Diel gene expression improves software prediction of cyanobacterial operons.

Cyanobacteria are important participants in global biogeochemical process, but their metabolic processes and genomic functions are incompletely understood. In particular, operon structure, which can provide valuable metabolic and genomic insight, is difficult to determine experimentally, and algorithmic operon predictions probably underestimate actual operon extent. A software method is presented for enhancing current operon predictions by incorporating information from whole-genome time-series expression studies, using a Machine Learning classifier. Results are presented for the marine cyanobacterium Crocosphaera watsonii. A total of 15 operon enhancements are proposed. The source code is publicly available.

A novel approach to teaching Hidden Markov Models to a diverse undergraduate population.

Hidden Markov Models (HMMs) are an essential tool for Bioinformatic analysis, with extensive success at finding patterns (e.g. CRISPR arrays or genes of interest) in DNA or protein sequences. HMMs are conceptually intricate, and the algorithms that make use of them are complicated. Thus they present a challenge to Bioinformatics instructors at the undergraduate level, particularly when the students' educational backgrounds are broadly diverse. At San Jose State University, many undergraduate Bioinformatics students are Biology majors with little or no prior coursework in mathematics, statistics, or programming. For this population a theory-based approach to teaching HMMs would be ineffective. To address this problem we have developed an active learning module that takes advantage of the similarity between HMMs and board games. Our materials include a physical game board for introducing concepts, a software implementation of the game, similar software for visualizing and manipulating HMMs that model proteins, in-class lab exercises, and homework assignments. We have observed high student engagement with these materials over 4 semesters in a diverse undergraduate Advanced Bioinformatics course. Here we present our materials, which are freely available to educators.

A database of metazoan cytochrome c oxidase subunit I gene sequences derived from GenBank with CO-ARBitrator.

The Cytochrome C Oxidase subunit I gene ("COI") is the de facto standard for animal DNA barcoding. Organism identification based on COI requires an accurate and extensive annotated database of COI sequences. Such a database can also be of value in reconstructing evolutionary history and in diversity studies. Two COI databases are currently available: BOLD and Midori. BOLD's submissions conform to stringent sequence and metadata requirements; BOLD is specific to COI but makes no attempt to be comprehensive. Midori, derived from GenBank, has more sequences but less stringent standards than BOLD, resulting in higher error rates. To address the need for a comprehensive and accurate COI database, we adapted the ARBitrator algorithm, which classifies based only on sequence properties and has successfully auto-curated bacterial genes mined from GenBank. The adapted algorithm, which we call CO-ARBitrator, built a database of over a million metazoan COI sequences. Sensitivity and specificity are significantly higher than Midori. Specificity is comparable to what BOLD achieves with data quality prerequisites. Results and software are publicly available.

Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria.

Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed 'diazotrophs') are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats.

ARBitrator: a software pipeline for on-demand retrieval of auto-curated nifH sequences from GenBank.

MOTIVATION: Studies of the biochemical functions and activities of uncultivated microorganisms in the environment require analysis of DNA sequences for phylogenetic characterization and for the development of sequence-based assays for the detection of microorganisms. The numbers of sequences for genes that are indicators of environmentally important functions such as nitrogen (N2) fixation have been rapidly growing over the past few decades. Obtaining these sequences from the National Center for Biotechnology Information's GenBank database is problematic because of annotation errors, nomenclature variation and paralogues; moreover, GenBank's structure and tools are not conducive to searching solely by function. For some genes, such as the nifH gene commonly used to assess community potential for N2 fixation, manual collection and curation are becoming intractable because of the large number of sequences in GenBank and the large number of highly similar paralogues. If analysis is to keep pace with sequence discovery, an automated retrieval and curation system is necessary. RESULTS: ARBitrator uses a two-step process composed of a broad collection of potential homologues followed by screening with a best hit strategy to conserved domains. 34 420 nifH sequences were identified in GenBank as of November 20, 2012. The false-positive rate is ∼0.033%. ARBitrator rapidly updates a public nifH sequence database, and we show that it can be adapted for other genes. AVAILABILITY AND IMPLEMENTATION: Java source and executable code are freely available to non-commercial users at http://pmc.ucsc.edu/∼wwwzehr/research/database/. CONTACT: zehrj@ucsc.edu SUPPLEMENTARY INFORMATION: SUPPLEMENTARY INFORMATION is available at Bioinformatics online.

Whole genome comparison of six Crocosphaera watsonii strains with differing phenotypes.

Crocosphaera watsonii, a unicellular nitrogen-fixing cyanobacterium found in oligotrophic oceans, is important in marine carbon and nitrogen cycles. Isolates of C. watsonii can be separated into at least two phenotypes with environmentally important differences, indicating possibly distinct ecological roles and niches. To better understand the evolutionary history and variation in metabolic capabilities among strains and phenotypes, this study compared the genomes of six C. watsonii strains, three from each phenotypic group, which had been isolated over several decades from multiple ocean basins. While a substantial portion of each genome was nearly identical to sequences in the other strains, a few regions were identified as specific to each strain and phenotype, some of which help explain observed phenotypic features. Overall, the small-cell type strains had smaller genomes and a relative loss of genetic capabilities, while the large-cell type strains were characterized by larger genomes, some genetic redundancy, and potentially increased adaptations to iron and phosphorus limitation. As such, strains with shared phenotypes were evolutionarily more closely related than those with the opposite phenotype, regardless of isolation location or date. Unexpectedly, the genome of the type-strain for the species, C. watsonii WH8501, was quite unusual even among strains with a shared phenotype, indicating it may not be an ideal representative of the species. The genome sequences and analyses reported in this study will be important for future investigations of the proposed differences in adaptation of the two phenotypes to nutrient limitation, and to identify phenotype-specific distributions in natural Crocosphaera populations.

Dose ratio is important in maximizing naloxone enhancement of nalbuphine analgesia in humans.

The analgesic effect of kappa partial agonist opioids (i.e. nalbuphine, pentazocine and butorphanol) is significantly greater in women. Recent evidence suggests that this sexual dimorphism may result from a naloxone-sensitive anti-analgesic effect that is activated along with, and summates with, the analgesic effect of these agents, resulting in decreased analgesia or increased pain. For example, nalbuphine (5 mg) produces profound anti-analgesia (i.e. enhanced pain) in men, but addition of a low dose of the opioid receptor antagonist naloxone (0.4 mg, opioid antagonist) induces significant analgesia in men and enhances nalbuphine analgesia in women. To further delineate the dose-dependent relationship of nalbuphine and naloxone, we recently evaluated the effect of a lower dose of nalbuphine (2.5 mg) with and without naloxone (0.4 mg) on dental postoperative pain. In women, nalbuphine alone induced modest short duration analgesia, which was antagonized by the addition of naloxone. In men, this dose of nalbuphine alone did not produce analgesia or anti-analgesia, and naloxone did not alter the response to nalbuphine. Thus, it appeared that the 2.5 mg dose of nalbuphine was not sufficient to induce anti-analgesia while the 0.4 mg dose of naloxone was able to antagonize the analgesic effect of nalbuphine, at least in women. In the current study, we tested the hypothesis that an important determinant of naloxone enhancement of nalbuphine analgesia is the dose ratio of nalbuphine to naloxone. Since a dose ratio of 12.5:1 (i.e. 5 mg nalbuphine:0.4 mg naloxone) resulted in analgesic enhancement, but a dose ratio of 6.25:1 (2.5 mg:0.4 mg) did not, we tested the same, lower, dose of nalbuphine (2.5 mg) in combination with a lower dose of naloxone (0.2 mg) to maintain the 12.5:1 dose ratio. This lower dose of naloxone significantly prolonged the analgesic effect of nalbuphine in both men and women, suggesting that the anti-analgesic effect of nalbuphine is present in both sexes at the 2.5 mg dose and that the dose ratio of nalbuphine to naloxone is an important determinant of the analgesic efficacy of this combination.

Sexual dimorphism in very low dose nalbuphine postoperative analgesia.

In recent studies we demonstrated that the analgesic effect of the kappa-like opioids is significantly greater in women, that low dose nalbuphine (5 mg) produces profound anti-analgesia (i.e. enhances pain) in men, and that addition of a low dose of the non-selective opioid receptor antagonist naloxone (0.4 mg) to nalbuphine (5 mg) abolishes the sex difference and results in significantly enhanced analgesia in both sexes. To further delineate the dose-dependent analgesic and anti-analgesic effects of nalbuphine, the present study evaluated the effect of a lower dose of nalbuphine (2.5 mg), with and without naloxone, on dental postoperative pain. In women, nalbuphine alone induced modest, short duration analgesia, which was antagonized rather than enhanced by the addition of naloxone (0.4 mg). In men, this dose of nalbuphine alone did not produce analgesia or anti-analgesia, and naloxone (0.4 mg) did not alter the response to nalbuphine. Thus, the anti-analgesic effect of nalbuphine, present in both sexes at the 5 mg dose disappears at the lower dose of nalbuphine. In addition, the mild analgesia in women produced by this lower dose of nalbuphine is antagonized by naloxone.

Interactions between fluoxetine and opiate analgesia for postoperative dental pain.

In a double-blind placebo-controlled study we investigated the analgesic efficacy of combinations of the serotonergic tricyclic antidepressant fluoxetine with either the mu-opiate morphine or the kappa-opiate pentazocine. Administration of oral fluoxetine (10 mg p.o. daily for 7 days pre-operatively) had no effect on the immediate postoperative pain level. However, pre-operative administration of fluoxetine was found, compared to placebo, to antagonize analgesia seen after administration of morphine (6 mg, i.v.) in the immediate postoperative period. Attenuation of morphine analgesia consisted essentially of a shortening of the duration of action of the dose of morphine administered. Similar administration of fluoxetine had no effect on the analgesia produced by the kappa-opiate pentazocine (45 mg, i.v.). This effect probably results from alteration in the known serotonergic circuits in endogenous pain-modulating systems.

Temporal factors in the enhancement of morphine analgesia by desipramine.

Administration of desipramine, the tricyclic noradrenergic agent, for 7 days pre-operatively, had been found to potentiate postoperative morphine analgesia. In this study we investigated the necessary timing of administration of desipramine in its action to potentiate morphine analgesia. We report that the administration of desipramine for only 3 days, starting 7 days before surgery, also potentiated postoperative morphine analgesia and that the analgesia observed was not different from that in patients receiving a full 7 days of desipramine pre-operatively. The potentiation of morphine analgesia observed was most evident as a prolongation of the analgesic response. Patients who also received desipramine for only 3 days, but starting 3 days pre-operatively had an analgesic response to postoperative morphine that was the same as that in patients receiving placebo. The ability of the administration of desipramine early in the pre-operative week to interact with postoperative morphine and the lack of response when desipramine was given late in the week does not have an explanation at present. However, it may reflect the known latency in humans to the onset of the central effects of tricyclic antidepressants (TCAs).

Enhancement of pentazocine analgesia by clonidine.

Opiate-adrenergic interactions were investigated by studying the effect of the selective alpha 2-adrenergic agonist, clonidine, on the analgesia produced by intravenous placebo and by the predominantly kappa-opiate agonist, pentazocine, in patients with dental postoperative pain. Clonidine did not affect the pain level when administered with intravenous placebo. When administered with pentazocine, clonidine caused a statistically significant increase in pentazocine analgesia. Comparison is made to other opiate-adrenergic interactions and possible mechanisms are discussed.

Lack of effect of ephedrine on opiate analgesia.

Opiate-adrenomimetic interaction was investigated by studying the effect of the adrenomimetic agent, ephedrine, on the analgesia produced by intravenous placebo and that produced by the predominantly kappa opiate agonist, pentazocine, in patients with dental postoperative pain. Ephedrine did not significantly affect the analgesia of intravenous placebo or of pentazocine. These results contrast with earlier studies demonstrating enhancement of opiate analgesia by other adrenomimetics. Further clinical studies are needed to delineate the specificity of opiate-adrenomimetic interaction.

A possible indicator of functional pain: poor pain scale correlation.

We studied correlations of pain measures in patients with either inflammatory bowel disease (IBD), a disease with a clear organic cause, or irritable bowel syndrome (IBS), a functional pain syndrome in which there is little demonstrable pathology. Correlations were determined between measures on the visual analogue scale (VAS) and on the McGill Pain Questionnaire (MPQ). The VAS score and present pain intensity scale (PPI) of the MPQ correlated well in the organic IBD but correlated poorly in the functional IBS. Differences in correlation between the VAS and PPI scores in functional versus organic disease did not appear to be due to altered sensory and affective pain components. This finding is similar to what we observed in our previous study of organic and functional pain syndromes in the musculoskeletal system. Correlations between the other measures are also discussed.

Altered autonomic function in patients with arthritis or with chronic myofascial pain.

Dynamic autonomic function tests (pupillary light reflex, Valsalva maneuver, and mental arithmetic) were used to evaluate autonomic function (pupil size, heart rate, and skin conductance) in patients with inflammatory arthritis or with chronic myofascial pain. Physiological responses in both groups of patients suggest concurrent increases in tonic pupillary autonomic activity (sympathetic and parasympathetic), with a relative sympathetic dominance, and a decrease in tonic parasympathetic cardiovascular activity. Furthermore, in the arthritis group, the data suggest decreased cardiovascular parasympathetic reaction and enhanced sudomotor reaction.

Differing correlations between pain measures in syndromes with or without explicable organic pathology.

We investigated the use of pain measures in chronic pain syndromes with and without explicable organic cause by correlating scores obtained from the Visual Analog Scale and from subscales of the McGill Pain Questionnaire in patients with either inflammatory arthritis or primary fibrositis. We confirmed the finding that patients with fibrositis, despite having significantly less demonstrable pathology, report more pain on a significant number of measures. In patients with arthritis, we observed large positive correlations between scores on different pain measures, supporting the hypothesis that these scores validly measure pain secondary to tissue injury. In the fibrositis group, however, significantly lower correlations, and one even strongly negative, were found between scores on different measures. We suggest that commonly used pain measures which have been shown to be valid to study patients with demonstrable pathology may be less valid for measurement of pain in patients with syndrome such as fibrositis, whose pain may be different or more complex than that of patients with explicable organic basis.

Cardiovascular autonomic response during preoperative stress and postoperative pain.

Heart rate response to physiologic maneuvers was used to evaluate autonomic nervous system (ANS) function in normal control subjects and during the stress and pain experienced by patients before and after surgery. In preoperative patients (stressed without pain) and postoperative patients (stressed with pain), maneuvers which routinely increase activity in the parasympathetic or sympathetic divisions of the ANS produced only 50% of the response seen in control subjects. The heart rate response was not further reduced in patients with pain compared to patients with stress alone. The difference in heart rate response between surgical patients and control subjects was not accompanied by a difference in baseline heart rate. The data suggest that tonic stress impairs the ability of the ANS to respond fully to perturbing influences.

Altered electrodermal responsivity associated with clinical pain.

We used certain physiologic maneuvers to perturb the autonomic nervous system (ANS) in an attempt to detect a link between the ANS and pain. In the unperturbed state, we found no difference in the electrodermal response among normal controls, preoperative patients (increased stress without pain) and postoperative patients (increased stress and pain). The electrodermal response elicited by autonomic maneuvers was significantly attenuated in postoperative patients but not in preoperative patients or in normal control subjects.