Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

Background

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.

Results

Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.

Conclusions

While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.     

COMPASS II: extended coverage for polymer and drug-like molecule databases

The COMPASS II force field has been developed by extending the coverage of the COMPASS force field (J Phys Chem B 102(38):7338–7364, 1998) to polymer and drug-like molecules found in popular databases. Using a fragmentation method to systematically construct small molecules that exhibit key functional groups found in these databases, parameters applicable to database compounds were efficiently obtained. Based on the same parameterization paradigm as used in the development of the COMPASS force field, new parameters were derived by a combination of fits to quantum mechanical data for valence parameters and experimental liquid and crystal data for nonbond parameters. To preserve the quality of the original COMPASS parameters, a quality assurance suite was used and updated to ensure that additional atom-types and parameters do not interfere with the existing ones. Validation against molecular properties, liquid and crystal densities, and enthalpies, demonstrates that the quality of COMPASS is preserved and the same quality of prediction is achieved for the additional coverage.     

Halorespiring bacteria-molecular characterization and detection

Recently, a rapidly increasing number of bacteria has been isolated that is able to couple the reductive dehalogenation of various halogenated aromatic and aliphatic compounds like chlorophenols and tetrachloroethene to energy conservation by electron-transport-coupled phosphorylation. The potential of these halorespiring bacteria for innovative clean-up strategies of polluted anoxic environments has greatly stimulated efforts to unravel the molecular basis of the novel respiratory chains they possess. The thorough characterization of halorespiratory key components at the physiological, biochemical and molecular genetic level has revealed both structural and functional similarity of chloroaryl- and chloroalkyl-respiratory chains from different phylogenetically distinct microorganisms. The reductive dehalogenases from halorespiring bacteria were found to comprise a novel class of corrinoid-containing Fe/S-proteins. Sensitive molecular methods for monitoring both presence and fate of halorespiring bacteria have been developed, which will be instrumental for the design and maintenance of optimised in situ bioremediation processes.     

Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas.     

Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4⁺ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.     

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Neurophysiological evaluation of healthy human anorectal sensation

Patients with functional gastrointestinal disorders often demonstrate abnormal visceral sensation. Currently, rectal sensation is assessed by manual balloon distension or barostat. However, neither test is adaptable for use in the neurophysiological characterization of visceral afferent pathways by sensory evoked potentials. The aim of this study was to assess the reproducibility and quality of sensation evoked by electrical stimulation (ES) and rapid balloon distension (RBD) in the anorectum and to apply the optimum stimulus to examine the visceral afferent pathway with rectal evoked potentials. Healthy subjects (n = 8, median age 33 yr) were studied on three separate occasions. Variability, tolerance, and stimulus characteristics were assessed with each technique. Overall ES consistently invoked pain and was chosen for measuring rectal evoked potential whereas RBD in all cases induced the strong urge to defecate. Rectal intraclass correlation coefficient (ICC) for ES and RBD (0.82 and 0.72, respectively) demonstrated good reproducibility at pain/maximum tolerated volume but not at sensory threshold. Only sphincter ICC for ES at pain showed acceptable between-study reproducibility (ICC 0.79). Within studies ICC was good (>0.6) for anorectal ES and RBD at both levels of sensation. All subjects reported significantly more unpleasantness during RBD than ES (P < 0.01). This study demonstrates that ES and RBD are similarly reproducible. However, the sensations experienced with each technique differed markedly, probably reflecting differences in peripheral and/or central processing of the sensory input. This is of relevance in interpreting findings of neuroimaging studies of anorectal sensation and may provide insight into the physiological characteristics of visceral afferent pathways in health and disease.     

Lung function decline in relation to diagnostic criteria for airflow obstruction in respiratory symptomatic subjects

Background

Current COPD guidelines advocate a fixed < 0.70 FEV1/FVC cutpoint to define airflow obstruction. We compared rate of lung function decline in respiratory symptomatic 40+ subjects who were 'obstructive' or 'non-obstructive' according to the fixed and/or age and gender specific lower limit of normal (LLN) FEV1/FVC cutpoints.

Methods

We studied 3,324 respiratory symptomatic subjects referred to primary care diagnostic centres for spirometry. The cohort was subdivided into four categories based on presence or absence of obstruction according to the fixed and LLN FEV1/FVC cutpoints. Postbronchodilator FEV1 decline served as primary outcome to compare subjects between the respective categories.

Results

918 subjects were obstructive according to the fixed FEV1/FVC cutpoint; 389 (42%) of them were non-obstructive according to the LLN cutpoint. In smokers, postbronchodilator FEV1 decline was 21 (SE 3) ml/year in those non-obstructive according to both cutpoints, 21 (7) ml/year in those obstructive according to the fixed but not according to the LLN cutpoint, and 50 (5) ml/year in those obstructive according to both cutpoints (p = 0.004).

Conclusion

This study showed that respiratory symptomatic 40+ smokers and non-smokers who show FEV1/FVC values below the fixed 0.70 cutpoint but above their age/gender specific LLN value did not show accelerated FEV1 decline, in contrast with those showing FEV1/FVC values below their LLN cutpoint.     

Probiotics enhance pancreatic glutathione biosynthesis and reduce oxidative stress in experimental acute pancreatitis

Factors determining severity of acute pancreatitis (AP) are poorly understood. Oxidative stress causes acinar cell injury and contributes to the severity, whereas prophylactic probiotics ameliorate experimental pancreatitis. Our objective was to study how probiotics affect oxidative stress, inflammation, and acinar cell injury during the early phase of AP. Fifty-three male Sprague-Dawley rats were randomly allocated into groups: 1) control, 2) sham procedure, 3) AP with no treatment, 4) AP with probiotics, and 5) AP with placebo. AP was induced under general anesthesia by intraductal glycodeoxycholate infusion (15 mM) and intravenous cerulein (5 microg.kg(-1).h(-1), for 6 h). Daily probiotics or placebo were administered intragastrically, starting 5 days prior to AP. After cerulein infusion, pancreas samples were collected for analysis including lipid peroxidation, glutathione, glutamate-cysteine-ligase activity, histological grading of pancreatic injury, and NF-kappaB activation. The severity of pancreatic injury correlated to oxidative damage (r = 0.9) and was ameliorated by probiotics (1.5 vs. placebo 5.5; P = 0.014). AP-induced NF-kappaB activation was reduced by probiotics (0.20 vs. placebo 0.53 OD(450nm)/mg nuclear protein; P < 0.001). Probiotics attenuated AP-induced lipid peroxidation (0.25 vs. placebo 0.51 pmol malondialdehyde/mg protein; P < 0.001). Not only was AP-induced glutathione depletion prevented (8.81 vs. placebo 4.1 micromol/mg protein, P < 0.001), probiotic pretreatment even increased glutathione compared with sham rats (8.81 vs. sham 6.18 miccromol/mg protein, P < 0.001). Biosynthesis of glutathione (glutamate-cysteine-ligase activity) was enhanced in probiotic-pretreated animals. Probiotics enhanced the biosynthesis of glutathione, which may have reduced activation of inflammation and acinar cell injury and ameliorated experimental AP, via a reduction in oxidative stress.     

Molecular breakdown of corn starch by thermal and mechanical effects

The molecular weight reduction of corn starch at 30–43% moisture during thermal treatment at temperatures 90–160 °C and during well-defined thermomechanical treatment at temperatures 90–140 °C was investigated. Thermal treatment resulted, during the first 5 min in a decrease in molecular weight as measured by intrinsic viscosity, after which longer heating had no significant effect. Higher moisture contents and temperatures generally resulted in more breakdown, although the effect diminished at higher temperatures. The decrease in intrinsic viscosity during thermomechanical treatment at relatively low temperatures and moisture contents was shown to be only dependent on the maximal shear stress. At higher temperatures, thermomechanical breakdown could be split into a mechanical part depending on maximal shear stress and a thermal breakdown part, which was again time-dependent on the shorter time-scales only. Higher moisture content during thermomechanical treatment resulted in more thermal breakdown and lowered the shear stresses required for mechanical breakdown. Consequences for process design are discussed briefly.