they couple to ryanodine receptors, conduct ions or both.

Sequence comparisons using multiple sequences detect three times as many remote homologues as pairwise methods.

The sequences of related proteins can diverge beyond the point where their relationship can be recognised by pairwise sequence comparisons. In attempts to overcome this limitation, methods have been developed that use as a query, not a single sequence, but sets of related sequences or a representation of the characteristics shared by related sequences. Here we describe an assessment of three of these methods: the SAM-T98 implementation of a hidden Markov model procedure; PSI-BLAST; and the intermediate sequence search (ISS) procedure. We determined the extent to which these procedures can detect evolutionary relationships between the members of the sequence database PDBD40-J. This database, derived from the structural classification of proteins (SCOP), contains the sequences of proteins of known structure whose sequence identities with each other are 40% or less. The evolutionary relationships that exist between those that have low sequence identities were found by the examination of their structural details and, in many cases, their functional features. For nine false positive predictions out of a possible 432,680, i.e. at a false positive rate of about 1/50,000, SAM-T98 found 35% of the true homologous relationships in PDBD40-J, whilst PSI-BLAST found 30% and ISS found 25%. Overall, this is about twice the number of PDBD40-J relations that can be detected by the pairwise comparison procedures FASTA (17%) and GAP-BLAST (15%). For distantly related sequences in PDBD40-J, those pairs whose sequence identity is less than 30%, SAM-T98 and PSI-BLAST detect three times the number of relationships found by the pairwise methods.

A discriminative framework for detecting remote protein homologies.

A new method for detecting remote protein homologies is introduced and shown to perform well in classifying protein domains by SCOP superfamily. The method is a variant of support vector machines using a new kernel function. The kernel function is derived from a generative statistical model for a protein family, in this case a hidden Markov model. This general approach of combining generative models like HMMs with discriminative methods such as support vector machines may have applications in other areas of biosequence analysis as well.

Improved splice site detection in Genie.

We present an improved splice site predictor for the genefinding program Genie. Genie is based on a generalized Hidden Markov Model (GHMM) that describes the grammar of a legal parse of a multi-exon gene in a DNA sequence. In Genie, probabilities are estimated for gene features by using dynamic programming to combine information from multiple content and signal sensors, including sensors that integrate matches to homologous sequences from a database. One of the hardest problems in genefinding is to determine the complete gene structure correctly. The splice site sensors are the key signal sensors that address this problem. We replaced the existing splice site sensors in Genie with two novel neural networks based on dinucleotide frequencies. Using these novel sensors, Genie shows significant improvements in the sensitivity and specificity of gene structure identification. Experimental results in tests using a standard set of annotated genes showed that Genie identified 86% of coding nucleotides correctly with a specificity of 85%, versus 80% and 84% in the older system. In further splice site experiments, we also looked at correlations between splice site scores and intron and exon lengths, as well as at the effect of distance to the nearest splice site on false positive rates.

Response of CFU-GM (colony forming units for granulocytes and macrophages) from intact and pinealectomized rat bone marrow to murine recombinant interleukin-3 (rIl-3), recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) and human recombinant erythropoietin (rEPO).

The present study provides evidence, for the first time, of a role of the pineal gland in the proliferation of CFU-GM under the influence of different cytokines. The degree of colony formation in different cytokine groups was evaluated after 24 h, 3, 7 and 10 days. The colony growth in the present type of bone marrow cell cultures slows after the 10th day and ceases after the 14th day of incubation. The results show that rGM-CSF and the combination of rGM-CSF with rIl-3 and with rEPO stimulate the colony formation of granulocytes and macrophages. A 50% general reduction in the colony number was noted in the pinealectomy group. Their pattern of response to the different cytokines was similar to that of the intact group. It is suggested that the pineal activity has a physiological role in important aspects of host defence mechanisms, such as the proliferation of CFU-GM.

Integrating genomes.

As genomic sequencing projects attempt ever more ambitious integration of genetic, molecular, and phenotypic information, a specialization of genomics has emerged, embodied in the subdiscipline of computational genomics. Models inherited from population genetics, phylogenetics, and human disease genetics merge with those from graph theory, statistics, signal processing, and computer science to provide a rich quantitative foundation for genomics that can only be realized with the aid of a computer. Unleashed on a rapidly increasing sample of the planet's 10(30) organisms, these analyses will have an impact on diverse fields of science while providing an extraordinary new window into the story of life.

Using multiple alignments and phylogenetic trees to detect RNA secondary structure.

We describe a statistical method to determine if a pair of columns in a multiple alignment of a homologous family of RNA sequences shows evidence of being base paired. The method makes explicit use of a given phylogenetic tree for the sequences in the alignment. It is tested on a multiple alignment of 16S rRNA sequences with good results.

Effect of the pineal gland on circadian rhythmicity of colony forming units for granulocytes and macrophages (CFU-GM) from rat bone marrow cell cultures.

The present study provides evidence that the pineal gland has a physiological role in the proliferation of colony forming units for granulocytes and macrophages (CFU-GM). A biphasic circadian rhythm of CFU-GM proliferation in rat bone marrow cell cultures (BMC) from intact animals peaking at 0600 and 1800 was observed. Pinealectomy (Px) at 1600 obliterated the circadian rhythm patterns of CFU-GM. Afternoon injections of melatonin (1630, 20 micrograms/per day for 10 days) to Px animals restored the rhythmicity. When pinealectomy was done at 0800, the morning peak remained unaltered and the colony number at 1800 was higher than that found in the afternoon Px animals. In conclusion, the pineal gland or its main hormone melatonin seems to have a regulatory role in the proliferation of CFU-GM in rat BMC. Further, the expression of the activity of CFU-GM in rat BMC depends on the time when pinealectomy is done or melatonin is substituted.

Assembly of the working draft of the human genome with GigAssembler.

The data for the public working draft of the human genome contains roughly 400,000 initial sequence contigs in approximately 30,000 large insert clones. Many of these initial sequence contigs overlap. A program, GigAssembler, was built to merge them and to order and orient the resulting larger sequence contigs based on mRNA, paired plasmid ends, EST, BAC end pairs, and other information. This program produced the first publicly available assembly of the human genome, a working draft containing roughly 2.7 billion base pairs and covering an estimated 88% of the genome that has been used for several recent studies of the genome. Here we describe the algorithm used by GigAssembler.

Optimally parsing a sequence into different classes based on multiple types of evidence.

We consider the problem of parsing a sequence into different classes of subsequences. Two common examples are finding the exons and introns in genomic sequences and identifying the secondary structure domains of protein sequences. In each case there are various types of evidence that are relevant to the classification, but none are completely reliable, so we expect some weighted average of all the evidence to provide improved classifications. For example, in the problem of identifying coding regions in genomic DNA, the combined use of evidence such as codon bias and splice junction patterns can give more reliable predictions than either type of evidence alone. We show three main results: 1. For a given weighting of the evidence a dynamic programming algorithm returns the optimal parse and any number of sub-optimal parses. 2. For a given weighting of the evidence a dynamic programming algorithm determines the probability of the optimal parse and any number of sub-optimal parses under a natural Boltzmann-Gibbs distribution over the set of possible parses. 3. Given a set of sequences with known correct parses, a dynamic programming algorithm allows one to apply gradient descent to obtain the weights that maximize the probability of the correct parses of these sequences.