Discovery of lead compounds targeting the bacterial sliding clamp using a fragment-based approach

The bacterial sliding clamp (SC), also known as the DNA polymerase III β subunit, is an emerging antibacterial target that plays a central role in DNA replication, serving as a protein-protein interaction hub with a common binding pocket to recognize linear motifs in the partner proteins. Here, fragment-based screening using X-ray crystallography produced four hits bound in the linear-motif-binding pocket of the Escherichia coli SC. Compounds structurally related to the hits were identified that inhibited the E. coli SC and SC-mediated DNA replication in vitro. A tetrahydrocarbazole derivative emerged as a promising lead whose methyl and ethyl ester prodrug forms showed minimum inhibitory concentrations in the range of 21-43 μg/mL against representative Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for identifying bacterial sliding clamp inhibitors as lead compounds with broad-spectrum antibacterial activity. © 2014 American Chemical Society

Structure and function of a spectrin-like regulator of bacterial cytokinesis

© 2014 Macmillan Publishers Limited. All rights reserved. Bacterial cell division is facilitated by a molecular machine - the divisome - that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family

Gestational age influences the early microarchitectural changes in response to mechanical ventilation in the preterm lamb lung

Background: Preterm birth is associated with abnormal lung architecture, and a reduction in pulmonary function related to the degree of prematurity. A thorough understanding of the impact of gestational age on lung microarchitecture requires reproducible quantitative analysis of lung structure abnormalities. The objectives of this study were (1) to use quantitative histological software (ImageJ) to map morphological patterns of injury resulting from delivery of an identical ventilation strategy to the lung at varying gestational ages and (2) to identify associations between gestational age-specific morphological alterations and key functional outcomes. Method: Lung morphology was compared after 60 min of a standardized ventilation protocol (40 cm H2O sustained inflation and then volume-targeted positive pressure ventilation with positive end-expiratory pressure 8 cm H2O) in lambs at different gestations (119, 124, 128, 133, 140d) representing the spectrum of premature developmental lung states and the term lung. Age-matched controls were compared at 124 and 128d gestation. Automated and manual functions of Image J were used to measure key histological features. Correlation analysis compared morphological and functional outcomes in lambs aged ≤128 and >128d. Results: In initial studies, unventilated lung was indistinguishable at 124 and 128d. Ventilated lung from lambs aged 124d gestation exhibited increased numbers of detached epithelial cells and lung tissue compared with 128d lambs. Comparing results from saccular to alveolar development (120-140d), lambs aged ≤124d exhibited increased lung tissue, average alveolar area, and increased numbers of detached epithelial cells. Alveolar septal width was increased in lambs aged ≤128d. These findings were mirrored in the measures of gas exchange, lung mechanics, and molecular markers of lung injury. Correlation analysis confirmed the gestation-specific relationships between the histological assessments and functional measures in ventilated lambs at gestation ≤128 vs. >128d.Conclusion: Image J allowed rapid, quantitative assessment of alveolar morphology, and lung injury in the preterm lamb model. Gestational age-specific patterns of injury in response to delivery of an identical ventilation strategy were identified, with 128d being a transition point for associations between morphological alterations and functional outcomes. These results further support the need to develop individualized respiratory support approaches tailored to both the gestational age of the infant and their underlying injury response

Differences in the signaling pathways of α1A- and α1B-adrenoceptors are related to different endosomal targeting

Aims: To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods: Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions: Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Effect of Peer Health Workers on AIDS Care in Rakai, Uganda: A Cluster-Randomized Trial

Human resource limitations are a challenge to the delivery of antiretroviral therapy (ART) in low-resource settings. We conducted a cluster randomized trial to assess the effect of community-based peer health workers (PHW) on AIDS care of adults in Rakai, Uganda.15 AIDS clinics were randomized 2:1 to receive the PHW intervention (n = 10) or control (n = 5). PHW tasks included clinic and home-based provision of counseling, clinical, adherence to ART, and social support. Primary outcomes were adherence and cumulative risk of virologic failure (>400 copies/mL). Secondary outcomes were virologic failure at each 24 week time point up to 192 weeks of ART. Analysis was by intention to treat. From May 2006 to July 2008, 1336 patients were followed. 444 (33%) of these patients were already on ART at the start of the study. No significant differences were found in lack of adherence (<95% pill count adherence risk ratio [RR] 0.55, 95% confidence interval [CI] 0.23-1.35; <100% adherence RR 1.10, 95% CI 0.94-1.30), cumulative risk of virologic failure (RR 0.81, 95% CI 0.61-1.08) or in shorter-term virologic outcomes (24 week virologic failure RR 0.93, 95% CI 0.65-1.32; 48 week, RR 0.83, 95% CI 0.47-1.48; 72 week, RR 0.81, 95% CI 0.44-1.49). However, virologic failure rates >or=96 weeks into ART were significantly decreased in the intervention arm compared to the control arm (96 week failure RR 0.50, 95% CI 0.31-0.81; 120 week, RR 0.59, 95% CI 0.22-1.60; 144 week, RR 0.39, 95% CI 0.16-0.95; 168 week, RR 0.30, 95% CI 0.097-0.92; 192 week, RR 0.067, 95% CI 0.0065-0.71).A PHW intervention was associated with decreased virologic failure rates occurring 96 weeks and longer into ART, but did not affect cumulative risk of virologic failure, adherence measures, or shorter-term virologic outcomes. PHWs may be an effective intervention to sustain long-term ART in low-resource settings.ClinicalTrials.gov NCT00675389

A Comparison of rpoB and 16S rRNA as Markers in Pyrosequencing Studies of Bacterial Diversity

Background: The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. &lt;p/&gt;Methodology/Principal Findings: Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster. &lt;p/&gt;Conclusions/Significance: The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed

Age-related changes in rat bone-marrow mesenchymal stem cell plasticity

<p>Abstract</p> <p>Background</p> <p>The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients.</p> <p>Results</p> <p>We demonstrated that the expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs.</p> <p>Conclusion</p> <p>The results suggest age related differences in the differentiation capacity of BM-MSCs. These changes may affect the efficacy of BM-MSCs for use in stem cell therapy.</p

Using Phylogenetic, Functional and Trait Diversity to Understand Patterns of Plant Community Productivity

BACKGROUND:Two decades of research showing that increasing plant diversity results in greater community productivity has been predicated on greater functional diversity allowing access to more of the total available resources. Thus, understanding phenotypic attributes that allow species to partition resources is fundamentally important to explaining diversity-productivity relationships. METHODOLOGY/PRINCIPAL FINDINGS:Here we use data from a long-term experiment (Cedar Creek, MN) and compare the extent to which productivity is explained by seven types of community metrics of functional variation: 1) species richness, 2) variation in 10 individual traits, 3) functional group richness, 4) a distance-based measure of functional diversity, 5) a hierarchical multivariate clustering method, 6) a nonmetric multidimensional scaling approach, and 7) a phylogenetic diversity measure, summing phylogenetic branch lengths connecting community members together and may be a surrogate for ecological differences. Although most of these diversity measures provided significant explanations of variation in productivity, the presence of a nitrogen fixer and phylogenetic diversity were the two best explanatory variables. Further, a statistical model that included the presence of a nitrogen fixer, seed weight and phylogenetic diversity was a better explanation of community productivity than other models. CONCLUSIONS:Evolutionary relationships among species appear to explain patterns of grassland productivity. Further, these results reveal that functional differences among species involve a complex suite of traits and that perhaps phylogenetic relationships provide a better measure of the diversity among species that contributes to productivity than individual or small groups of traits