Author Correction: Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

"Candidatus Paraporphyromonas polyenzymogenes" encodes multi-modular cellulases linked to the type IX secretion system.

BACKGROUND: In nature, obligate herbivorous ruminants have a close symbiotic relationship with their gastrointestinal microbiome, which proficiently deconstructs plant biomass. Despite decades of research, lignocellulose degradation in the rumen has thus far been attributed to a limited number of culturable microorganisms. Here, we combine meta-omics and enzymology to identify and describe a novel Bacteroidetes family ("Candidatus MH11") composed entirely of uncultivated strains that are predominant in ruminants and only distantly related to previously characterized taxa. RESULTS: The first metabolic reconstruction of Ca. MH11-affiliated genome bins, with a particular focus on the provisionally named "Candidatus Paraporphyromonas polyenzymogenes", illustrated their capacity to degrade various lignocellulosic substrates via comprehensive inventories of singular and multi-modular carbohydrate active enzymes (CAZymes). Closer examination revealed an absence of archetypical polysaccharide utilization loci found in human gut microbiota. Instead, we identified many multi-modular CAZymes putatively secreted via the Bacteroidetes-specific type IX secretion system (T9SS). This included cellulases with two or more catalytic domains, which are modular arrangements that are unique to Bacteroidetes species studied to date. Core metabolic proteins from Ca. P. polyenzymogenes were detected in metaproteomic data and were enriched in rumen-incubated plant biomass, indicating that active saccharification and fermentation of complex carbohydrates could be assigned to members of this novel family. Biochemical analysis of selected Ca. P. polyenzymogenes CAZymes further iterated the cellulolytic activity of this hitherto uncultured bacterium towards linear polymers, such as amorphous and crystalline cellulose as well as mixed linkage ß-glucans. CONCLUSION: We propose that Ca. P. polyenzymogene genotypes and other Ca. MH11 members actively degrade plant biomass in the rumen of cows, sheep and most likely other ruminants, utilizing singular and multi-domain catalytic CAZymes secreted through the T9SS. The discovery of a prominent role of multi-modular cellulases in the Gram-negative Bacteroidetes, together with similar findings for Gram-positive cellulosomal bacteria (Ruminococcus flavefaciens) and anaerobic fungi (Orpinomyces sp.), suggests that complex enzymes are essential and have evolved within all major cellulolytic dominions inherent to the rumen.

Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus.

Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A, and characterization of its enzymatic activities. The enzyme possesses the archetypical (ß/α)8-barrel found throughout the GH5 family, and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of ß-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear ß-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the -3 to -1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic "clamp", potentially interacting with sugars at the +1 and +2 subsites. The enzyme's active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.

Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against ß-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward ß-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

Do rumen Bacteroidetes utilize an alternative mechanism for cellulose degradation?

Uncultured and therefore uncharacterized Bacteroidetes lineages are ubiquitous in many natural ecosystems which specialize in lignocellulose degradation. However, their metabolic contribution remains mysterious, as well-studied cultured Bacteroidetes have been shown to degrade only soluble polysaccharides within the human distal gut and herbivore rumen. We have interrogated a reconstructed genome from an uncultured Bacteroidetes phylotype that dominates a switchgrass-associated community within the cow rumen. Importantly, this characterization effort has revealed the first preliminary evidence for polysaccharide utilization locus (PUL)-catalyzed conversion of cellulose. Based on these findings, we propose a further expansion of the PUL paradigm and the saccharolytic capacity of rumen Bacteroidetes species to include cellulose, the most abundant terrestrial polysaccharide on Earth. Moreover, the perspective of a cellulolytic PUL lays the foundation for PULs to be considered an alternative mechanism for cellulose degradation, next to cellulosomes and free-enzyme systems.

QT interval abnormalities are often present at diagnosis in diabetes and are better predictors of cardiac death than ankle brachial pressure index and autonomic function tests.

OBJECTIVES: To study serial measures of maximum QT interval corrected for heart rate (QTc) and QT dispersion (QTD) and their association with cardiac mortality patients with non-insulin dependent diabetes and to compare QT abnormalities with other mortality predictors (ankle brachial pressure index (ABPI) and autonomic function tests) in their ability to predict cardiac death. SETTING: Teaching hospital. METHODS AND PATIENTS: QT interval analysis, heart rate (RR) variation in response to deep breathing and standing, and ABPI were analysed in 192 patients with non-insulin dependent diabetes. Cardiac death was the primary end point. RESULTS: Mean (SD) follow up was 12.7 (3.2) years (range 1.2-17.1 years). There were 48 deaths, of which 26 were cardiac. QTc and QTD were individually significant predictors of cardiac mortality throughout the follow up period (p < 0.001). The predictability of QT parameters was superior to the predictability of ABPI and RR interval analysis. Temporal changes in QT parameters showed that the mean absolute QT parameter was a significant predictor of cardiac death (p < 0.001), whereas an intraindividual change in QT parameter over time was not predictive. CONCLUSION: QT abnormalities seem to exist at the point of diagnosis of diabetes and do not appear to change between then and the subsequent cardiac death. Furthermore, the analysis of QT interval is superior to ABPI and the RR interval in identifying diabetic patients at high risk of cardiac death.

Immune response to Encephalitozoon cuniculi infection in laboratory mice.

The study was performed to determine the immune response to Encephalitozoon cuniculi infection in immunocompetent mice during 120 days of experiment. Mice infected with E. cuniculi had an increased number of CD4+ T cells up to Day 20 post infection (p.i.), but counts of CD8+ T lymphocytes were significantly lower up to Day 90 p.i. in peripheral blood. Blood monocytes were significantly increased on the Day 60 and Day 120 of infection. A lack of significant decrease of CD4+ T cells may be considered as an important event in the immune response to E. cuniculi infection in immunocompetent mice.

Irbesartan reduces QT dispersion in hypertensive individuals.

Angiotensin type 1 receptor antagonists have direct effects on the autonomic nervous system and myocardium. Because of this, we hypothesized that irbesartan would reduce QT dispersion to a greater degree than amlodipine, a highly selective vasodilator. To test this, we gathered electrocardiographic (ECG) data from a multinational, multicenter, randomized, double-blind parallel group study that compared the antihypertensive efficacy of irbesartan and amlodipine in elderly subjects with mild to moderate hypertension. Subjects were treated for 6 months with either drug. Hydrochlorothiazide and atenolol were added after 12 weeks if blood pressure (BP) remained uncontrolled. ECGs were obtained before randomization and at 6 months. A total of 188 subjects (118 with baseline ECGs) were randomized. We analyzed 104 subjects who had complete ECGs at baseline and after 6 months of treatment. Baseline characteristics between treatments were similar, apart from a slight imbalance in diastolic BP (irbesartan [n=53] versus amlodipine [n=51], 99.2 [SD 3. 6] versus 100.8 [3.8] mm Hg; P=0.03). There were no significant differences in BP normalization (diastolic BP <90 mm Hg) between treatments at 6 months (irbesartan versus amlodipine, 80% versus 88%; P=0.378). We found a significant reduction in QT indexes in the irbesartan group (QTc dispersion mean, -11.4 [34.5] milliseconds, P=0.02; QTc max, -12.8 [35.5] milliseconds, P=0.01), and QTc dispersion did not correlate with the change in BP. The reduction in QT indexes with amlodipine (QTc dispersion, -9.7 [35.4] milliseconds, P=0.06; QTc max, -8.6 [33.2] milliseconds, P=0.07) did not quite reach statistical significance, but there was a correlation between the change in QT indexes and changes in systolic BP. In conclusion, irbesartan improved QT dispersion, and this effect may be important in preventing sudden cardiac death in at-risk hypertensive subjects.

Enalapril reduces QTc dispersion in mild congestive heart failure secondary to coronary artery disease.

This study was designed to investigate the possible mechanisms whereby enalapril improves cardiac function and mortality in chronic heart failure. We explored potential mechanisms by following 41 patients with early heart failure over the course of 1 year. These patients were randomized in a prospective triple-blind manner to receive either enalapril or placebo. Over the 1 year, repeated measurements were obtained of echocardiographic parameters, glomerular filtration rate, renal blood flow, hematocrit, plasma neurohormones, and QTc dispersion. Echocardiographic parameters improved with enalapril but deteriorated with placebo (cardiac output 4.6 +/- 1.6 to 3.7 +/- 1.5 L/min with placebo, and 4.5 +/- 1.3 to 5.8 +/- 2.0 L/min with enalapril; p <0.01). In contrast, there were no significant changes in renal blood flow (518 +/- 185 to 509 +/- 180 ml/min/1.73 m2 with placebo, and 541 +/- 142 to 504 +/- 162 ml/min/1.73 m2 with enalapril). Glomerular filtration rate changed from 79 +/- 20 to 78 +/- 19 ml/min/1.73 m2 with placebo, and from 85 +/- 21 to 73 +/- 27 ml/min/1.73 m2 with enalapril (p = 0.051). Enalapril reduced hematocrit (0.414 +/- 0.041 to 0.377 +/- 0.040%) significantly more than placebo (0.420 +/- 0.029 to 0.411 +/- 0.023 l/l; p <0.01). In addition, enalapril produced a marked reduction in QTc dispersion (93 +/- 36 to 88 +/- 28 ms with placebo and 93 +/- 35 to 60 +/- 22 ms with enalapril; p <0.05). Thus, enalapril significantly reduced hematocrit and reduced QTc dispersion in early heart failure. Both of these effects, but especially the latter, could be an important mechanism for the reduced mortality seen with angiotensin-converting enzyme inhibitors in heart failure. In contrast, renal hemodynamics did not parallel either the placebo-induced deterioration in cardiac function or the enalapril-induced improvements in cardiac function.

Comparison of atrial natriuretic peptide B-type natriuretic peptide, and N-terminal proatrial natriuretic peptide as indicators of left ventricular systolic dysfunction.

We have directly compared atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and N-terminal pro-ANP (N-ANP) as markers of patients with left ventricular ejection fraction (LVEF) < or = 35%, as measured by radionuclide ventriculography. Venous blood samples were obtained from an unselected group of 87 patients who had been referred for assessment of ventricular function. ANP, BNP, and N-ANP were measured by radioimmunoassay using commercial kits. Receiver-operating characteristic analysis was used for the objective assessment of the diagnostic performance of each assay. There was a weak negative correlation between LVEF and plasma levels of ANP-li (r = -0.50,), BNP-li (r = -0.57), and N-ANP-li (r = -0.49) (p <0.01 for each peptide). Areas under the receiver-operating characteristic curves for BNP (0.880) and N-ANP (0.832) were not significantly different from each other, but were both significantly greater than the value for ANP (0.761): BNP versus ANP, p <0.01; and N-ANP versus ANP, p <0.05. The optimal sensitivity and specificity of each assay for the detection of patients with LVEF < or = 35% were: BNP > 4 pmol/L-sensitivity 1.0, specificity 0.58; N-ANP >200 pmol/L-sensitivity 0.95, specificity 0.35; and ANP >10 pmol/L-sensitivity 0.90, specificity 0.30. Plasma concentrations of BNP and N-ANP provide sensitive indicators of moderate to severe LV dysfunction; both peptides, are objectively superior to ANP for identifying patients with LVEF < or = 35%. These simple tests could be used to screen patients with suspected ventricular dysfunction to reduce the demand for further cardiac investigations.

QT dispersion in essential hypertension.

Increased QT dispersion is associated with sudden cardiac death in congestive heart failure, hypertrophic cardiomyopathy, and following acute myocardial infarction. Patients with hypertension, in particular those with left ventricular hypertrophy, are also at greater risk of sudden cardiac death. We examined whether QT dispersion, which is easily obtained from a routine ECG, correlates with echo LVH. Sixty-nine untreated patients with essential hypertension had QT dispersion measured from a surface 12-lead electrocardiogram, and two-dimensional echocardiography performed to measure interventricular septal thickness, posterior wall thickness, and left ventricular internal diameter. Office blood pressure was recorded, and in 56 patients, 24 h ambulatory blood pressure monitoring was also done. Multivariate analysis demonstrated significant relationships between QT dispersion and office systolic blood pressure, and left ventricular mass index. Similar findings were obtained when QT dispersion was corrected for heart rate (QTc dispersion). After patients with electrocardiographic left ventricular hypertrophy (n = 5) were excluded from the analysis, the above relationships persisted. Increased QT dispersion is thus found in those essential hypertensives at greatest risk of sudden death. Since this relationship persists even in the absence of electrocardiographic left ventricular hypertrophy, measurement of QT dispersion might be a simple, non-invasive screening procedure to identify those hypertensives at greatest risk of sudden death.

QT dispersion and sudden unexpected death in chronic heart failure.

Death in chronic heart failure (CHF) can be from progression of disease or sudden and unexpected. We have attempted to identify factors that predict sudden death in CHF. We followed up 44 patients with CHF for 12-50 (mean 36) months. 4 patients died of non-cardiovascular causes and were excluded from analysis. There were 7 sudden deaths (symptoms for less than 1 h in a previously stable patient) and 12 from progressive CHF. Patients who died of progressive CHF had lower left-ventricular ejection fractions and higher concentrations of atrial natriuretic factor than the 21 survivors, but there were no differences in these variables between survivors and those who died suddenly. However, the sudden death group had significantly (p < 0.05) greater inter-lead variability in the QT interval on the electrocardiogram (QT dispersion; 98.6 [95% CI 79.1-118] ms1/2) than survivors (53.1 [41.9-64.3] ms1/2) or the group who died from progressive CHF (66.7 [51.8-81.6] ms1/2). QT dispersion is a marker of myocardial electrical instability. The association of increased QT dispersion with sudden death suggests that patients at high risk of such death could be identified by means of this simple, reproducible test. This group might benefit from more intensive treatment.