Saliva sampling method influences oral microbiome composition and taxa distribution associated with oral diseases.

Saliva is a readily accessible and inexpensive biological specimen that enables investigation of the oral microbiome, which can serve as a biomarker of oral and systemic health. There are two routine approaches to collect saliva, stimulated and unstimulated; however, there is no consensus on how sampling method influences oral microbiome metrics. In this study, we analyzed paired saliva samples (unstimulated and stimulated) from 88 individuals, aged 7-18 years. Using 16S rRNA gene sequencing, we investigated the differences in bacterial microbiome composition between sample types and determined how sampling method affects the distribution of taxa associated with untreated dental caries and gingivitis. Our analyses indicated significant differences in microbiome composition between the sample types. Both sampling methods were able to detect significant differences in microbiome composition between healthy subjects and subjects with untreated caries. However, only stimulated saliva revealed a significant association between microbiome diversity and composition in individuals with diagnosed gingivitis. Furthermore, taxa previously associated with dental caries and gingivitis were preferentially enriched in individuals with each respective disease only in stimulated saliva. Our study suggests that stimulated saliva provides a more nuanced readout of microbiome composition and taxa distribution associated with untreated dental caries and gingivitis compared to unstimulated saliva.

Red Coloration in an Anchialine Shrimp: Carotenoids, Genetic Variation, and Candidate Genes.

Red coloration is a widely distributed phenotype among animals, yet the pigmentary and genetic bases for this phenotype have been described in relatively few taxa. Here we show that the Hawaiian endemic anchialine shrimp Halocaridina rubra is red because of the accumulation of astaxanthin. Laboratory colonies of phylogenetically distinct lineages of H. rubra have colony-specific amounts of astaxanthin that are developmentally, and likely genetically, fixed. Carotenoid supplementation and restriction experiments failed to change astaxanthin content from the within-colony baseline levels, suggesting that dietary limitation is not a major factor driving coloration differences. A possible candidate gene product predicted to be responsible for the production of astaxanthin in H. rubra and other crustaceans is closely related to the bifunctional cytochrome P450 family 3 enzyme CrtS found in fungi. However, homologs to the enzyme thought to catalyze ketolation reactions in birds and turtles, CYP2J19, were not found. This work is one of the first steps in linking phenotypic variation in red coloration of H. rubra to genotypic variation. Future work should focus on (1) pinpointing the genes that function in the bioconversion of dietary carotenoids to astaxanthin, (2) examining what genomic variants might drive variation in coloration among discrete lineages, and (3) testing more explicitly for condition-dependent carotenoid coloration in crustaceans.

Using Video Telehealth to Facilitate Inhaler Training in Rural Patients with Obstructive Lung Disease.

BACKGROUND: Proper inhaler technique is important for effective drug delivery and symptom control in chronic obstructive pulmonary disease (COPD) and asthma, yet not all patients receive inhaler instructions. INTRODUCTION: Using a retrospective chart review of participants in a video telehealth inhaler training program, the study compared inhaler technique within and between monthly telehealth visits and reports associated with patient satisfaction. MATERIALS AND METHODS: Seventy-four (N = 74) rural patients prescribed ≥1 inhaler participated in three to four pharmacist telehealth inhaler training sessions using teach-to-goal (TTG) methodology. Within and between visit inhaler technique scores are compared, with descriptive statistics of pre- and postprogram survey results including program satisfaction and computer technical issues. Healthcare utilization is compared between pre- and post-training periods. RESULTS: Sixty-nine (93%) patients completed all three to four video telehealth inhaler training sessions. During the initial visit, patients demonstrated improvement in inhaler technique for metered dose inhalers (albuterol, budesonide/formoterol), dry powder inhalers (formoterol, mometasone, tiotropium), and soft mist inhalers (ipratropium/albuterol) (p < 0.01 for all). Improved inhaler technique was sustained at 2 months (p < 0.01). Ninety-four percent of participants were satisfied with the program. Although technical issues were common, occurring among 63% of attempted visits, most of these visits (87%) could be completed. There was no significant difference in emergency department visits and hospitalizations pre- and post-training. DISCUSSION: This study demonstrated high patient acceptance of video telehealth training and objective improvement in inhaler technique. CONCLUSIONS: Video telehealth inhaler training using the TTG methodology is a promising program that improved inhaler technique and access to inhaler teaching for rural patients with COPD or asthma.

Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-ß and cognitive function in APP/PS1 mice.

Activation of the inflammasome is implicated in the pathogenesis of an increasing number of inflammatory diseases, including Alzheimer's disease (AD). Research reporting inflammatory changes in post mortem brain tissue of individuals with AD and GWAS data have convincingly demonstrated that neuroinflammation is likely to be a key driver of the disease. This, together with the evidence that genetic variants in the NLRP3 gene impact on the risk of developing late-onset AD, indicates that targetting inflammation offers a therapeutic opportunity. Here, we examined the effect of the small molecule inhibitor of the NLRP3 inflammasome, MCC950, on microglia in vitro and in vivo. The findings indicate that MCC950 inhibited LPS+Aß-induced caspase 1 activation in microglia and this was accompanied by IL-1ß release, without inducing pyroptosis. We demonstrate that MCC950 also inhibited inflammasome activation and microglial activation in the APP/PS1 mouse model of AD. Furthermore, MCC950 stimulated Aß phagocytosis in vitro, and it reduced Aß accumulation in APP/PS1 mice, which was associated with improved cognitive function. These data suggest that activation of the inflammasome contributes to amyloid accumulation and to the deterioration of neuronal function in APP/PS1 mice and demonstrate that blocking assembly of the inflammasome may prove to be a valuable strategy for attenuating changes that negatively impact on neuronal function.

Epigenetic differences in monozygotic twins discordant for major depressive disorder.

Although monozygotic (MZ) twins share the majority of their genetic makeup, they can be phenotypically discordant on several traits and diseases. DNA methylation is an epigenetic mechanism that can be influenced by genetic, environmental and stochastic events and may have an important impact on individual variability. In this study we explored epigenetic differences in peripheral blood samples in three MZ twin studies on major depressive disorder (MDD). Epigenetic data for twin pairs were collected as part of a previous study using 8.1-K-CpG microarrays tagging DNA modification in white blood cells from MZ twins discordant for MDD. Data originated from three geographical regions: UK, Australia and the Netherlands. Ninety-seven MZ pairs (194 individuals) discordant for MDD were included. Different methods to address non independently-and-identically distributed (non-i.i.d.) data were evaluated. Machine-learning methods with feature selection centered on support vector machine and random forest were used to build a classifier to predict cases and controls based on epivariations. The most informative variants were mapped to genes and carried forward for network analysis. A mixture approach using principal component analysis (PCA) and Bayes methods allowed to combine the three studies and to leverage the increased predictive power provided by the larger sample. A machine-learning algorithm with feature reduction classified affected from non-affected twins above chance levels in an independent training-testing design. Network analysis revealed gene networks centered on the PPAR-γ (NR1C3) and C-MYC gene hubs interacting through the AP-1 (c-Jun) transcription factor. PPAR-γ (NR1C3) is a drug target for pioglitazone, which has been shown to reduce depression symptoms in patients with MDD. Using a data-driven approach we were able to overcome challenges of non-i.i.d. data when combining epigenetic studies from MZ twins discordant for MDD. Individually, the studies yielded negative results but when combined classification of the disease state from blood epigenome alone was possible. Network analysis revealed genes and gene networks that support the inflammation hypothesis of MDD.

Does vitamin D protect against respiratory viral infections?

The active form of vitamin D has effects on both innate and adaptive immune responses that may influence the outcome in many infectious diseases. Observational studies conclusively show that a low vitamin D status is associated with an increased occurrence of respiratory viral infections, which globally represent significant health and financial burdens. However, no consistent protective effects are evident in prospective clinical trials carried out to date where vitamin D was provided as a dietary supplement, except possibly in cases where the starting vitamin D status of the individual was considered deficient. Thus far, vitamin D has not been found to enhance the immune response to vaccines. The design of future prospective clinical trials assessing a role for vitamin D in respiratory viral infections requires very careful planning to avoid the uncertainties associated with the data available currently.

Protein annotation and modelling servers at University College London.

The UCL Bioinformatics Group web portal offers several high quality protein structure prediction and function annotation algorithms including PSIPRED, pGenTHREADER, pDomTHREADER, MEMSAT, MetSite, DISOPRED2, DomPred and FFPred for the prediction of secondary structure, protein fold, protein structural domain, transmembrane helix topology, metal binding sites, regions of protein disorder, protein domain boundaries and protein function, respectively. We also now offer a fully automated 3D modelling pipeline: BioSerf, which performed well in CASP8 and uses a fragment-assembly approach which placed it in the top five servers in the de novo modelling category. The servers are available via the group web site at http://bioinf.cs.ucl.ac.uk/.

AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system.

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license.

The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Prediction of novel and analogous folds using fragment assembly and fold recognition.

A number of new and newly improved methods for predicting protein structure developed by the Jones-University College London group were used to make predictions for the CASP6 experiment. Structures were predicted with a combination of fold recognition methods (mGenTHREADER, nFOLD, and THREADER) and a substantially enhanced version of FRAGFOLD, our fragment assembly method. Attempts at automatic domain parsing were made using DomPred and DomSSEA, which are based on a secondary structure parsing algorithm and additionally for DomPred, a simple local sequence alignment scoring function. Disorder prediction was carried out using a new SVM-based version of DISOPRED. Attempts were also made at domain docking and "microdomain" folding in order to build complete chain models for some targets.

Public-private partnerships. Public pain, private gain.

Using private hospitals over 11 weeks this winter enabled a health authority to remove more than 1,000 patients from waiting lists. Prices were comparable to, and sometimes cheaper than, the NHS. The average cost was 1,120 Pounds per treatment. Patient satisfaction seemed high. Consultant productivity was higher in the private sector.

What are the baselines for protein fold recognition?

MOTIVATION: What constitutes a baseline level of success for protein fold recognition methods? As fold recognition benchmarks are often presented without any thought to the results that might be expected from a purely random set of predictions, an analysis of fold recognition baselines is long overdue. Given varying amounts of basic information about a protein-ranging from the length of the sequence to a knowledge of its secondary structure-to what extent can the fold be determined by intelligent guesswork? Can simple methods that make use of secondary structure information assign folds more accurately than purely random methods and could these methods be used to construct viable hierarchical classifications? EXPERIMENTS PERFORMED: A number of rapid automatic methods which score similarities between protein domains were devised and tested. These methods ranged from those that incorporated no secondary structure information, such as measuring absolute differences in sequence lengths, to more complex alignments of secondary structure elements. Each method was assessed for accuracy by comparison with the Class Architecture Topology Homology (CATH) classification. Methods were rated against both a random baseline fold assignment method as a lower control and FSSP as an upper control. Similarity trees were constructed in order to evaluate the accuracy of optimum methods at producing a classification of structure. RESULTS: Using a rigorous comparison of methods with CATH, the random fold assignment method set a lower baseline of 11% true positives allowing for 3% false positives and FSSP set an upper benchmark of 47% true positives at 3% false positives. The optimum secondary structure alignment method used here achieved 27% true positives at 3% false positives. Using a less rigorous Critical Assessment of Structure Prediction (CASP)-like sensitivity measurement the random assignment achieved 6%, FSSP-59% and the optimum secondary structure alignment method-32%. Similarity trees produced by the optimum method illustrate that these methods cannot be used alone to produce a viable protein structural classification system. CONCLUSIONS: Simple methods that use perfect secondary structure information to assign folds cannot produce an accurate protein taxonomy, however they do provide useful baselines for fold recognition. In terms of a typical CASP assessment our results suggest that approximately 6% of targets with folds in the databases could be assigned correctly by randomly guessing, and as many as 32% could be recognised by trivial secondary structure comparison methods, given knowledge of their correct secondary structures.

The PSIPRED protein structure prediction server.

SUMMARY: The PSIPRED protein structure prediction server allows users to submit a protein sequence, perform a prediction of their choice and receive the results of the prediction both textually via e-mail and graphically via the web. The user may select one of three prediction methods to apply to their sequence: PSIPRED, a highly accurate secondary structure prediction method; MEMSAT 2, a new version of a widely used transmembrane topology prediction method; or GenTHREADER, a sequence profile based fold recognition method. AVAILABILITY: Freely available to non-commercial users at http://globin.bio.warwick.ac.uk/psipred/

Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A- and B-DNA located by simulated annealing.

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na+ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.

Successful recognition of protein folds using threading methods biased by sequence similarity and predicted secondary structure.

Analysis of our fold recognition results in the 3rd Critical Assessment in Structure Prediction (CASP3) experiment, using the programs THREADER 2 and GenTHREADER, shows an encouraging level of overall success. Of the 23 submitted predictions, 20 targets showed no clear sequence similarity to proteins of known 3D structure. These 20 targets can be divided into 22 domains, of which, 20 domains either entirely match a previously known fold, or partially match a substantial region of a known fold. Of these 20 domains, we correctly assigned the folds in 10 cases.

CAFASP-1: critical assessment of fully automated structure prediction methods.

The results of the first Critical Assessment of Fully Automated Structure Prediction (CAFASP-1) are presented. The objective was to evaluate the success rates of fully automatic web servers for fold recognition which are available to the community. This study was based on the targets used in the third meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP-3). However, unlike CASP-3, the study was not a blind trial, as it was held after the structures of the targets were known. The aim was to assess the performance of methods without the user intervention that several groups used in their CASP-3 submissions. Although it is clear that "human plus machine" predictions are superior to automated ones, this CAFASP-1 experiment is extremely valuable for users of our methods; it provides an indication of the performance of the methods alone, and not of the "human plus machine" performance assessed in CASP. This information may aid users in choosing which programs they wish to use and in evaluating the reliability of the programs when applied to their specific prediction targets. In addition, evaluation of fully automated methods is particularly important to assess their applicability at genomic scales. For each target, groups submitted the top-ranking folds generated from their servers. In CAFASP-1 we concentrated on fold-recognition web servers only and evaluated only recognition of the correct fold, and not, as in CASP-3, alignment accuracy. Although some performance differences appeared within each of the four target categories used here, overall, no single server has proved markedly superior to the others. The results showed that current fully automated fold recognition servers can often identify remote similarities when pairwise sequence search methods fail. Nevertheless, in only a few cases outside the family-level targets has the score of the top-ranking fold been significant enough to allow for a confident fully automated prediction. Because the goals, rules, and procedures of CAFASP-1 were different from those used at CASP-3, the results reported here are not comparable with those reported in CASP-3. Nevertheless, it is clear that current automated fold recognition methods can not yet compete with "human-expert plus machine" predictions. Finally, CAFASP-1 has been useful in identifying the requirements for a future blind trial of automated served-based protein structure prediction.

Role of cellular thiol status in tocopheryl hemisuccinate cytoprotection against ethyl methanesulfonate-induced toxicity.

Suspensions of rat hepatocytes treated with the alkylating agent ethyl methanesulfonate (EMS) exhibited extensive lipid peroxidation as well as rapid and near complete depletion of cellular reduced glutathione (GSH) levels prior to cell death. Pretreatment of hepatocytes with medium deficient in sulfur amino acids accelerated cell death induced by EMS, confirming the previously reported cytoprotective role for GSH in this toxic event. Nearly all of the cellular GSH lost following 50 mM EMS treatment was accounted for as S-ethyl glutathione (GS-Et). No significant formation of glutathione disulfide was observed. The GS-Et formed was not exported from the cell but remained at high intracellular concentrations throughout the course of the experiment. In addition, EMS treatment inhibited the efflux of intracellular GSH and inhibited the cellular accumulation of glutamate (Glu). Supplementation of hepatocytes with 25 microM d-alpha-tocopheryl hemisuccinate (TS) protected these cells against EMS-induced lipid peroxidation and cell death. Cytoprotection with TS had no effect on EMS-induced depletion of intracellular GSH or intracellular levels of GS-Et or Glu. However, TS supplementation did prevent EMS-induced depletion of cellular protein thiols. Interestingly, the pretreatment of hepatocytes with 1 mM dithiothreitol promoted EMS toxicity. The results of this study suggest that the cytoprotective abilities of TS are related to the prevention of both EMS-induced lipid peroxidation and protein thiol depletion. Thus, the onset of lipid peroxidation and the loss of protein thiols in hepatocytes appear to be critical cellular events leading to EMS-induced cell death.

Postoperative carbon pigment granuloma: a report of eight cases involving the ovary.

Eight cases of ovarian granuloma containing carbon pigment are described. In all cases, this distinctive lesion occurred at sites of prior laser or fulguration surgery. Three types of carbon pigment granuloma were noted, the most common being the necrobiotic granuloma. Although postoperative necrobiotic granulomas have been described in a variety of clinical sites, the carbon pigment granuloma has not been well described in the pathology literature. Recognition of this lesion seems to be of value in determining its postoperative, noninfectious nature.

Synthesis and evaluation of 7 alpha-iodo-5 alpha-dihydrotestosterone as a potential radioligand for androgen receptor.

7 alpha-Iodo-17 beta-hydroxy-5 alpha-androstan-3-one (7 alpha-iodo-5 alpha-dihydrotestosterone, 7 alpha-IDHT) has been synthesized as a potential radioligand for the detection and measurement of androgen receptor and for imaging of androgen-receptor-containing tissues when labeled with the gamma-emitting radionuclides 125I and 123I, respectively. In vitro binding studies show that 7 alpha-IDHT binds with high affinity to the rat and human androgen receptor (RBA = 74) compared to R1881 (RBA = 100). Further, this compound showed high specificity for the androgen receptor. 7 alpha-IDHT showed only a marginal affinity for the progestin receptor and even less affinity for the estrogen receptor. No binding was detected to the glucocorticoid receptor. These characteristics make 7 alpha-IDHT a potentially ideal agent for imaging and evaluation of androgen-receptor-containing tissues.

Protection against carbon tetrachloride-induced hepatotoxicity by pretreating rats with the hemisuccinate esters of tocopherol and cholesterol.

Previous studies have demonstrated that alpha-tocopheryl hemisuccinate (TS) protects hepatocyte suspensions from chemical-induced toxicity. It has been suggested that TS cytoprotection is related to unique properties of the TS molecule or is dependent on the cellular release and activity of unesterified alpha-tocopherol (T). To test the unique cytoprotective nature of TS in vivo, the protective ability of T and tocopherol esters against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats was examined. Hepatoprotection [determined by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and histopathology] was not observed after T (or tocopheryl acetate and tocopheryl nicotinate) administration, even though this treatment resulted in a fivefold elevation in hepatic T content. Only pretreatment with TS (100 mg/kg, intraperitoneally) resulted in partial hepatoprotection against CCl4 (2.9 g/kg, orally) toxicity. These findings suggest that hepatoprotection results not from the cellular accumulation of T but rather from the intact TS molecule. To test this hypothesis, the hepatoprotective capacity of cholesteryl hemisuccinate (CS), unesterified cholesterol, and cholesteryl acetate (CA) was examined against CCl4 toxicity. As observed with the tocopherol derivatives, pretreatment with unesterified cholesterol or CA demonstrated no protective ability. However, when rats were pretreated with CS (100 mg/kg), the hepatotoxic effects of CCl4 (elevated serum AST and ALT levels and centrilobular necrosis) were completely prevented. The prevention of CCl4-induced hepatotoxicity by CS and TS do not appear to result from an alteration in hepatic drug metabolism. These data clearly demonstrate that CS and TS are unique and powerful cytoprotective agents against CCl4 hepatotoxicity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)